JPS5959620A - Preparation of blood serum - Google Patents

Abstract

PURPOSE:To obtain serum having proliferation promoting action to hepatic cells and exhibiting excellent culture effect in the cultivation of other tissue cells, by ligating the main portal vein or the branch of the vein connected to the hepatic lobus of a warm-blooded vertebrate, collecting the blood from the animal, and separating the serum from the blood. CONSTITUTION:At least a part of the main portal vein or the branch of the portal vein connected to the hepatic lobus of a warm-blooded vertebrate, e.g. about 1/2-2/3 of the branch, is ligated, and after a definite time interval, the blood is collected from the animal, and the objective serum is prepared from the blood. The obtained serum is further heated and/or dialyzed for purification. The serum can be eluted by concanavalin A-sephalose chromatography, exhibits three bands at the molecular range of about 16,000-48,000 by the electrophoresis with SDS polyamide gel, and has no specificity to animal species.

Description

[Detailed description of the invention] The present invention relates to a method for producing serum.

Conventionally, treatment of rats with partial liver resection (regenerated liver serum)
In order to obtain a serum that has the positive effect of promoting the proliferation of hepatocytes, the present invention was achieved as a result of various efforts.
.

That is, the object of the present invention is to ligate at least a part of the portal vein trunk of a warm-blooded animal or its branching part that enters the liver lobe, and then collect the blood of the animal, The method for producing the serum is characterized by the fact that it is extracted from this blood.

The present invention will be explained in detail below.

Examples of warm-sensitive animals in the present invention include rats, mice, chickens, goats, cows, rabbits, pigs, horses, and the like.

In the method of the present invention, at least a portion of the portal vein trunk of these animals or the branch lτli that enters the liver lobe is ligated.

The portal vein carries blood from the digestive system and spleen! It is a venous system that collects k and transports it to the liver, and branches into the fragrant duct of the liver 11FI.

In the method of the present invention, this +1A entry portion, that is, the branching portion where the portal vein enters each liver organ, or a part or all of the portal vein tree trunk is ligated with silk thread or the like. If the ligation is to be carried out partially, for example, the isthmus is ligated, 2 to 2 minutes. An example of this method is to ligate about 2/3 of the tissue.

For example, to ligate , 2/, 3 and 7
T. List the method of ligating the backs of the three parts that enter the left lobe, middle lobe, and right lobe from the portal vein trunk.

After a predetermined period of several hours or more has elapsed after the ligation, the blood of the animal such as the cow whose portal vein has been ligated at least in part is collected by a conventional method.

Next, serum was prepared from the collected blood.

For example, after the blood is coagulated by drying, the supernatant 7. C1112i! The desired serum amount is obtained by $-f. Tokirima 1 normal r, θOθ~/, 2. by Urashin Bunga. It is carried out for several minutes or more at a culm degree of θθ09.

In the present invention, Tff can be produced by further heating and/or dialysis of the obtained serum.

Heating is generally carried out for 1 minute or more at approximately 1 OOC, and thermally denatured high molecular weight proteins are precipitated and removed.

Alternatively, dialysis is carried out in a saline solution that is substantially free of calcium, preferably also substantially free of magnesium.

As this salt solution, a solution containing potassium and sodium, particularly one containing all potassium, is suitable.

Examples of the above-mentioned salts include Tyrode's solution (Tyr
oae'8Pflutton) (hereinafter referred to as "CMF
solution) is used.

The composition of this solution Vi1 is as follows.

NapH, 00Na2HPO4/ , / jKO/
0.20 KH2]”0409.20(g/l
) The above dialysis treatment removes low molecular weight components in the serum.

The serum according to the present invention has the following properties.

a) OOnA (Concanavalin A) - eluted by Sepharose chromatography.

That is, it does not exhibit properties of polysaccharides or glycoproteins.

b) Electropherogram obtained by SDS polyamide gel has three bands at 1 molecule interval/A, 000 to III, 000.

C) Animal species 4? Do not show the opposite sex.

d) It has a proliferation-promoting effect on hepatocytes, and also exhibits the same culture effect as bovine fetal serum, quasi-fetal serum, or calf serum in culturing other tissue cells.

As described above, according to the method of the present invention, it is possible to obtain serum with excellent culture effects in a simpler manner than in the past, and it is also expected to be used as a liver function promoter.

Hereinafter, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to these examples as long as the gist thereof is not exceeded.

Example: Sprauge-Dawley male rats approximately 3 months old;
The serum obtained by dialysis was added to neonatal rat hepatocyte culture to induce proliferation.

([) After the portal vein was ligated, the animal was slightly acidified with ether, and after laparotomy, the part that enters the middle lobe and left lobe of the main portal vein ti+ (3 at 21') is shown in Figure -/.
It was ligated with silk thread. After ligation, the tissue was disinfected with UMk 70% ethanol, and the original tissue was sutured in two layers on the abdominal wall.

