CN116694792A - Prkcb基因的应用 - Google Patents
Prkcb基因的应用 Download PDFInfo
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- CN116694792A CN116694792A CN202310670308.6A CN202310670308A CN116694792A CN 116694792 A CN116694792 A CN 116694792A CN 202310670308 A CN202310670308 A CN 202310670308A CN 116694792 A CN116694792 A CN 116694792A
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- prkcb
- nontuberculosis
- prkcb gene
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Abstract
本发明涉及PRKCB基因的应用；本发明提供检测PRKCB基因表达水平的试剂在制备诊断非结核分枝杆菌病试剂盒中的应用；本发明针对NTM病提出了PRKCB基因水平可以作为NTM病的诊断标志物；PRKCB基因表达水平参与了NTM病的发病，并可作为NTM病的药物治疗靶点。
Description
技术领域
本发明涉及生物医药技术领域，尤其涉及PRKCB基因的应用。
背景技术
非结核分枝杆菌(non-tuberculous mycobacteria，NTM)是指结核分枝杆菌复合群和麻风分枝杆菌以外的其他分枝杆菌。NTM最常侵犯人体肺脏，引起肺结核分枝杆菌肺病(NTM病)，在免疫力低下人群中甚至可引起全身播散性疾病。近年来，随着环境的改变，NTM病的发病率在不断升高，正在快速成为一个严重的公共卫生问题，危害人类的健康，加强NTM感染过程中重要免疫分子的鉴定和分析，对于理解NTM病的发病机制、开发新的干预以及治疗手段而言十分重要。
蛋白激酶C(PKC)是细胞内信号转导途径中的一种关键酶，是细胞外信号与细胞核反应间信号转导的共同通路，广泛参与细胞信息传递、分泌，离子通道调节，细胞增殖、分化及癌变等一系列过程。PRKCB是PKC家族的重要成员，是其β亚型，又叫PKCβ。PRKCB基因编码PKCβI、PKCβII，定位于16p11.2，全长375kb，包括18个外显子。PRKCB的结构包括具有高度同源性的四个保守区(C1-C4)和低度同源性的五个可变区(VI-V5)。C1、C2区可结合Ca2+、磷脂、DAG和佛波酯(phorbol ester，PMA)，与V1-V3位于N末端的调节区；C3、C4区分别含有ATP结合位点和底物结合位点，与V4、V5共同位于C末端的催化区。正常情况下，N末端作为假底物与C末端催化区的底物结合位点结合形成“罩”，使PRKCB处于失活状态，存在于胞质内；受到外界刺激时受其特异性底物蛋白吸引，PRKCB自胞质向胞膜转移，通过多种膜蛋白的磷酸化作用使构象改变，假底物与催化区解离，导致PRKCB激活。始动因子PRKCB的膜转移现象为PRKCB激活的重要标志。PRKCB激活后通过多种蛋白质(底物)磷酸化，介导广泛的生物学效应。
NTM对经典的抗结核分枝杆菌药物反应欠佳，总体应答率大约为50％，常常导致疾病进展和扩散。目前临床上常用的治疗方案通常是基于大环内酯类药物的三药及三药以上联合治疗方案，治疗18-24个月，但治疗成功率平均也只有66％，并且有一定的复发率和转为耐药的概率。
发明内容
本发明的目的是针对现有技术中的不足，提供PRKCB基因的应用。
