JPH0148886B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a substance that promotes proliferation of hepatocytes. The present inventor has arrived at the present invention as a result of various studies to obtain a substance that promotes the proliferation of hepatocytes from the intestinal mucosa of warm-blooded animals. That is, the gist of the present invention is to collect the intestinal mucosa of a warm-blooded cough animal, treat it with a saline solution that is substantially free of calcium to obtain a homogenate, and then heat the supernatant and further dialyze it. It is a non-dialyzable substance obtained by ConA-sepharose chromatography, and the eluate is
The electropherogram obtained by SDS-polyacrylamide gel has three bands with molecular weights of approximately 48,000 to 16,000.
The present invention is a hepatocyte proliferation promoting substance characterized by having a hepatocyte proliferation promoting effect. The present invention will be explained in detail below. Examples of warm-blooded animals in the present invention include rats, mice, chickens, goats, cows, rabbits, pigs, and horses. The types of intestinal mucosa in these animals include small intestine,
Examples include the cecum, duodenum, jejunum, stentum, and large intestine, but usually the small intestine and cecum of cows, pigs, chickens, etc. are preferably used. The promoting substance according to the present invention can be obtained, for example, by the following method. First, the intestinal mucosa of the above animal is collected. The collection is
Intestinal epithelial mucosal tissue is usually obtained by scraping. This is treated with a saline solution substantially free of calcium to obtain a homogenate. These steps are usually carried out at a temperature of about 0 to 2°C. As the above-mentioned salt solution, Tyrode's solution (hereinafter referred to as "CMF") is used, for example. The composition of this solution is as follows. NaCl 8.00 Na ₂ HPO ₄ 1.15 KCl 0.20 KE ₂ PO ₄ 0.20 (g/l) That is, as a salt solution, one containing K, Na, especially K is suitable. The intended purpose of the invention cannot be achieved if a solution containing calcium is used. Furthermore, a salt solution that does not substantially contain magnesium is most preferable. In other words, it is optimal to use one that does not contain calcium or magnesium. A supernatant is then obtained from the homogenate by centrifugation or the like. When using centrifugation, it is usually 8000 ~
The test is carried out at 12,000×g for several minutes or more. The obtained supernatant is heated, and the denatured polymeric protein is precipitated and removed. Heating is performed at about 60 to 100°C.
The supernatant is then dialyzed against the above-mentioned saline solution, such as CMF, to remove low molecular weight components. The substance obtained in this way has the following properties. B. Shows good thermal stability and is non-dialyzable. B ConA (concanavalin A) - eluted by sepharose chromatography. That is, it does not exhibit properties of polysaccharides or glycoproteins. C. The electropherogram obtained by SDS polyacrylamide gel has three bands with molecular weights of about 48,000 to 16,000. D. Does not exhibit species specificity depending on the animal. E) It has the effect of promoting proliferation of hepatocytes, and by adding this substance, it is possible to culture hepatic parenchymal cells even in a serum-free medium. As described above, the substance according to the present invention is expected to be applied as a liver function promoter and the like. Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these examples. Example 1 An extract of the small intestine mucosa of a male Sprauge-Dawley rat approximately 3 months old was added to a neonatal rat hepatocyte culture, and its proliferation was observed. (1) Collection and extraction method of small intestine mucosa The rat was lightly anesthetized with ether, the carotid artery was cut, and the carotid artery was cut.
After exsanguination, the whole small intestine was removed through laparotomy, and the contents of the small intestine were expressed and washed with cold CMF solution. This was cut lengthwise with surgical scissors, the small intestine mucosa was scraped and collected with tweezers, homogenized with approximately 10 times the volume of CMF solution, centrifuged at 12,000 x g for 30 minutes, and the supernatant was used as a small intestine mucosal extract. (2) Heating and dialysis treatment The obtained small intestinal mucosal extract was heated at 100°C for 2 minutes, then centrifuged at 12,000 x g for 20 minutes, and the supernatant was
Dialysis was performed with CMF solution for 24 hours. (3) Test Rats that underwent regenerative liver surgery (Note 1) or regenerative liver sham surgery (Note 2) using Eagle MEM (composition shown in Table 1) as the basic medium (24 hours after surgery)
Heated, dialyzed small intestine mucosal extract (1.3mg
protein/ml) was added at 10%. The results are shown in Table 2. The method for culturing neonatal rat hepatocytes and the method for measuring cell number are as follows. a Neonatal rat hepatocyte culture method 8 to 10 newborn rats on the 6th day of life were decapitated, exsanguinated, sterilized with 70% ethanol, and placed in a sterile box. From then on, all operations except centrifugation were performed in the sterile box. did. The abdomen was incised with scissors and the entire liver was removed.
Wash thoroughly with CMF solution, and use a razor blade to remove approximately 1 mm.
Cut into square pieces. The minced liver was incubated with 0.05% collagenase (dissolved in HanKs solution; the composition of the solution is shown in Table 3) at 37°C for 1 hour, then filtered through double nylon gauze, and incubated with CMF solution. After washing, it was filtered again through double nylon gauze. Filtered crude cell suspension 40x
g, low-speed centrifugation for 2 minutes to collect hepatic parenchymal cells;
Remove the supernatant, add CMF solution again,
The cells were gently pipetted and centrifuged in the same manner, and the same centrifugation was repeated twice to thoroughly wash the cells. The washed cells were placed in the desired medium, and 1.5 cc of each cell was sown in a plastic jar (35 x 15 mm), and cultured at 37°C in a gas phase of 95% air and 5% CO ₂ . b Method for measuring cell number After culturing, on the day of measurement, the petals were taken out, the medium was removed, and the cells were incubated with 0.25% trypsin (dissolved in CMF solution) at 37°C for 30 minutes, and the cells were peeled off from the petals. 500x the cell suspension
g, Centrifuge for 10 minutes, collect the cells, dissolve them in a known amount of CMF solution, and stain with trypan blue.
Only viable cells in the liver parenchyma were measured using a hematometer (those whose nuclei were not stained by trypan blue staining were considered viable cells). In addition, three shears were each measured, and the average value was taken as the cell number.

