201224148 六、發明說明： 【發明所屬之技術領立成】 [0001] 本發明涉及一種神經移植體的製備方法，尤其係涉及一 種可供生物體移植的神經移植體的製備方法。 [先前技術] [0002] 神經系統主要由神經元（neurons)以及神經膠質細胞 (neuron glial cells)構成一複雜且特異的溝通網域 ，用以與其他組織或器官建立連結以進行功能協調。神 經系統中，係由神經元來執劈争收刺激、通過傳導並輸 〇 出神經遞質（neuron transmit ter)以進行組織或器官 間訊息溝通，.而神經膠質細岭則執行神經元物理性支援 、營養提供以及調節溝通訊息速度等功能。每一神經元 依據型態包含胞體（cel 1 body)與神經寒舞（neurite) 兩部分’神經突起自胞體延伸並朝向其他神經元或係其 他細胞（例如··肌肉細胞）生長，其中神'經突起又分為轴 突（axon)與樹突（dendrite)兩種、一般I說，刺激由 樹突接收並將衝動傳向胞體，衝動經過轴突傳導至軸突 末端，並釋放#導物質來觸動其他細胞。 [0003] 由於神經系統扮演生物體内各組織與器官之間的協調作 用，其重要性不言可喻。先前，因神經系統受損而導致 的神經缺損係臨床常見的致殘性疾病。其中，通過植入 神經移植體來修復受損的神經系統，係神經外科手術用 來修復因各種情況引㈣神經系統損傷的—種重要手段 。先前的神經移植體通常為“橋接”在神經系統受損： 位兩端的神經管，該神經管由生物降解材料製成的管狀 099143856 表單編號A0101 第3頁/共38頁 201224148 結構。神經系統受損部位—端的神經元沿所述神經管内 另 ^生長出神經犬起以到達所述神經系統受損部位的 端0 [0004] …，心呷羥管的方式進行修復的的受 ^部位的長度較長、，而所述神經突起的生長過程非常緩 又,J用所述神經管來修復受損的神經系統所需的 修復時間較長。 【發明内容】 [0005] [0006] [0007] 099143856 有鑒於糾種神經移植體的製備方法實為必要， 由》亥裝備方法t備的神經移植體能夠減少受損的神經系 統的修復時間。 -種神經移植體的製備方法，其包括如下步驟：提供一 培育層’所述培育層包括_奈米碳管㈣構及設置在該 奈米碳管膜結構表面的—蛋白質層；在該蛋白質層表面 種植多個神經細胞；以及培育該多個神經細胞直到該多 個神經細胞生長出多個神經突起並該多個神經細胞之間 的神經突起相互連接形成一神經網路。 相較於先前技術，所述神經移植體的製備方法通過在該 奈米碳管膜結構表面設置蛋白制形成培育層並在所 述培育層的表面形成所述神經網路。所述奈米碳管膜結 構具有彈性佳、延展性良好及密度低等優點故，所述 神經移植體可根據受損神經系統的受損部位的形狀、大 小進行裁剪、拉伸並植入受損部位。所述神經網路具有 生物活性及仏號傳遞能力，從而使得包括所述神經網路 的神經移植體也具有生物活性及信號傳遞能力。當所述 表單編號A0101 第4頁/共38頁 0992075911-0 201224148 珅經移植體植人生物體中的受損部位時，由於所述神經 植入體中的神經^與所述受損部位兩端或邊緣的神經元 的距離較近，故可通過直接缝合所述神經植入體中的神 經元與受損部位邊緣的神經元的方式使所述受損部位的 兩端建立起信號傳遞能力，完成受損部位的神經修復，、 從而節省所述神經突起的生長時間，減少受損的神= 統的修復時間。 [0008] Ο [0009] [0010] [0011] 【實施方式】 請參閱圖1，本發明提供一種神唑移植體的製備方法 包括： ，其 S10 ’提供-培育層，所述培育層包括—奈米碳管膜結構 及設置在該奈米碳管膜結構表面Ρ一蛋白質層. S20，在該蛋白質層表面種植複數神經細胞；以及201224148 VI. Description of the Invention: [Technology of the Invention] [0001] The present invention relates to a method for preparing a nerve graft, and more particularly to a method for preparing a nerve graft for transplantation of an organism. [Prior Art] [0002] The nervous system is mainly composed of neurons and neuron glial cells, which constitute a complex and specific communication domain for establishing links with other tissues or organs for functional coordination. In the nervous system, neurons are used to compel and stimulate, transmit and transmit neurotransmitters (neuron transmit ter) for tissue or organ communication, while glial ridges perform neuronal physicality. Support, nutrition, and the ability to adjust the speed of communication messages. Each neuron depends on the type including the cel 1 body and the neurite 'neurites' extending from the cell body and growing toward other neurons or other cells (eg, muscle cells), The god's protrusion is divided into two types: axon and dendrite. Generally speaking, the stimulus is received by the dendrite and transmits the impulse to the cell body. The impulse is transmitted through the axon to the end of the axon and released. The substance is used to touch other cells. [0003] Since the nervous system plays a coordinating role between tissues and organs in a living body, its importance is self-evident. Previously, neurological deficits due to impaired nervous system were clinically common disabling diseases. Among them, the implantation of a nerve graft to repair the damaged nervous system is an important means for neurosurgery to repair the damage of the nervous system caused by various conditions. Previous nerve grafts are usually "bridged" in the nervous system: the neural tube at both ends, the tube is made of biodegradable material. 099143856 Form No. A0101 Page 3 of 38 201224148 Structure. The damaged part of the nervous system - the end of the neuron grows out of the neural tube to reach the end of the damaged part of the nervous system [0004] ..., the heart of the hydroxy tube is repaired by the ^ The length of the site is long, and the growth process of the neurite is very slow, and the repair time required for the nerve tube to repair the damaged nervous system is longer. SUMMARY OF THE INVENTION [0006] [0007] [0007] 099143856 In view of the fact that the preparation method of the nerve graft is necessary, the nerve graft prepared by the method can reduce the repair time of the damaged nervous system. - a method for preparing a nerve graft, comprising the steps of: providing a culture layer comprising: a carbon nanotube (four) structure and a protein layer disposed on a surface of the carbon nanotube membrane structure; Planting a plurality of nerve cells on the surface of the layer; and cultivating the plurality of nerve cells until the plurality of nerve cells grow a plurality of neurites and the neurites between the plurality of nerve cells are connected to each other to form a neural network. In contrast to the prior art, the preparation method of the nerve graft is formed by forming a culture layer on the surface of the carbon nanotube membrane structure and forming the neural network on the surface of the growth layer. The carbon nanotube membrane structure has the advantages of good elasticity, good ductility and low density, and the nerve graft can be cut, stretched and implanted according to the shape and size of the damaged part of the damaged nervous system. Damaged part. The neural network has biological activity and nickname transmission ability, so that the nerve graft including the neural network also has biological activity and signal transmission ability. When the form number A0101 page 4/38 page 0992075911-0 201224148 is transplanted to the damaged part of the human body, due to the nerve in the nerve implant and the two ends of the damaged part The distance between the neurons of the edge or the edge is relatively close, so that the signal transmission capability can be established at both ends of the damaged portion by directly suturing the neurons in the nerve implant and the neurons at the edge of the damaged portion. The nerve repair of the damaged part is completed, thereby saving the growth time of the neurite, and reducing the repair time of the damaged god. [0008] [0010] [Embodiment] Referring to FIG. 1, the present invention provides a method for preparing a dizolium implant comprising: a S10 'providing-cultivating layer, the cultivating layer comprising - a carbon nanotube membrane structure and a protein layer disposed on the surface of the carbon nanotube membrane structure. S20, implanting a plurality of nerve cells on the surface of the protein layer;