+21 Blood collection and serum preparation Two hours after the operation, the animals were anesthetized with ether, the carotid artery was cut, and the whole blood was collected.

The collected blood was allowed to coagulate at room temperature, incubated overnight with GAC, and then centrifuged for 1 minute with J00X9 to obtain the supernatant of total serum. The serum obtained as described above should be used only once before use! θ
It was stored frozen at C.

+31.770 Heat and dialysis treatment Serum obtained as described above was heated for 2 minutes in a 1oor2 water bath and the heat-denatured protein was heated at 2000 x 9
The supernatant was centrifuged for 20 minutes and removed as a precipitate, and the supernatant was dialyzed against CMF ordinary solution for 24 hours to obtain purified serum.

(4) Test The basic medium used was EaglθMFjM (composition shown in Table 1), and 70% of heated, dialyzed, and ligated rat phlegm was added to the experimental area.

In the control group, 10% sham-operated rat serum) was added. The results are shown in Kin-2 and 1-3.

In addition, the method of culturing liver cells/Ifii of newborn rats and the method of measuring the number of whole cells are as follows.

a) Neonatal rat liver #411 cell culture method: Decapitate and exsanguinate 10 newborn rat liver #411 cells, disinfect them with 70% ethanol, and place them in a sterile box. After that, except for centrifugation, All operations are performed in a sterile box. I made an incision with J Fukube's scissors, and the whole liver was taken out by JIS, cMF? Wash thoroughly with dfG and cut into pieces approximately the size of a square inch with a razor blade. Shredded liver #! Total 0.0
! Incubate with ticolagenase (dissolved in Hanks solution; the composition of the solution is shown in Table 1) for 1 hour.
After that, filter through double nylon gauze, wash with cMF solution, and filter again through double nylon gauze.

The filtered crude cell suspension was centrifuged at low speed for 2 minutes at G to collect all the liver particles, remove all the supernatant, dissolve in CMF again, and pipette into the medium. , Similarly, centrifuge the molecule flI L, and the same centrifugation operation is carried out 2 times.
Repeat several times (~h + r(It cells? Wash well.

Washed old man's cyst? Place the cells in the desired culture medium and sow 7°j cc of cells in each plastic petri dish (JJtX/jryu), J7c.

The cells were cultured in a gas phase of 1% air-1%002.

b) How to measure the number of cells After culturing * 1111 Take out the Petri dish on a regular day and check the culture medium.
Remove 06λ! Tidrypsin (C).The cell suspension was centrifuged for 10 minutes at jθO x 9 at 1'l'F.
[,,Cells were dissolved in OMlr H5H solution of a well-known serum, and after staining with trypan blue, only living cells of liver T substance were added using a hematometer (nuclei were not stained by trivan blue staining). Monoke living cells).

In addition, each 3 petri dishes were measured and used as the 117-average total cell number.

Note/) Regeneration liver sham surgery method: All animals were anesthetized with ether and fixed in supine position on a firm foot platform. Shaving, disinfecting with 70% ethanol, above the white line? A 3 to 3 mm incision was made up to the xiphoid process, and then the anterior thorax was compressed with fingers to expose the 111 J portion of the liver outside the peritoneal cavity, which was then disinfected with 70% ethanol and placed back into the peritoneal cavity. Double sutures were made to the abdominal wall using No. 43 silk thread, and the suture area was disinfected with acriflavin (surgery was always performed between 1:30 a.m. and 7:2 a.m.).

Table - Composition of 1°MEM (17/l) Table 2, Composition of Hanks solution (9/l) pH 7, Hirotu - λ, -3 Sera from rats whose veins were ligated showed significant proliferation compared to serum from sham surgery.

[Brief explanation of the drawing]

Figure-/ is a diagram showing an example of a ligated site of a rat portal vein.IJ-,2 is a change in the number of Yll cells in neonatal rat hepatocytes. Figure 3 shows the growth curve of hepatocytes. In Figure 1, the portal vein trunk shows the entire ligated portion. Applicant: Mitsubishi Chemical Industries, Ltd. Agent: Patent attorney: Yo Hase - and one other figure - 1 - 2

Claims (1)

[Claims] After ligating at least a portion of the portal vein trunk of a bloodthirsty animal or its branching portion that enters the liver lobe, the animal's blood is collected, and from this blood serum is extracted. A method for producing serum, which comprises: (2) After ligating at least a portion of the portal vein main trunk or its branching portion that enters the liver lobe of a warm-tempered animal, the whole blood of the animal was collected, and serum was prepared from this blood. A method for producing serum, the entire process comprising heating and/or dialysis the serum.