为实现上述目的，本发明采取的技术方案是：
本发明的第一方面是提供检测PRKCB基因表达水平的试剂在制备诊断非结核分枝杆菌病试剂盒中的应用。
本发明的第二方面是提供检测PRKCB基因表达产物的表达水平的试剂在制备诊断非结核分枝杆菌病试剂盒中的应用。
优选地，所述PRKCB基因的核苷酸序列如SEQ ID NO:1(GAGGGACACATCAAGATTGCCGATTTTGGCATGTGTAAGGAAAACATCT GGGATGGGGTGACAACCAAGACATTCTGTGGCACTCCAGACTACATCGCC CCCGAGATAATTGCTTATCAGCCCTATGGGAAGTCCGTGGATTGGTG)所示。
本发明的第三方面是提供抑制PRKCB基因表达的试剂在制备治疗非结核分枝杆菌病试剂盒中的应用。
优选地，所述抑制PRKCB基因表达的试剂包括：化学阻断剂、siRNA、shRNA、单克隆抗体或多肽中的至少一种。
更优选地，所述化学阻断剂包括：恩扎妥林或鲁伯斯塔中的至少一种。
本发明的第四方面是提供抑制PRKCB基因的表达产物表达的试剂在制备治疗非结核分枝杆菌病试剂盒中的应用。
优选地，所述治疗非结核分枝杆菌病药物还包括：药学上可接受的一种或多种助剂。
优选地，所述治疗非结核分枝杆菌病药物的剂型包括：口服液剂、胶囊剂、片剂或注射剂。
优选地，所述治疗非结核分枝杆菌病药物的给药途径包括：口服给药、舌下给药、经肌肉或皮下给药或静脉给药。
本发明采用以上技术方案，与现有技术相比，具有如下技术效果：
本发明针对NTM病提出了PRKCB基因水平可以作为NTM病的诊断标志物；PRKCB基因表达水平参与了NTM病的发病，并可作为NTM病的药物治疗靶点。
附图说明
图1为荧光定量PCR检测PRKCB在NTM病人和健康人外周血单核细胞中的表达；
图2为免疫组化检测PRKCB在鸟分枝杆菌病人肉芽肿组织中的表达；
图3为免疫组化检测PRKCB在堪萨斯分枝杆菌病人肉芽肿组织中的表达；
图4为免疫组化检测PRKCB在脓肿分枝杆菌病人肉芽肿组织中的表达；
图5为PRKCB基因敲除和野生型小鼠鸟分枝杆菌感染小鼠肺组织HE染色；其中，图5A为PRKCB基因敲除和野生型小鼠正常肺组织HE染色；图5B为PRKCB基因敲除和野生型小鼠鸟分枝杆菌感染肺组织HE染色；
图6为PRKCB基因敲除和野生型小鼠鸟分枝杆菌感染小鼠肺组织荷菌量；
图7为WT和PRKCB基因敲除小鼠腹腔巨噬细胞胞内存活情况；其中，图7A为鸟分枝杆菌感染WT和PRKCB基因敲除小鼠腹腔巨噬细胞胞内存活情况；图7B为堪萨斯分枝杆菌感染WT和PRKCB基因敲除小鼠腹腔巨噬细胞胞内存活情况；图7C为脓肿分枝杆菌感染WT和PRKCB基因敲除小鼠腹腔巨噬细胞胞内存活情况；
图8为PRKCB抑制剂处理组和未处理组非结核分杆菌巨噬细胞胞内存活情况；其中，图8A为PRKCB抑制剂处理组和未处理组鸟分枝杆菌感染巨噬细胞胞内存活情况；图8B为PRKCB抑制剂处理组和未处理组堪萨斯分枝分杆菌感染巨噬细胞胞内存活情况；图8C为PRKCB抑制剂处理组和未处理组脓肿分枝杆菌感染巨噬细胞胞内存活情况；
图9为PRKCB抑制剂处理组和未处理组鸟分枝杆菌感染小鼠肺组织荷菌量；
图10为PRKCB抑制剂处理组和未处理组鸟分枝杆菌感染小鼠肺组织HE染色。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。
下面结合附图和具体实施例对本发明作进一步说明，但不作为本发明的限定。
实施例
一、PRKCB在NTM病人中的表达水平