[0012] S30，培育該複數神經細胞直到該複數神經細胞生長出複 數神經突起且該複數神經細胞之間的神經突起相 ,||· ,,：-··^ |··''· , V^· 形成一神經網路。 t Jllli (¾ 'ί' Γ ·· Ϊ； Ιτ 在所述步驟S10中，所述奈米碳管膜結構由複數 組成。所述複數奈米碳管的延伸方向可基本平行於 奈米碳管膜結構的表面。優選地，所述複數奈米I ^ 間通過凡得瓦力（Van der Waals . attract iv ^ force)連接，從而形成一自支稽結構。所謂‘‘自支律’’ 即該奈米碳管膜結構無需通過設置於一基體表面 ’亦能 保持自身特定的形狀。由於該自支撐的奈米碳;^ 興結構 中大量的奈米碳管通過凡得瓦力相互吸引，從而使#^ 099143856 表單編號Α0101 第5頁/共38頁 〇992〇7591 201224148 米碳管膜結構具有特定的形狀，形成一自支撐結構。所 述奈米碳管膜結構為自支撐結構時，該奈米碳管膜結構 可為由至少一奈米碳管膜形成的膜狀結構，當所述奈米 碳管膜結構包括複數奈米碳管膜時，該複數奈米碳管膜 層疊設置，相鄰的奈米碳管膜之間通過凡得瓦力相結合 。由於所述奈米碳管膜基本由奈米碳管組成且奈米碳管 之間通過凡得瓦力連接，故所述奈米碳管膜結構具有彈 性佳、延展性良好及密度低等優點，便於裁剪和拉伸。 [0013] 請參閱圖2，所述奈米碳管膜可為一奈米碳管絮化膜，該 奈米碳管絮化膜為將一奈米碳管原料絮化處理獲得的一 自支撐的奈米碳管膜。該奈米碳管絮化膜包括相互纏繞 且均勻分佈的奈米碳管。奈米碳管的長度大於10微米， 優選為200微米到900微米，從而使奈米碳管相互纏繞在 一起。所述奈米碳管之間通過凡得瓦力相互吸引、分佈 ，形成網路狀結構。由於該自支撐的奈米碳管絮化膜中 大量的奈米碳管通過凡得瓦力相互吸引並相互纏繞，從 而使該奈米碳管絮化膜具有特定的形狀，形成一自支撐 結構。所述奈米碳管絮化膜各向同性。所述奈米碳管絮 化膜中的奈米碳管為均勻分佈，無規則排列，形成大量 尺寸在1奈米到500奈米之間的間隙或微孔。所述間隙或 微孔能夠增加所述奈米碳管膜的比表面積及浸潤更多的 蛋白質。 [0014] 所述奈米碳管膜可為一奈米碳管碾壓膜，該奈米碳管碾 壓膜為通過碾壓一奈米碳管陣列獲得的一種具有自支撐 性的奈米碳管膜。該奈米碳管碾壓膜包括均勻分佈的奈 099143856 表單編號A0101 第6頁/共38頁 0992075911-0 201224148 米碳管，奈米碳管沿同一方向或不同方向擇優取向排列 。所述奈米碳管碾壓膜中的奈米碳管相互部分交迭，並 通過凡得瓦力相互吸引，緊密結合，使得該奈米碳管膜 具有很好的柔韌性，可彎曲折迭成任意形狀而不破裂。 - 且由於奈米碳管碾壓膜中的奈米碳管之間通過凡得瓦力 ' 相互吸引，緊密結合，使奈米碳管碾壓膜為一自支撐的 結構。所述奈米碳管碾壓膜中的奈米碳管與形成奈米碳 管陣列的生長基底的表面形成一夾角/3，其中，召大於 等於0度且小於等於15度，該夾角万與施加在奈米碳管陣 〇 列上的壓力有關，壓力越大，該夾角越小，優選地，該 奈米碳管碾壓膜中的奈米碳管平行於該生長基底排列。 該奈米碳管碾壓膜為通過碾壓一奈米碳管陣列獲得，依 據碾壓的方式不同，該奈米碳管碾壓膜中的奈米碳管具 有不同的排列形式。具體地，奈米碳管可無序排列；請 參閱圖3，當沿不同方向碾壓時，奈米碳管沿不同方向擇 優取向排列；當沿同一方向碾壓時，奈米碳管沿一固定 方向擇優取向排列。該奈米碳管碾壓膜中奈米碳管的長 ❹ 度大於50微米。 [0015] 該奈米碳管碾壓膜的面積與奈米碳管陣列的尺寸基本相 同。該奈米碳管碾壓膜厚度與奈米碳管陣列的高度以及 碾壓的壓力有關，可為0. 5奈米到10 0微米之間。可以理 解，奈米碳管陣列的高度越大而施加的壓力越小，則製 備的奈米碳管碾壓膜的厚度越大；反之，奈米碳管陣列 的高度越小而施加的壓力越大，則製備的奈米碳管碾壓 膜的厚度越小。所述奈米碳管碾壓膜之中的相鄰的奈米 099143856 表單編號A0101 第7頁/共38頁 0992075911-0 201224148 碳管之間具有一定間隙，從而在奈米碳管碾壓膜中形成 複數尺寸在1奈米到500奈米之間的間隙或微孔。所述間 隙或微孔能夠增加所述奈米碳管膜的比表面積及浸潤更 多的蛋白質。 [0016] 所述奈米碳管膜可為一奈米碳管拉膜，所述奈米碳管拉 膜係由若干奈米碳管組成的自支撐結構。請參閱圖4，所 述若干奈米碳管為沿該奈米碳管拉膜的長度方向擇優取 向排列。所述擇優取向係指在奈米碳管拉膜中大多數奈 米碳管的整體延伸方向基本朝同一方向。而且，所述大 多數奈米碳管的整體延伸方向基本平行於奈米碳管拉膜 的表面。 [0017] 進一步地，所述奈米碳管拉膜中多數奈米碳管係通過凡 得瓦力首尾相連。具體地，所述奈米碳管拉膜中基本朝 同一方向延伸的大多數奈米碳管中每一奈米碳管與在延 伸方向上相鄰的奈米碳管通過凡得瓦力首尾相連。當然 ，所述奈米碳管拉膜中存在少數偏離該延伸方向的奈米 碳管，這些奈米碳管不會對奈米碳管拉膜中大多數奈米 碳管的整體取向排列構成明顯影響。所述自支撐為奈米 碳管拉膜不需要大面積的載體支撐，而僅相對兩邊提供 支撐力即能整體上懸空而保持自身膜狀狀態，即將該奈 米碳管拉膜置於（或固定於）間隔一定距離設置的兩個 支撐體上時，位於兩個支撐體之間的奈米碳管拉膜能夠 懸空保持自身膜狀狀態。所述自支撐主要通過奈米碳管 拉膜中存在連續的通過凡得瓦力首尾相連延伸排列的奈 米碳管而實現。具體地，所述奈米碳管拉膜中基本朝同 099143856 表單編號A0101 第8頁/共38頁 0992075911-0 201224148 一方向延伸❹數奈米碳管，並非絕對的直線狀，可適 當的彎H者齡完全按料伸方向上顧，可適當 的偏離延伸方向。故’不能排除奈米碳管拉膜的基本朝 二-方向延伸的多數奈米碳管中並列的奈米碳管 之間可 能存在部分接觸。具體地，該奈㉔管_包括複數連 續且的奈米碳管諸。該複數奈树管片段通 過凡得瓦力首尾減。每碳管片段錢數相互平 灯的奈米碳管組成。該奈米碳管片段具有任意的長度、 Ο [0018] 厚度、均勻性及形狀。該奈来碳管_具有較好的透光 性’可見光透過率可達到75%以上。 當該奈米奸職構包括複_米奸拉_，所述複 數奈来碳管拉媒層疊設置形成—層狀結構。該層狀結構 的厚度不限’相鄰的奈米碳管_通過凡得瓦力結合。 優選地所述層狀結構包括的奈来碳管膜的唐數小於或 等於10層，從而使單位面積内的奈米碳管數量較少，使 S不米反管自身的拉曼光強保持在較小的範園，從而減 小拉义光譜中奈米想的拉曼蜂強。該層狀Μ構中相鄰 的奈来碳管拉膜中的奈米碳管之間具有-交又角度α， 且該α大於〇度且小於等於9〇度。當相鄰的奈米碳管拉膜 中的奈来&管之間具有一交又角度α時，所述複數奈米 碳管拉膜中的奈米碳管相互交織形成-神經移植體，使 所述奈米碳以結構的機械性能增加。在本實施例中， 所述奈米碳管騎構包滅數層奈/切管㈣層叠設置 ，相鄰的奈米碳管膜中的奈米碳管之間的交又角度“大 致等於90度，即’相鄰奈米碳管拉膜中的奈米碳管的延 099143856 表Α0101 第9貢/共38頁 0992075911-0 201224148 伸方向大致平行。 [0019] 所述蛋白質層設置在所述奈米碳管膜結構的一個表面或 兩個相對的表面，用於使所述奈米碳管層具有親水性及 生物相容性，從而使得所述培育層能夠為所述神經細胞 的種植及生長提供一個合適的環境。優選地，所述蛋白 質層中的蛋白質（prote iη)為可溶性蛋白質。所述蛋白 質選自纖維狀蛋白質、血漿蛋白質及酶蛋白質等。在本 實施例中，所述蛋白質為哺乳動物的血清，如緒血清、 牛金清或人血清等。所述血清不僅能夠為所述神經細胞 的種植及生長提供一個合適的環境，還能夠在所述神經 細胞生長時，為所述神經細胞提供生長因數。 [0020] 所述培育層的製備方法不限，僅能夠使蛋白質層與所述 奈米碳管膜結構混合在一起即可。譬如，可通過將所述 奈米碳管膜結構浸泡在一蛋白質溶液中，使所述蛋白質 溶液浸潤所述奈米碳管膜結構，從而使得所述蛋白質溶 液中的蛋白質附著在所述奈米碳管膜結構的一個表面或 者兩個相對設置的表面形成蛋白質層。亦可將所述蛋白 質溶液喷塗在所述奈米碳管膜結構一個表面或者兩個相 對設置的表面，使所述蛋白質層設置在奈米碳管膜結構 一個表面或者兩個相對設置的表面。還可將蛋白質溶液 滴在所述奈米碳管膜結構一個表面或者兩個相對設置的 表面，再採用甩膜的方式使所述蛋白質層設置在奈米碳 管膜結構一個表面或者兩個相對設置的表面。通常，當 所述奈米碳管膜結構的厚度較大時，可控制所述蛋白質 僅設置在所述奈米碳管膜結構的一個表面。而當所述奈 099143856 表單編號A0101 第10頁/共38頁 0992075911-0 201224148 Ο [0021] Ο [0022] 米碳管膜結構的厚度較小時，所述蛋”通常設置在所 述奈米碳管膜結構相對的兩個表面。所述蛋白質溶液除 所述蛋白質外’還可包括溶解所述蛋白質的生物媒介 (biological media)，所述生物媒介的種類不限可 根據蛋白質的種類的不同而調製。通常，所述蛋白質溶 液中的蛋白質的濃度大於等於5〇%小於等於麵。在本實 施例中’所述蛋白質溶液中的蛋白質濃度為議，即， 所述蛋白質,錢為純蛋自質，無需溶劑溶解。 T所述奈米碳管膜結構氣括嫌數奈米碳管且複數奈来 =官之間存在間隙形成複數微孔，當所述蛋白質溶液浸 :::奈:碳管膜結構時’所述蛋白質溶液可滲透入所 洛^、，管臈結構内部浸潤.所述.複數条求碳管的表面。 :财P培育層中’並不倾有奈W管的表面均 乎二：腔自質層’然，僅需位於需培育神經細胞的奈 構的表_部分奈米碳管㈣有蛋白質溶液 P可在所述$米碳伽結構表面形成蛋白質層使所 ^来碳管膜結構具有親水性及生物相紐，實現使培 :==Γ胞生長的載體的功能。這係因為， :於不未碳管具有疏水性，由奈米射組成的夺米碳管 ΓΓ不能為神經細胞生長提供適合的親水性環境。 而“米碳管表面覆蓋有具有親水性及無毒性的蛋白質 層後，由覆蓋有蛋白曾Μ 的奈米碳管組成的結構即能為細 胞生長提㈣合m㈣境。在本實施财，所述培 育層的製備方法可進—步包括如下步驟： sl1，提供所述奈来碳營膜結構； 099143856 表單編號A0101 第11頁/共38頁 0992075911-0 201224148 [0023] S12，使所述奈米碳管膜結構浸潤有蛋白質溶液；以及 [0024] S1 3，對浸潤有蛋白質溶液的奈米碳管膜結構進行滅菌處 理形成所述培育層。 [0025] 在步驟S12中，使所述奈米碳管膜結構浸潤有蛋白質溶液 的方式不限，僅使蛋白質溶液中的蛋白質附著在奈米碳 管膜結構表面形成一蛋白質層即可。譬如，可通過將所 述奈米碳管膜結構浸泡在所述蛋白質溶液中，實現蛋白 質溶液的浸潤。亦可通過在所述奈米碳管膜結構喷塗所 述蛋白質溶液，實現蛋白質溶液的浸潤。在本實施例中 ，為實現蛋白質溶液的浸潤，選擇將所述奈米碳管膜結 構浸泡在純蛋白質中。所述奈米碳管膜結構的浸泡時間 依奈米碳管膜結構的具體結構及蛋白質溶液的具體組分 而定，僅能使所述奈米碳管膜結構中的大部分奈米碳管 浸潤有蛋白質溶液即可。通常，所述奈米碳管膜結構的 浸泡時間在2小時以上。所述奈米碳管膜結構的浸泡的環 境不限，僅不使所述蛋白質變質即可。通常，所述浸泡 過程可在常溫、常壓環境下進行。 [0026] 在步驟S12中，由於所述奈米碳管膜結構為一自支撐結構 ，從而該奈米碳管膜結構直接浸泡在所述蛋白質溶液中 。當然，為減少所述奈米碳管膜結構浸泡在蛋白質溶液 中時受液體表面張力影響而產生破損的概率，所述步驟 S1 2還可進一步包括如下步驟：S1 21，將所述奈米碳管膜 結構預先設置在一疏水性基底表面。為使所述奈米碳管 膜結構與所述疏水性基底結合緊密，還可對設置在所述 疏水性基底表面上的奈米碳管膜結構進行有機溶劑處理 099143856 表單編號A0101 第12頁/共38頁 0992075911-0 201224148 。所述疏水性基底還必須對生物體沒有毒性’從而不破 壞所述蛋白質的活性。優選地，所述疏水性基底還可具 有良好的延展性，如矽膠基底。 [0027] 在歩驟S13中，對浸潤有蛋白質溶液的奈米碳管膜結構進 订滅菌處理的方式不限，僅能夠殺死蛋白質溶液中的大 分細菌即可。譬如可採用高溫滅菌或紫外光滅菌的方 式對所述蛋白質溶液進行滅菌。在本實施例中’採用高 溫殺菌的方式對該蛋白質溶液進行滅菌。當然’為使所 Ο 述蛋白質溶液中的蛋白質不暴於被破壞，高溫滅菌時的 . .......:... ... .. 溫度不得超過220度。在本實施例中，所述高溫滅菌時的 溫度大致為120度。可以理解，當所述蛋白質溶液中本身 細菌較少，則該步驟S13則可省略當所述在步驟S13中 ’浸潤有蛋白質溶液的奈米碳管膜結構進行滅菌處理時 ::... ’蛋白質溶液中的溶劑或水分將減少。通常地，浸潤在 所述奈米碳管膜結構中的蛋白質溶液隨著溶劑或水分的 ❹ [0028] 減少而固化’從而在所述奈米碳管膜結構的表面形成所 述蛋白質層》 在步驟S10中’為増加該培育層對神經細胞的附著性及提 供更適合神經細胞的生長環境，在形成蛋白質層後，該 步驟Sl〇還可進一步包括如下步驟：sl4 ,在所述培育層 中蛋白質層的表面形成一聚賴氨酸（pjy—D—iysine， PDL )層。具體地，可將所述培育層浸泡在一聚賴氨酸溶 液令’所述聚賴氨酸溶液中聚賴氨酸的濃度大致為2〇微 克每毫升。 在步驟S20中’所述神經細胞包括哺乳動物的神經細胞， 099143856 表單編Sfe A0101 第 13 真/共 38 f 0992075911-0 [0029] 201224148 優選地，所述神經細胞為海馬神經元。在該培育層表面 種植複數神經細胞的方法不限，可採用在該培育層表面 喷射或塗覆含有該神經細胞的溶液，亦可採用將該培育 層浸泡在所述含神經細胞的神經細胞液中，僅使所述神 經細胞液覆蓋所述培育層即可。為使所述神經細胞液覆 蓋所述培育層，所述神經細胞液可盛放在一培養皿中。 所述培育層可懸空設置在所述培養孤中。亦可設置在所 述培養皿的一底面上，僅能使所述培養液覆蓋所述神經 細胞即可。當所述培育層設置在所述培養胍的底面上時 ，所述培育層與培養皿的底面之.間必須設置有一疏水性 基體上，以避免所述神經細胞在所述培養皿的底面生長 〇 [0030] 在步驟S30中，所述神經細胞的培育環境不限，僅能夠生 長出神經突起即可。通常，所述神經細胞在常溫、常壓 環境中即可生長。即，將所述神經細胞放置在室内環境 中，所述神經細胞即可生長，而培育層中的蛋白質如牛 血清可提供生長因數，促進該神經細胞生長。當然，亦 可使該培育環境接近提供該神經細胞的生物體的體内生 長環境亦可。譬如，當所述神經細胞為取自老鼠的海馬 神經細胞時，可模擬所述老鼠體内的生長環境。 [0031] 所述神經細胞在培育時，能夠長出複數神經突起（Neur-ite)。所述神經突起包括樹突（Dendrite)與軸突（ Axon)。當所述培育層表面僅有一個神經細胞時，所述 神經細胞的神經突起沿培育層的表面朝各個方向隨機生 長。然由於神經細胞本身會釋放出誘導神經突起定向生 099143856 表單編號A0101 第14頁/共38頁 0992075911-0 201224148 長的因數’故，當所述培育層表面設置有複數神經細胞 時’該神經細胞的神經突起具有將沿向相鄰的神經細胞 生長的趨勢，從而使相鄰的神經細胞得以連接溝通。故 ，控制神經細胞在所述培育層的分佈，即可控制所述神 經突起的生長方向。譬如，如果所述神經細胞係隨機均 勻分佈在所述培育層的表面，所述神經細胞將各自生長 出神經突起與相鄰的細胞連接’當所述複數神經細胞的 全部或大多數神經細胞均生長出連接在相鄰的神經細胞 之間的神經突起時，所述複數神經細胞借由所述神經突 ..... ..... . 起形成所述神經網崦，使該複數神，經細胞之間能相互溝 通》相鄰的神經細胞的神經突起知果相;遇，則會合為同 一個神經突起^再譬如，當所述神經細胞在該培育層表 面以線狀或者陣列的方式排列時，且沿縱佝方向的神經 細胞相距較近，而沿橫向方向的神經細胞相距較遠，此 時，所述神經細胞所生長的神經細胞可基本沿所述縱向 方向延伸。為使所述神經細胞能夠在所述培育層表面以 線狀或者陣列的方式排列，可選擇使所述奈米碳管膜中 的奈米碳管基本沿同一方向延伸i(>，通過培育，彼此相鄰 的神經細胞大多通過神經突起建立起連接，從而形成所 述神經網路。所述神經網路與所述培育層—起形成所述 神經移植體。 [0032]所述奈米妷管膜結構為一宏觀的膜狀結構，其面積一般 都<達到15毫米χΐ5毫米以上’具體地，該奈米碳管膜結 構的長度可達300米以上，寬度可達〇 5米以上。且該奈 米破管膜結構具有雜佳、職性良好、不含金屬及密 099143856 表箪編號A0101 第15頁/共38頁 0992075911-0 201224148 度低等優點，可直接植入生物體。故，由所述奈米碳管 膜結構做主要載體的神經移植體可根據受損神經系統的 受損部位的形狀、大小進行裁剪、拉伸並植入受損部位 。所述神經網路具有生物活性及信號傳遞能力，從而使 得包括所述神經網路的神經移植體亦具有生物活性及信 號傳遞能力。當所述神經移植體植入生物體中的受損部 位時，由於所述神經植入體中的神經元與所述受損部位 兩端或邊緣的神經元的距離較短，故可通過直接缝合所 述神經植入體中的神經元與受損部位邊緣的神經元的方 式使所述受損部位的兩端建立起信號傳遞能力，完成受 損部位的神經修復，從而節省所述神經突起的生長時間 ，減少受損的神經系統的修復時間。可以理解，即便係 在所述神經植入體植入受損部位時，不進行直接縫合， 由於所述神經植入體中的神經元所述受損部位邊緣的神 經元的距離小於所述受損部位兩端的神經元的距離，故 ，通過植入所述神經植入體，亦能減少神經突起的生長 時間，從而減少受損的神經系統的修復時間。 [0033] 需要指出的係，通常情況下，所述奈米碳管膜結構中的 奈米碳管係指未經過化學或物理處理的奈米碳管，如未 經過表面親水性處理的奈米碳管，即，所述奈米碳管為 純奈米碳管。