【table】

【table】

【table】 The following parts were sutured and disinfected in the same way as in regenerative liver surgery.

[Table] That is, by adding the above small intestine mucosal extract,
Significant proliferation of cultured hepatocytes from neonatal rats is observed.

[Table] Example 2 A small intestine mucosal extract was added to a culture of adult rat hepatocytes, and cell adhesion rate, proliferation, and liver function were observed (basic medium: Eagle MEM). The animals used for culture are approximately 2 months old.
Male Sprauge-Dawley rats were used. a Measurement of cell number The cell number was measured in the same manner as in Example 1. 10% of heat-dialyzed small intestine mucosal extract (0.3 mg protein/ml) from normal rats was added and compared with 10% of normal rat serum. The results are shown in Table 4.

[Table] It can be seen that in the medium to which the small intestine mucosal extract was added, the cell number was almost maintained for 7 days, but in the medium to which normal rat serum was added, it gradually decreased. b Cell adhesion rate Heat-dialyzed small intestinal mucosal extract from normal rats
0.8mg protein/ml with 10% addition and 0.3mg
A solution containing 10% protein/ml was used. As a control, one with 10% normal rat serum added and one with no addition were used. The results are shown in Table 5. The method for measuring the adhesion rate of cell numbers is as follows. That is, after 24 hours of culture, the shears were taken out, the medium was removed, and after washing with CMF solution, 0.25%
The cells were incubated with trypsin at 37°C for 30 minutes, the cells were peeled off, and the number of cells was measured using trypan blue. It is expressed as the percentage of cells attached to the bottom of the shear dish relative to the number of cells initially seeded.

【table】

[Table] That is, when the above small intestine mucosal extract was added, the adhesion rate was significantly increased. c Measurement of the amino acids urea and ammonia in the medium.
After culturing for 3 days in Eagle MEM medium containing It was measured. The results are shown in Table 6. Amino acid nitrogen: Based on ninhydrin color method. Urea nitrogen: Based on the measurement reagent using the diacetyl monooxime method. Ammonia nitrogen: Berthelot reaction was used.

【table】

[Table] In other words, the liver function in nitrogen metabolism of hepatocytes when cultured with the above small intestine mucosal extract added is as follows:
Since the production of amino acids, urea, and ammonia changes depending on the different medium conditions of MEM and CMF solutions, it seems that sufficient liver function is maintained. Example 3 Heat-dialyzed small intestinal mucosal extracts from goats, rabbits, and chickens were added to the culture medium, and the adhesion rate and proliferation of rat hepatocytes were observed. The goat is a female Saanen breed, about 3 years old, and the rabbit is about 10 years old.
A male New Zealand White, approximately 2 months old, was used as the chicken; a white Leghorn male, approximately 2 months old, was used as the chicken. a Adhesion rate of mature rat hepatocytes Heat-dialyzed small intestinal mucosal extracts of goats, rabbits, and chickens were obtained by the method described in Example 1, and 10% of this was added (0.8 mg protein/ml). .
Table 7 shows the adhesion rate of the mature rat hepatocytes after 24 hours of culture.

[Table] b Number of neonatal rat hepatocytes 10% heat-dialyzed small intestine mucosal extract (0.4 mg protein/ml) of goat, rabbit, and chicken was added.
The results are shown in Table 8.

[Table] Example 4 a A normal rat small intestine mucosa extract subjected to heat dialysis was
Purified by ConA-Sepharose chromatography. i.e. 0.5M-NaCl-10mM
After equilibration with Tris-HCl buffer PH7.5, add the above extract, and
It eluted at a rate of ml/hr. Figure 1 shows the elution pattern.
Shown below. Only one fraction was eluted. b This eluted fraction was concentrated in a collodion bag, added to a culture medium for neonatal rat hepatocytes, cultured for 5 days, and observed for proliferation.
Remarkable proliferation was observed (Figure 2). In Figure 2,
-●- is the addition of extract (0.04mg/ml) before elution,
-〇- indicates addition of eluted extract (0.04 mg/ml). c The eluted small intestine mucosal extract was run on a 5.6% SDS polyacrylamide gel in 0.4M Tris-acetic acid buffer at pH 7.4. Staining was done with Coomassie blue. The electropherogram is
It had a molecular weight of about 48,000 and three bands at 16,000. Example 5 A heat-dialyzed normal rat small intestine mucosal extract was fractionated by Sheaf Detection chromatography.
The fraction obtained in the fraction collector is
UV absorption spectra were measured at 280 nm. The results are shown in Figure 3.

[Brief explanation of drawings]

FIG. 1 shows the elution fraction of heat-dialyzed small intestine mucosal extract of normal rats by ConA-sepharose chromatography, and FIG. 2 shows the proliferation of neonatal rat hepatocytes when the elution fraction was added. FIG. 3 shows the UV absorption spectrum of the fraction obtained by SiF chromatography.

Claims (1)

[Claims] 1. A substance obtained by collecting the intestinal mucosa of a warm-blooded coughing animal, treating it with a saline solution that does not substantially contain calcium to obtain a homogenate, then heating this supernatant, and further dialysis. It is non-dialyzable, eluted by ConA-Sepharose chromatography, and the eluate is
The electropherogram obtained by SDS-polyacrylamide gel has three bands with a molecular weight of approximately 48,000 to 16,000.
A hepatocyte proliferation-promoting substance characterized by having a hepatocyte proliferation-promoting effect.