用密度梯度离心法从外周血中提取外周血单个核细胞，加入1mL Trizol试剂，混匀，冰上孵育10min。4℃，12000rpm离心10min，转移上清液到新的EP管中，加入200μL氯仿，剧烈振荡15s，室温孵育5min。4℃，12000rpm离心15min，转移上层水相到新的EP管，加入等体积的异丙醇，轻轻地颠倒混匀约10次，室温放置10min。4℃，12000rpm离心10min，弃上清，1mL 75％乙醇洗涤RNA沉淀两次。4℃，12000rpm离心5min，弃上清，室温下风干5min-10min。将RNA溶于15μL-50μL DEPC水中。利用逆转录酶进行逆转录反应获得cDNA，荧光定量PCR检测NTM病人和健康人外周血PBMC中PRKCB的mRNA表达水平。
PRKCB基因扩增的引物序列为：
PRKCB上游引物：GAGGGACACATCAAGATTGCCG(SEQ ID NO:2)
PRKCB下游引物：CACCAATCCACGGACTTCCCAT(SEQ ID NO:3)
PCR扩增体系为：cDNA模板2μL，2×SYBR Green qPCR mix 10μL，上下游引物各0.4μL；ddH2O 7.2μL；
PCR扩增条件为：在94℃下进行预热5min；在95℃下保持30s进行变性、65℃下保持30s进行退火、72℃下保持30s进行扩增和延伸，循环该过程40次，在72℃下保持10min。
结果如图1所示，与健康人相比，PRKCB的表达水平在NTM病人中显著升高。
用免疫组化的方法检测了PRKCB在NTM病人肉芽肿组织中的表达水平。
将病人肉芽肿标本的石蜡切片脱蜡，依次将载玻片放入二甲苯、二甲苯、100％酒精、100％酒精、95％酒精、90％酒精、80％酒精以及70％酒精中进行脱蜡，脱蜡后将载玻片置于清水中冲洗一段时间，加入3％ H2O2浸泡10min，从而除去内源性的过氧化氢酶，然后倒掉H2O2，在清水中洗两次，进行抗原修复，再进行血清封闭，然后加PRKCB一抗，对照的组织上加PBS，加完一抗后于4℃冰箱中保存过夜，将载玻片从冰箱中取出，放入PBS中洗3次，每次5min，擦干组织周围的PBS后加上二抗，然后置于37℃温箱中0.5h，再依次经过加显色剂、复染、脱水、封片，最后在显微镜下观测。
结果如图2-图4所示，与肉芽肿组织旁边的正常肺组织相比，PRKCB在鸟分枝杆菌和堪萨斯分枝杆菌感染的病人肉芽肿组织中表达水平显著升高，在脓肿分枝杆菌感染的病人肉芽肿组织中几乎检测不到，提示PRKCB参与NTM病的发生。
二、PRKCB促进NTM的感染致病过程
PRKCB基因敲除小鼠采购自赛业(苏州)生物科技有限公司，小鼠品系名称C57BL/6J-Prkcbem1cyagen。利用小鼠感染鸟分枝杆菌模型，明确PRKCB在NTM发病过程中的作用。
培养至对数生长期的鸟分枝杆菌经离心收集后，PBS洗2次，磨菌比浊，离心沉淀重悬于PBS中。异氟烷麻醉PRKCB基因敲除小鼠和野生型小鼠，将麻醉后的小鼠握在手上，吸取20μL混匀后菌悬液，tip头稍微插入小鼠上鼻孔，缓慢注入，随着小鼠的呼吸，鸟分枝杆菌悬液被吸入。滴鼻感染鸟分枝杆菌4周后处死小鼠，检测小鼠肺组织的病理损伤情况和荷菌量。
肺组织病理切片HE染色结果(图5)显示PRKCB基因敲除感染小鼠肺组织病理损伤较野生型感染小鼠明显减轻。PRKCB基因敲除小鼠肺组织中的荷菌数显著低于野生型小鼠(p<0.05)(图6)。以上结果表明PRKCB基因调控了鸟分枝杆菌感染过程中的宿主的免疫反应，可以抑制宿主对病原菌的清除，降低了宿主抵抗鸟分枝杆菌感染的能力，PRKCB分子在分枝杆菌感染的过程中发挥负调的作用。
三、PRKCB促进非结核分枝杆菌在巨噬细胞内的胞内存活