當然，奈米碳管膜結構中的奈米碳管如果 係經過改性的奈米碳管，僅係對神經細胞沒有毒性，亦 應在在本發明的保護範圍之内，只係，所述奈米碳管的 改性並不會對實現本發明有任何實質性貢獻，因為，當 所述蛋白質層覆蓋該奈米碳管後，所述神經細胞與所述 099143856 表單編號A0101 第16頁/共38頁 0992075911-0 201224148 奈米碳管衫直接朗1奈米碳管 係可忽略的。 的表面結構實際上 [0034] 本發明提供的神經移植體可 備方法在包__結構及==體的製 培養由複數神經細胞及神經突層的培育層表面 .„ 大起形成的神經網路所得到 產品 [0035] Ο [0036] [0037] Ο 明參閱圖5，所述神經移植體 @1η. 〇包括一培育層ίο及分佈 在以培月層1G表面的—神經網路20。 所^培育㈣包括—奈料Μ結㈣及設置 米碳管膜結構12的一個表&# L不 哲品" 或者相對的兩個表面的蛋白 質層14。在本實施例中，所 .^ 所述蛋白質層14僅設置在所述 不米碳管膜結構12的一個表面'。： V 所述奈«管賴㈣包括複衫㈣管基本平行於所 述奈米碳管膜結構的表面，且相鄰的奈米破管之間通過 凡得瓦力相互連接形成-自切結構。所述奈米碳管膜 結構12包括至少—奈料管媒^«管财為如圖2 中的奈米碳管絮化膜、圖3中的奈米碳管礒壓膜及圖4中 的奈米碳管減。在本實，所述奈米碳管膜社構 12包括複數層疊設置的拉膜，相鄰的㈣通過凡彳|以 相互結合4相_拉财，奈^㈣的延伸方向可 具有-個交又角度，優選地，所述交又角度物度。所 述奈求碳管膜結構12的厚度可根據具㈣求而設置。通 常，所述奈米碳管膜結構12厚度大於〇. 3微米小於6〇微米 。在本實施例中，所述奈米碳管膜結構12的厚度大致為 099143856 表單編號A0101 第17頁/共38頁 0992075911-0 201224148 0. 6微米。 [0038] 所述蛋白質層1 4為由可溶性蛋白質組成。所謂可溶性蛋 白質即該蛋白質具有較好的親水性。所述蛋白質層14的 厚度不限，僅能夠提供一個親水性環境即可。通常，所 述蛋白質層14的厚度為0. 3微米到2微米。在本實施例中 ，所述蛋白質層14的厚度大致為0. 5微米。在宏觀上，所 述蛋白質層14可選擇僅設置在所述奈米碳管膜結構12的 一個表面或者相對的兩個表面。在微觀上，所述蛋白質 層14中的蛋白質容易滲透到所述奈米碳管膜結構12的内 部，並包覆所述奈米碳管膜結構12中的部分或者全部奈 米碳管，此時，所述蛋白質層14與該奈米碳管膜結構12 之間並沒有明顯的分介面。通常，當所述奈米碳管膜結 構12的厚度較薄時，譬如，所述奈米碳管膜結構12的厚 度小於等於3微米時，所述蛋白質層14中的蛋白質容易滲 透到所述奈米碳管膜結構12的内部，並基本包覆所述奈 米碳管膜結構12中的所有的奈米碳管。而當所述奈米碳 管膜結構12的厚度較厚時，譬如，所述奈米碳管膜結構 12的厚度大於等於3微米時，所述蛋白質層14中的蛋白質 雖然亦可滲透到所述奈米碳管膜結構12内部，然通常僅 包覆所述奈米碳管膜結構12靠近所述神經網路20的奈米 碳管。在本實施例中，所述蛋白質層14中的蛋白質基本 包覆所述奈米碳管膜結構12中的所有的奈米碳管。 [0039] 所述神經網路20設置在所述蛋白質層14遠離所述奈米碳 管膜結構12的一個表面。當所述神經移植體100僅包括一 個蛋白質14設置在所述奈米碳管膜結構1 2的一個表面時 099143856 表單編號Α0101 第18頁/共38頁 0992075911-0 201224148 [0040] ❹ [0041] [0042] ’所述神經移植體100僅包括一個神經網路20。當所述蛋 白質層14包括兩個蛋白質層14分別設置在所述奈米碳管 膜結構12的相對的兩個表面時，所述神經移植體1〇〇可包 括兩個神經網路20分別設在所述兩個蛋白質層14遠離所 述奈米碳管膜結構12的表面，亦可僅包括一個神經網路 20設置在其中一個蛋白質層14的表面。在本實施例中， 所述神經移植體1〇〇僅包括一個神經網路20。 可以理解’為提高所述生物移植體1〇〇的抑菌性，提高該 神經移植體100的壽命，所述神經移植體100還可進一步 包括一多聚賴氨酸層設置在所述神經網路20與所述蛋白 質層14之間。 請參閱圖6，所述神經網路20包括複數神經細胞22及自所 述複數神經細胞22延伸出來的複數神經突起24。每一個 神經細胞22延伸出來的神經突起24的個數不限，僅能夠 使所述複數神經細胞22之間建立起生物連接使所述複數 神經細胞22能夠相互溝通卽可.β譬如__，其中一個神經細 胞22可延伸出複數神經突起24或不延伸出任何神經突起 24 -本發明中的神經移植體100，具有修復生物體中神經系統 中的神經網路20設置在該培育層1〇表面。而所述培育層 10中的奈米碳管膜結構12為基本由奈米碳管組成的自支 撐結構，具有不含金屬、彈性佳、不易腐蝕、延展性良 好及低密度等優點。故，該奈米碳管膜結構12可隨同該 由複數神經犬起14連接的複數神經細胞12 —起植入到生 物體中，用於修復生物體中受損的神經系統，且可根據 099143856 表單編號Α0101 第19頁/共38頁 0992075911-0 201224148 生物體中神經系統的創傷面積對所述神經移植體1 00進行 裁剪或拉伸。 [0043] 以下將結合附圖並以具體實施例方式詳細說明本發明的 神經移植體的製備方法及神經移植體。 [0044] 本發明提供一種神經移植體的製備方法，其包括如下步 驟： [0045] S210，將一奈米碳管膜結構浸泡在一蛋白質溶液中。 [0046] 在步驟S210中，所述奈米碳管膜結構包括複數層奈米碳 管拉膜，相鄰的奈米碳管拉膜之間的奈米碳管的延伸方 向具有一交叉角度。請參閱圖7及圖8,優選地，所述交 叉角度大致等於90度。所述蛋白質溶液純牛血清溶液。 請參見圖9，當所述奈米碳管膜結構從所述蛋白質溶液中 浸泡1. 5個小時左右，所述奈米碳管膜結構中的大部分奈 米碳管表面可浸潤有蛋白質溶液。 [0047] S220，從所述蛋白質溶液中取出所述奈米碳管膜結構， 在120攝氏度下進行高溫滅菌處理。 [0048] 在步驟S220中，將所述奈米碳管膜結構從所述蛋白質溶 液中取出後，可在一乾燥箱中進行加熱滅菌處理。所述 乾燥箱的滅菌溫度大致在120度左右，滅菌處理後後，所 述蛋白質溶液中的蛋白質基本固化，在所述奈米碳管膜 結構表面形成一蛋白質層，從而形成一培育層。 [0049] S230，在所述培育層浸泡在一聚賴氨酸溶液中。 [0050] 在步驟S230中，所述聚賴氨酸溶液中的聚賴氨酸的濃度 099143856 表單編號A0101 第20頁/共38頁 0992075911-0 201224148 [0051] [0052] [0053] Ο [0054] 〇 [0055] 大致為5G%。通過浸泡’所述培育層表面附著有聚賴氨酸 ，並提供一個水性環境。 S24 0在所述&:泡後的培育層滴加一神經細胞液直到該 神經細胞液覆蓋該培育層。 在步驟S240中，所述培育層可懸空放置在一培養皿中， 或設置在矽膠基底上。 S250，培育所述附著在所述培育層的複數神經細胞，使 έ亥複數神經細胞生長出複數神經突起連接在所述複數神 經細胞之間，從而在所述培育層形成一神經網路。 所述神經細胞的培-育環境為普通的室内環境’培育時間 可根據實際需求而定。故，.在步驟s24〇的環境下請參 閱圖ίο ’保持各種條件不變，在室内魏培養15天左右 ’即可使所述神經細胞分化出多個神經突起。所述神經 細胞生長時’所述蛋白質畸牛血清，能夠提供供所述神 經細胞生長的生長因數。所述多個神經細胞上的多個神 經突起相互料後，形成所料經娜及移植體，請參 閱圖11及圖12，為所述神經移植體未經染色的掃描電鏡 照片及經過染色後的透㈣制n述投射電鏡照 片可以清晰看出，所述神經移㈣中的多個神經細胞通 過神經突起連接在-起。同時，如圖i i所示，部分神經 細胞雖然延伸出多個神經突起，但並未通過該多個神經 突起與其他神經細胞連接在-起，但這並不影響該神經 移植體在整體上具有生物活性的性質。 綜上所述，本發明私符合發明專利之要件，遂依法提 099143856 表單編號A0101 第21頁/共38頁 0992075911-0 201224148 出專利申請。惟，以上所述者僅為本發明之較佳實施例 ，自不能以此限制本案之申請專利範圍。舉凡習知本案 技藝之人士援依本發明之精神所作之等效修飾或變化， 皆應涵蓋於以下申請專利範圍内。 【圖式簡單說明】 [0056] 圖1為本發明實施例所提供的一神經移植體的製備方法的 流程不意圖。 [0057] 圖2為一奈米碳管絮化膜的掃描電鏡照片。 [0058] 圖3為一奈米碳管碾壓膜的掃描電鏡照片。 [0059] 圖4為一奈米碳管拉膜的掃描電鏡照片。 [0060] 圖5為本發明實施例所提供的神經移植體的侧視示意圖。 [0061] 圖6為本發明實施例所提供的神經移植體的俯視示意圖。 [0062] 圖7為本發明實施例所提供的奈米碳管膜結構的掃描電鏡 照片。 [0063] 圖8為本發明實施例所提供的奈米碳管膜結構的透射電鏡 照片。 [0064] 圖9為本發明實施例所提供的培育層的透射電鏡照片。 [0065] 圖1 0為本發明實施例所提供的種植在所述培育層上的神 經細胞分化出多個神經突起時的掃描電鏡照片。 [0066] 圖11為本發明實施例所提供的未經染色的神經移植體的 掃描電鏡照片。 [0067] 圖1 2為本發明實施例所提供的神經移植體染色後的掃描 099143856 表單編號A0101 第22頁/共38頁 0992075911-0 201224148 電鏡照片。 【主要元件符號說明】 [0068] 神經移植體：100 [0069] 培育層：10 [0070] 奈米碳管膜結構：12 [0071] [0072] 〇 _3] [0074] 蛋白質層：14 神經網路：20 神經細胞：22 神經突起：24[0012] S30, cultivating the plurality of nerve cells until the plurality of nerve cells grow a plurality of neurites and the neurite phase between the plurality of nerve cells, ||· , , :-··^ |··''·, V ^· Form a neural network. t Jllli (3⁄4 'ί' Γ ·· Ϊ; Ιτ In the step S10, the carbon nanotube film structure is composed of a plurality of plural. The extending direction of the plurality of carbon nanotubes may be substantially parallel to the carbon nanotubes The surface of the membrane structure. Preferably, the plurality of nanometers I ^ are connected by Van der Waals. attract iv ^ force to form a self-supporting structure. The so-called ''self-discipline'' The carbon nanotube film structure can maintain its own specific shape without being disposed on the surface of a substrate. Due to the self-supporting nano carbon, a large number of carbon nanotubes in the structure are attracted to each other by van der Waals force. Thus, #^ 099143856 Form No. Α0101 Page 5 of 38 〇992〇7591 201224148 The carbon nanotube film structure has a specific shape to form a self-supporting structure. When the carbon nanotube film structure is a self-supporting structure, The carbon nanotube film structure may be a film structure formed by at least one carbon nanotube film, and when the carbon nanotube film structure comprises a plurality of carbon nanotube films, the plurality of carbon nanotube films are stacked , adjacent to the carbon nanotube membrane between the van der Waals In combination, since the carbon nanotube film is basically composed of a carbon nanotube and the carbon nanotubes are connected by van der Waals, the carbon nanotube film structure has good elasticity, good ductility and low density. The advantages are that it is easy to cut and stretch. [0013] Referring to FIG. 2, the carbon nanotube film can be a carbon nanotube film, and the carbon nanotube film is a carbon nanotube. a self-supporting carbon nanotube film obtained by flocculation of the raw material. The carbon nanotube flocculation membrane comprises intertwined and uniformly distributed carbon nanotubes. The length of the carbon nanotubes is greater than 10 micrometers, preferably 200 micrometers. Up to 900 microns, so that the carbon nanotubes are entangled with each other. The carbon nanotubes are attracted and distributed by van der Waals to form a network structure. Due to the self-supporting carbon nanotube flocculation A large number of carbon nanotubes in the membrane are mutually attracted and intertwined by van der Waals force, so that the carbon nanotube flocculation membrane has a specific shape to form a self-supporting structure. Isotropic. The carbon nanotubes in the carbon nanotube flocculation membrane are evenly divided. The cloth, randomly arranged, forms a large number of gaps or micropores having a size between 1 nm and 500 nm. The gap or micropores can increase the specific surface area of the carbon nanotube film and infiltrate more protein. [0014] The carbon nanotube film may be a carbon nanotube rolled film, and the carbon nanotube film is a self-supporting nano carbon obtained by rolling a carbon nanotube array. The tube film. The carbon nanotube rolled film includes a uniform distribution of Nai 099143856 Form No. A0101 Page 6 / 38 pages 0992075911-0 201224148 Meter carbon tube, the carbon nanotubes are arranged in the same direction or in different directions. The carbon nanotubes in the carbon nanotube rolled film partially overlap each other and are attracted to each other by the van der Waals force, so that the carbon nanotube film has good flexibility and can be bent and folded. Any shape without breaking. - And because the carbon nanotubes in the carbon nanotube laminate film are attracted to each other by the van der Waals', they are tightly bonded, so that the carbon nanotube film is a self-supporting structure. The carbon nanotubes in the carbon nanotube rolled film form an angle /3 with the surface of the growth substrate forming the carbon nanotube array, wherein the sum is greater than or equal to 0 degrees and less than or equal to 15 degrees. The pressure applied to the array of carbon nanotubes is related. The larger the pressure, the smaller the angle. Preferably, the carbon nanotubes in the carbon nanotube film are aligned parallel to the growth substrate. The carbon nanotube rolled film is obtained by rolling a carbon nanotube array, and the carbon nanotubes in the carbon nanotube rolled film have different arrangements depending on the manner of rolling. Specifically, the carbon nanotubes can be arranged in disorder; referring to FIG. 3, when rolling in different directions, the carbon nanotubes are arranged in different orientations; when crushed in the same direction, the carbon nanotubes are along a The orientation is preferred and the orientation is preferred. The carbon nanotubes in the carbon nanotube rolled film have a long twist of more than 50 μm. [0015] The area of the carbon nanotube rolled film is substantially the same as the size of the carbon nanotube array. The thickness of the carbon nanotube film is related to the height of the carbon nanotube array and the pressure of the rolling, and may be between 0.5 nm and 100 μm. It can be understood that the larger the height of the carbon nanotube array and the smaller the applied pressure, the larger the thickness of the prepared carbon nanotube rolled film; on the contrary, the smaller the height of the carbon nanotube array, the more the applied pressure Large, the smaller the thickness of the prepared carbon nanotube rolled film. Adjacent nano 099143856 in the carbon nanotube rolled film Form No. A0101 Page 7 / 38 pages 0992075911-0 201224148 There is a certain gap between the carbon tubes, so that it is in the carbon nanotube film A gap or micropore having a plurality of sizes ranging from 1 nm to 500 nm is formed. The gap or micropores can increase the specific surface area of the carbon nanotube membrane and infiltrate more protein. [0016] The carbon nanotube film may be a carbon nanotube film, and the carbon nanotube film is a self-supporting structure composed of a plurality of carbon nanotubes. Referring to Figure 4, the plurality of carbon nanotubes are preferably aligned along the length of the carbon nanotube film. The preferred orientation means that the majority of the carbon nanotubes in the carbon nanotube film are oriented in substantially the same direction. Moreover, the overall direction of extension of the majority of the carbon nanotubes is substantially parallel to the surface of the carbon nanotube film. [0017] Further, most of the carbon nanotubes in the carbon nanotube film are connected end to end by van der Waals force. Specifically, each of the carbon nanotubes of the majority of the carbon nanotubes extending in the same direction in the carbon nanotube film is connected end to end with the carbon nanotubes adjacent in the extending direction by van der Waals force . Of course, there are a few carbon nanotubes in the carbon nanotube film that deviate from the extending direction. These carbon nanotubes do not constitute an obvious alignment of the majority of the carbon nanotubes in the carbon nanotube film. influences. The self-supporting carbon nanotube film does not require a large-area carrier support, but only provides support force on both sides, and can be suspended as a whole to maintain its own film state, that is, the carbon nanotube film is placed (or When fixed on two supports arranged at a certain distance, the carbon nanotube film located between the two supports can be suspended to maintain its own film state. The self-supporting is mainly achieved by the presence of continuous carbon nanotubes extending through the end-to-end extension of the van der Waals force in the carbon nanotube film. Specifically, the carbon nanotube film is substantially oriented in the same direction as 099143856 Form No. A0101, page 8 / 38 pages 0992075911-0 201224148, and is not an absolute straight line, and can be appropriately bent. The age of the H is completely in the direction of the material extension, and can be appropriately deviated from the extending direction. Therefore, there may be some partial contact between the carbon nanotubes juxtaposed in the majority of the carbon nanotubes extending substantially in the two-direction of the carbon nanotube film. Specifically, the tube 24 includes a plurality of consecutive carbon nanotubes. The plurality of nai tree segments are reduced by the first end of the van der Waals force. The carbon nanotubes of each carbon tube segment are composed of carbon nanotubes that are flat with each other. The carbon nanotube segments have an arbitrary length, thickness, uniformity, and shape. The carbon nanotubes have better light transmittance, and the visible light transmittance can reach 75% or more. When the nano-personal structure includes a complex, the plurality of carbon nanotubes are laminated to form a layered structure. The thickness of the layered structure is not limited to 'adjacent carbon nanotubes' combined by van der Waals force. Preferably, the layered structure comprises a carbon nanotube film having a number of ten or less layers, so that the number of carbon nanotubes per unit area is small, and the Raman light intensity of the S-meter is maintained. In the smaller fan garden, the nano-like Raman bee strength in the Rain spectrum is reduced. The carbon nanotubes in the adjacent carbon nanotubes in the layered structure have an intersection angle α, and the α is greater than the twist and less than or equal to 9 degrees. When there is an intersection angle α between the Neil & tubes in the adjacent carbon nanotube film, the carbon nanotubes in the plurality of carbon nanotube films are interwoven to form a nerve graft. The nanocarbon is increased in