小鼠腹腔注射5％淀粉肉汤，连续刺激3天后处死小鼠，消毒，用注射器将10mL PBS注射到小鼠腹腔，几分钟后再用注射器回收腹腔中的PBS，离心洗涤，获得小鼠腹腔巨噬细胞。采用上述方法分别获取野生型小鼠和PRKCB基因敲除小鼠腹腔巨噬细胞，在体外用鸟分枝杆菌、堪萨斯分枝杆菌和脓肿分枝杆菌感染野生型小鼠和PRKCB基因敲除小鼠腹腔巨噬细胞24h，检测菌落形成单位。
结果如图7所示，敲除PRKCB基因后，鸟分枝杆菌和堪萨斯分枝杆菌在巨噬细胞内的存活能力降低，脓肿分枝杆菌无差异，提示PRKCB可促进非结核分枝杆菌在宿主巨噬细胞内的生长，尤其是鸟分枝杆菌和堪萨斯分枝杆菌，在抗分枝杆菌感染过程中发挥重要作用。
四、PRKCB抑制剂抑制非结核分枝杆菌在巨噬细胞内的胞内存活
用恩扎妥林(Enzastaurin)和鲁伯斯塔(Ruboxistaurin)两种PRKCB的化学阻断剂进行体外实验。取小鼠腹腔巨噬细胞，用PRKCB抑制剂处理1小时后，再用鸟分枝杆菌、堪萨斯分枝杆菌和脓肿分枝杆菌感染24小时检测菌落形成单位，
结果如图8所示，与未经PRKCB抑制剂处理组相比，阻断PRKCB可以显著抑制鸟分枝杆菌、堪萨斯分枝杆菌和脓肿分枝杆菌三种分枝杆菌在巨噬细胞内的生长。
五、PRKCB抑制剂可作为NTM病治疗的潜在靶点
将培养至对数生长期的鸟分枝杆菌离心收集后制备为菌悬液，用异氟烷麻醉野生型小鼠，滴鼻感染鸟分枝杆菌20μL/只小鼠，在鸟分枝杆菌感染后的第二周开始给予治疗，每周两次给予小鼠PRKCB抑制剂灌胃，PRKCB抑制剂的剂量为1mg/kg，28天后处死小鼠，检测小鼠肺组织中的荷菌量。
结果显示，与未治疗组相比，PRKCB抑制剂治疗组小鼠肺组织中的荷菌数显著下降(图9)，肺组织冰冻切片HE染色也显示，PRKCB抑制剂治疗组感染小鼠肺组织病理损伤较未治疗组感染小鼠肺组织病理损伤明显减轻(图10)，表明PRKCB抑制剂可增强小鼠抵抗鸟分枝杆菌感染的能力，可作为NTM病治疗的潜在靶点。
综上所述，本发明针对NTM病提出了PRKCB基因水平可以作为NTM病的诊断标志物；PRKCB基因表达水平参与了NTM病的发病，并可作为NTM病的药物治疗靶点。
以上所述仅为本发明较佳的实施例，并非因此限制本发明的实施方式及保护范围，对于本领域技术人员而言，应当能够意识到凡运用本发明说明书及图示内容所作出的等同替换和显而易见的变化所得到的方案，均应当包含在本发明的保护范围内。
Claims (10)
1.检测PRKCB基因表达水平的试剂在制备诊断非结核分枝杆菌病试剂盒中的应用。
2.检测PRKCB基因表达产物的表达水平的试剂在制备诊断非结核分枝杆菌病试剂盒中的应用。
3.根据权利要求1或2所述的应用，其特征在于，所述PRKCB基因的核苷酸序列如SEQ IDNO:1所示。
4.抑制PRKCB基因表达的试剂在制备治疗非结核分枝杆菌病试剂盒中的应用。
5.根据权利要求4所述的应用，其特征在于，所述抑制PRKCB基因表达的试剂包括：化学阻断剂、siRNA、shRNA、单克隆抗体或多肽中的至少一种。
6.根据权利要求5所述的应用，其特征在于，所述化学阻断剂包括：恩扎妥林或鲁伯斯塔中的至少一种。
7.抑制PRKCB基因的表达产物表达的试剂在制备治疗非结核分枝杆菌病试剂盒中的应用。
8.根据权利要求4或7所述的应用，其特征在于，所述治疗非结核分枝杆菌病药物还包括：药学上可接受的一种或多种助剂。
9.根据权利要求4或7所述的应用，其特征在于，所述治疗非结核分枝杆菌病药物的剂型包括：口服液剂、胶囊剂、片剂或注射剂。
10.根据权利要求4或7所述的应用，其特征在于，所述治疗非结核分枝杆菌病药物的给药途径包括：口服给药、舌下给药、经肌肉或皮下给药或静脉给药。
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