structural mechanical properties. In this embodiment, the carbon nanotubes are arranged in a stack of several layers of nai/cut tubes (four), and the angle between the carbon nanotubes in the adjacent carbon nanotube film is "substantially equal to 90". Degree, ie, the extension of the carbon nanotubes in the adjacent carbon nanotube film, 099143856, Α0101, 9th tribute/total 38 pages 0992075911-0 201224148, the stretching direction is substantially parallel. [0019] The protein layer is disposed in the One surface or two opposite surfaces of the carbon nanotube membrane structure for imparting hydrophilicity and biocompatibility to the carbon nanotube layer, thereby enabling the cultivation layer to be capable of planting the nerve cells and Growth provides a suitable environment. Preferably, the protein in the protein layer is a soluble protein. The protein is selected from the group consisting of a fibrous protein, a plasma protein, an enzyme protein, etc. In this embodiment, the protein a mammalian serum, such as serum, bovine serum or human serum, etc. The serum not only provides a suitable environment for the growth and growth of the nerve cells, but also enables the nerve cells to grow. The nerve cells provide a growth factor. [0020] The preparation method of the incubation layer is not limited, and only the protein layer can be mixed with the carbon nanotube membrane structure. For example, the nanocarbon can be The membrane structure is immersed in a protein solution, and the protein solution is infiltrated into the carbon nanotube membrane structure such that proteins in the protein solution adhere to one surface or two of the carbon nanotube membrane structure The oppositely disposed surface forms a protein layer. The protein solution may also be sprayed on one surface or two oppositely disposed surfaces of the carbon nanotube membrane structure, such that the protein layer is disposed in the carbon nanotube membrane structure. a surface or two oppositely disposed surfaces. The protein solution may also be dropped on one surface or two oppositely disposed surfaces of the carbon nanotube membrane structure, and the protein layer is placed on the nanocarbon by means of a ruthenium film. The tubular membrane structure has one surface or two oppositely disposed surfaces. Generally, when the thickness of the carbon nanotube membrane structure is large, the protein can be controlled to be set only. On the surface of the carbon nanotube membrane structure. And when the Nai 099143856 Form No. A0101 Page 10 / 38 pages 0992075911-0 201224148 Ο [0021] Ο [0022] The thickness of the carbon nanotube membrane structure is small The eggs are typically disposed on opposite surfaces of the carbon nanotube membrane structure. The protein solution may include, in addition to the protein, a biological medium that dissolves the protein, and the type of the biological medium is not limited to be modulating depending on the kind of the protein. Usually, the concentration of the protein in the protein solution is 大于% or more and less than or equal to the surface. In the present embodiment, the protein concentration in the protein solution is negotiable, i.e., the protein, the money is pure egg self-contained, and does not require solvent dissolution. The carbon nanotube membrane structure gas includes a plurality of carbon nanotubes and a plurality of pores are formed between the plurality of nanotubes, and a plurality of micropores are formed when the protein solution is immersed:::na: carbon tube membrane structure The protein solution can be infiltrated into the column, and the inside of the tube structure is infiltrated. The plurality of sheets are for the surface of the carbon tube. : In the P layer of the financial P, there is no surface of the tube. The surface of the tube is two: the layer of the cavity is self-quality. It only needs to be located in the table of the nephron that needs to be cultivated. Some of the carbon nanotubes (4) have a protein solution P. A protein layer may be formed on the surface of the $m carbon gamma structure to make the carbon nanotube membrane structure hydrophilic and biologically complex, and to realize the function of a carrier for growing:== cell growth. This is because, because the carbon tube is not hydrophobic, the rice-carbonized tube composed of nano-rays cannot provide a suitable hydrophilic environment for nerve cell growth. On the other hand, after the surface of the carbon nanotube is covered with a hydrophilic and non-toxic protein layer, the structure consisting of a carbon nanotube covered with protein has been able to raise (4) and m (4) the cell growth. The preparation method of the cultivating layer may further comprise the following steps: sl1, providing the nanocarbon film structure; 099143856 Form No. A0101, page 11 / 38 pages 0992075911-0 201224148 [0023] S12, making the nai The rice carbon nanotube membrane structure is infiltrated with a protein solution; and [0024] S1 3, the carbon nanotube membrane structure infiltrated with the protein solution is sterilized to form the cultivation layer. [0025] In step S12, the nai is made The manner in which the carbon nanotube membrane structure is infiltrated with the protein solution is not limited, and only the protein in the protein solution is attached to the surface of the carbon nanotube membrane structure to form a protein layer. For example, the carbon nanotube membrane structure can be Soaking in the protein solution to achieve infiltration of the protein solution. The protein solution can also be infiltrated by spraying the protein solution on the carbon nanotube membrane structure. In this embodiment In order to achieve the infiltration of the protein solution, the nanocarbon tube membrane structure is selected to be immersed in the pure protein. The soaking time of the carbon nanotube membrane structure is specific to the structure of the nanotube membrane structure and the specificity of the protein solution. Depending on the composition, only most of the carbon nanotubes in the structure of the carbon nanotube membrane can be infiltrated with a protein solution. Generally, the soaking time of the carbon nanotube membrane structure is more than 2 hours. The environment in which the carbon nanotube membrane structure is immersed is not limited, and the protein is not deteriorated. Generally, the soaking process can be carried out under normal temperature and normal pressure environment. [0026] In step S12, The carbon nanotube membrane structure is a self-supporting structure, so that the carbon nanotube membrane structure is directly immersed in the protein solution. Of course, in order to reduce the structure of the carbon nanotube membrane when immersed in a protein solution The surface tension may affect the probability of damage, and the step S12 may further include the following steps: S1 21, presetting the carbon nanotube film structure on a surface of the hydrophobic substrate. The membrane structure is tightly bound to the hydrophobic substrate, and the carbon nanotube membrane structure disposed on the surface of the hydrophobic substrate can be subjected to an organic solvent treatment. 099143856 Form No. A0101 Page 12 of 38 0992075911-0 201224148. The hydrophobic substrate must also be non-toxic to the organism so as not to disrupt the activity of the protein. Preferably, the hydrophobic substrate may also have good ductility, such as a silicone substrate. [0027] In step S13 The method of ordering and sterilizing the carbon nanotube membrane structure infiltrated with the protein solution is not limited, and only the large-sized bacteria in the protein solution can be killed. For example, the protein can be sterilized by high temperature sterilization or ultraviolet light sterilization. The solution is sterilized. In the present embodiment, the protein solution was sterilized by high temperature sterilization. Of course, in order to prevent the protein in the protein solution from being destroyed, the temperature at the time of high temperature sterilization should not exceed 220 degrees. In the present embodiment, the temperature at the time of high temperature sterilization is approximately 120 degrees. It can be understood that when the protein solution itself has less bacteria, the step S13 can omit the sterilization process when the carbon nanotube membrane structure infiltrated with the protein solution in the step S13 is sterilized::... The solvent or moisture in the protein solution will decrease. Generally, the protein solution infiltrated in the carbon nanotube membrane structure is solidified as the solvent or moisture enthalpy [0028] is reduced to form the protein layer on the surface of the carbon nanotube membrane structure. In step S10, in order to increase the adhesion of the culturing layer to the nerve cells and provide a growth environment more suitable for the nerve cells, after forming the protein layer, the step S1 〇 may further include the following steps: sl4, in the cultivating layer A polylysine (pjy-D-iysine, PDL) layer is formed on the surface of the protein layer. Specifically, the incubation layer may be immersed in a polylysine solution to cause the concentration of polylysine in the polylysine solution to be approximately 2 μg per ml. In step S20, the nerve cells include mammalian nerve cells, 099143856, Form Sfe A0101, 13th True/Total 38 f 0992075911-0 [0029] 201224148 Preferably, the neural cells are hippocampal neurons. The method of implanting a plurality of nerve cells on the surface of the layer is not limited, and a solution containing the nerve cells may be sprayed or coated on the surface of the layer, or the layer may be immersed in the nerve cell containing nerve cells. In the above, only the nerve cell fluid may be covered by the culture layer. In order to cover the culture layer with the nerve cell fluid, the nerve cell fluid may be contained in a culture dish. The incubation layer may be suspended in the culture orphan. It may also be disposed on a bottom surface of the culture dish to allow only the culture solution to cover the nerve cells. When the cultivating layer is disposed on the bottom surface of the culture bowl, a hydrophobic substrate must be disposed between the cultivating layer and the bottom surface of the culture dish to prevent the nerve cells from growing on the bottom surface of the culture dish. [0030] In step S30, the culture environment of the nerve cells is not limited, and only neurites can be grown. Generally, the nerve cells can grow in a normal temperature and a normal pressure environment. That is, by placing the nerve cells in an indoor environment, the nerve cells can grow, and proteins such as bovine serum in the culture layer can provide a growth factor to promote the growth of the nerve cells. Of course, the incubation environment can also be brought close to the growth environment of the organism providing the nerve cells. For example, when the nerve cell is a hippocampal nerve cell taken from a mouse, the growth environment in the mouse can be simulated. [0031] The nerve cells are capable of growing a plurality of neurites when incubated. The neurites include dendrites and axons (Axon). When there is only one nerve cell on the surface of the growth layer, the nerve cells of the nerve cell grow randomly along the surface of the growth layer in various directions. However, since the nerve cells themselves will release the induced neurite outgrowth, 099143856, Form No. A0101, Page 14 of 38, 0992075911-0 201224148, a long factor, so when the surface of the layer is provided with a plurality of nerve cells, the nerve cell The neurites have a tendency to grow along adjacent nerve cells, allowing adjacent nerve cells to communicate. Therefore, by controlling the distribution of nerve cells in the growth layer, the growth direction of the nerve protrusions can be controlled. For example, if the neural cell lines are randomly and evenly distributed on the surface of the incubation layer, the nerve cells will each grow a neurite out of contact with an adjacent cell' when all or most of the nerve cells of the plurality of nerve cells are When growing a neurite protruding between adjacent nerve cells, the plurality of nerve cells form the neural network by the neurites, so that the plural god The neurites of the adjacent nerve cells can communicate with each other through the cells; if they meet, they will merge into the same neurite. For example, when the nerve cells are in the shape of a line or array on the surface of the layer When the modes are arranged, the nerve cells in the longitudinal direction are relatively close, and the nerve cells in the lateral direction are far apart. At this time, the nerve cells grown by the nerve cells may extend substantially in the longitudinal direction. In order to enable the nerve cells to be arranged in a line or array on the surface of the incubation layer, the carbon nanotubes in the carbon nanotube membrane may be selected to extend substantially in the same direction i (> The neural cells adjacent to each other are mostly connected by neurites to form the neural network. The neural network forms the nerve graft together with the cultivating layer. [0032] The tubular membrane structure is a macroscopic membrane-like structure, and its area is generally <15 mm χΐ 5 mm or more. Specifically, the nano carbon membrane membrane structure has a length of more than 300 m and a width of more than 5 m. And the nano membrane structure has good compatibility, good occupation, no metal and dense 099143856, No. A0101, page 15 / 38 pages 0992075911-0 201224148 low degree, can be directly implanted into the organism. The nerve graft having the carbon nanotube membrane structure as a main carrier can be cut, stretched and implanted into the damaged portion according to the shape and size of the damaged portion of the damaged nervous system. Activity and Transfer ability, such that the nerve graft including the neural network also has biological activity and signal transmission capability. When the nerve graft is implanted in a damaged part of the organism, due to the nerve implant The distance between the neurons and the neurons at the ends or edges of the damaged portion is short, so that the neurons can be directly sutured by the neurons in the nerve implant and the neurons at the edge of the damaged portion. The two ends of the damaged part establish a signal transmission capability to complete the nerve repair of the damaged part, thereby saving the growth time of the neurite and reducing the repair time of the damaged nervous system. It can be understood that even if the nerve is implanted When the body is implanted into the damaged part, no direct suturing is performed, because the distance of the neurons at the edge of the damaged part of the neuron in the nerve implant is smaller than the distance of the neurons at both ends of the damaged part, By implanting the nerve implant, the growth time of the neurites can also be reduced, thereby reducing the repair time of the damaged nervous system. [0033] The system to be pointed out, usually In the case, the carbon nanotube in the carbon nanotube membrane structure refers to a carbon nanotube that has not been subjected to chemical or physical treatment, such as a carbon nanotube that has not been subjected to surface hydrophilic treatment, that is, the nanometer The carbon tube is a pure carbon nanotube. Of course, if the carbon nanotube in the carbon nanotube membrane structure is a modified carbon nanotube, it is only non-toxic to nerve cells, and should also be protected in the present invention. Within the scope, only the modification of the carbon nanotubes does not have any substantial contribution to the realization of the present invention, because when the protein layer covers the carbon nanotubes, the nerve cells and 099143856 Form No. A0101 Page 16 / Total 38 Page 0992075911-0 201224148 Nano carbon tube shirt straight Lang 1 nm carbon tube system can be ignored. The surface structure of the present invention is actually provided in the method of preparing a nerve graft in a __ structure and a body of a cultivating layer of a plurality of nerve cells and a neurite layer. The product obtained by the method [0035] Referring to Fig. 5, the nerve graft body @1η. 〇 includes a seed layer ίο and a neural network 20 distributed on the surface of the 1G layer. The cultivating (four) includes - a sputum knot (four) and a table of the carbon nanotube film structure 12 &# L 不哲品" or the opposite two surface protein layers 14. In this embodiment, ^ The protein layer 14 is disposed only on one surface of the carbon nanotube film structure 12.: V. The tube (four) comprises a double-coat (four) tube substantially parallel to the surface of the carbon nanotube film structure And the adjacent nano-tubes are connected to each other by a van der Waals force to form a self-cut structure. The carbon nanotube membrane structure 12 includes at least a tube material, and the tube is as shown in FIG. The carbon nanotube flocculation membrane, the carbon nanotube membrane of FIG. 3, and the carbon nanotube of FIG. 4 are reduced. The carbon nanotube film structure 12 includes a plurality of laminated films arranged in a plurality of layers, and the adjacent (four) through the 彳 彳 以 相互 相互 相互 相互 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The thickness of the carbon film structure 12 can be set according to (4). Generally, the thickness of the carbon nanotube film structure 12 is greater than 〇. 3 microns and less than 6 〇 microns. In this embodiment, the thickness of the carbon nanotube film structure 12 is approximately 099143856. Form No. A0101 Page 17 / Total 38 Page 0992075911-0 201224148 0. 6 microns. [0038] The protein layer 14 is soluble 3微米。 The thickness of the protein layer 14 is 0.3 micron. The thickness of the protein layer 14 is 0. 3 microns 5微米。 In this embodiment, the thickness of the protein layer 14 is substantially 0.5 microns. In macroscopic view, the protein layer 14 can be selected only on one surface of the carbon nanotube film structure 12 Or the opposite two surfaces. At the microscopic Above, the protein in the protein layer 14 easily penetrates into the interior of the carbon nanotube membrane structure 12 and coats part or all of the carbon nanotubes in the carbon nanotube membrane structure 12, at this time, There is no distinct interface between the protein layer 14 and the carbon nanotube film structure 12. Typically, when the thickness of the carbon nanotube film structure 12 is thin, for example, the carbon nanotube film When the thickness of the structure 12 is less than or equal to 3 μm, the protein in the protein layer 14 easily penetrates into the interior of the carbon nanotube film structure 12 and substantially covers all of the carbon nanotube film structures 12 Carbon nanotubes. When the thickness of the carbon nanotube film structure 12 is relatively thick, for example, when the thickness of the carbon nanotube film structure 12 is greater than or equal to 3 micrometers, the protein in the protein layer 14 can penetrate into the tissue. The inside of the carbon nanotube membrane structure 12 is generally only coated with the carbon nanotube membrane structure 12 near the carbon nanotubes of the neural network 20. In this embodiment, the protein in the protein layer 14 substantially coats all of the carbon nanotubes in the carbon nanotube membrane structure 12. [0039] The neural network 20 is disposed on a surface of the protein layer 14 that is remote from the carbon nanotube film structure 12. When the nerve graft 100 includes only one protein 14 disposed on one surface of the carbon nanotube membrane structure 12, 099143856 Form No. 1010101 Page 18/38 Page 0992075911-0 201224148 [0040] ❹ [0041] [0042] The nerve graft 100 includes only one neural network 20. When the protein layer 14 includes two protein layers 14 respectively disposed on opposite surfaces of the carbon nanotube membrane structure 12, the nerve graft 1 may include two neural networks 20 respectively. The surface of the two protein layers 14 away from the surface of the carbon nanotube membrane structure 12 may also include only one neural network 20 disposed on the surface of one of the protein layers 14. In the present embodiment, the neural implant 1 includes only one neural network 20. It can be understood that the nerve graft 100 may further include a polylysine layer disposed on the neural network in order to improve the bacteriostasis of the biological graft and improve the life of the nerve graft 100. The road 20 is between the protein layer 14. Referring to Figure 6, the neural network 20 includes a plurality of neural cells 22 and a plurality of neurites 24 extending from the plurality of neural cells 22. The number of neurites 24 extending from each of the nerve cells 22 is not limited, and only the biological connection between the plurality of nerve cells 22 can be established to enable the plurality of nerve cells 22 to communicate with each other. β譬, such as __, One of the nerve cells 22 may extend out of the plurality of neurites 24 or not to extend any of the neurites 24 - the nerve graft 100 of the present invention, having a neural network 20 in the nervous system in the repairing organism set in the cultivating layer 1 surface. The carbon nanotube membrane structure 12 in the incubation layer 10 is a self-supporting structure consisting essentially of carbon nanotubes, and has the advantages of no metal, good elasticity, corrosion resistance, good ductility and low density. Therefore, the carbon nanotube membrane structure 12 can be implanted into the organism along with the plurality of nerve cells 12 connected by a plurality of nerve dogs for repairing the damaged nervous system in the organism, and can be based on 099143856. Form No. 1010101 Page 19 of 38 0992075911-0 201224148 The wound area of the nervous system in the living body is cut or stretched by the nerve graft 100. [0043] Hereinafter, a method for preparing a nerve graft of the present invention and a nerve graft will be described in detail with reference to the accompanying drawings and specific embodiments. [0044] The present invention provides a method for preparing a nerve graft, comprising the following steps: [0045] S210, immersing a carbon nanotube membrane structure in a protein solution. [0046] In step S210, the carbon nanotube film structure comprises a plurality of layers of carbon nanotube film, and the extending direction of the carbon nanotubes between the adjacent carbon nanotube films has an angle of intersection. Referring to Figures 7 and 8, preferably, the cross angle is substantially equal to 90 degrees. The protein solution is pure bovine serum solution. Referring to FIG. 9, when the carbon nanotube membrane structure is immersed from the protein solution for about 1.5 hours, most of the surface of the carbon nanotube membrane structure may be infiltrated with a protein solution. . [0047] S220, taking out the carbon nanotube membrane structure from the protein solution, and performing high temperature sterilization treatment at 120 degrees Celsius. [0048] In step S220, after the carbon nanotube membrane structure is taken out from the protein solution, it may be subjected to heat sterilization treatment in a dry box. The sterilization temperature of the drying oven is approximately 120 degrees. After the sterilization treatment, the protein in the protein solution is substantially solidified, and a protein layer is formed on the surface of the carbon nanotube membrane structure to form a seed layer. [0049] S230, immersing in the poly-lysine solution in the incubation layer. [0050] In step S230, the concentration of polylysine in the polylysine solution is 099143856. Form No. A0101 Page 20 / Total 38 Page 0992075911-0 201224148 [0051] [0053] 005 [0054] ] 〇[0055] is roughly 5G%. Polylysine is attached to the surface of the layer by soaking and providing an aqueous environment. S24 0 is added with a nerve cell solution in the &: bubbled layer until the nerve cell solution covers the layer. In step S240, the incubation layer may be suspended in a petri dish or placed on a silicone substrate. S250, cultivating the plurality of nerve cells attached to the cultivating layer, so that the plurality of neurites of the έhai plural cells grow between the plurality of nerve cells to form a neural network in the cultivating layer. The culture environment of the nerve cells is an ordinary indoor environment. The incubation time can be determined according to actual needs. Therefore, in the environment of step s24〇, please refer to the figure ίο' to keep the various conditions unchanged, and to culture the cells for about 15 days in the room to make the nerve cells differentiate into multiple neurites. When the nerve cells are grown, the protein malformed serum can provide a growth factor for the growth of the neural cells. The plurality of neurites on the plurality of nerve cells are mixed with each other to form a stalk and a transplant body. Referring to FIG. 11 and FIG. 12, the SEM image of the nerve graft is not stained and after staining. It can be clearly seen from the photographs of the four electron microscopes that the nerve cells in the nerve shift (four) are connected by neurites. Meanwhile, as shown in FIG. ii, although some nerve cells extend out of a plurality of neurites, they are not connected to other nerve cells through the plurality of neurites, but this does not affect the nerve graft as a whole. The nature of biological activity. In summary, the invention is in accordance with the requirements of the invention patent, and the patent application is filed according to law 099143856 Form No. A0101 Page 21/38 Page 0992075911-0 201224148. However, the above description is only a preferred embodiment of the present invention, and it is not possible to limit the scope of the patent application of the present invention. Equivalent modifications or variations made by those skilled in the art in light of the spirit of the invention are intended to be included within the scope of the following claims. BRIEF DESCRIPTION OF THE DRAWINGS [0056] FIG. 1 is a schematic flow chart of a method for preparing a nerve graft according to an embodiment of the present invention. 2 is a scanning electron micrograph of a carbon nanotube flocculation film. 3 is a scanning electron micrograph of a carbon nanotube rolled film. 4 is a scanning electron micrograph of a carbon nanotube film. 5 is a side view of a nerve graft according to an embodiment of the present invention. 6 is a schematic top view of a nerve graft according to an embodiment of the present invention. 7 is a scanning electron micrograph of a carbon nanotube film structure provided by an embodiment of the present invention. 8 is a transmission electron micrograph of a carbon nanotube film structure provided by an embodiment of the present invention. 9 is a transmission electron micrograph of a cultivating layer according to an embodiment of the present invention. 10 is a scanning electron micrograph of a neuron implanted on the culture layer to differentiate into a plurality of neurites according to an embodiment of the present invention. 11 is a scanning electron micrograph of an unstained nerve graft according to an embodiment of the present invention. 12 is a scanning of a nerve graft after staining according to an embodiment of the present invention. 099143856 Form No. A0101 Page 22 of 38 0992075911-0 201224148 Electron micrograph. [Main component symbol description] [0068] Nerve graft: 100 [0069] Cultivated layer: 10 [0070] Nano carbon tube membrane structure: 12 [0071] [0072] 〇_3] [0074] Protein layer: 14 nerve Network: 20 nerve cells: 22 neurites: 24
099143856 表單編號A0101 第23頁/共38頁 0992075911-0099143856 Form No. A0101 Page 23 of 38 0992075911-0