JPS5862116A - Multiplication promoting substance for hepatic cell - Google Patents

Abstract

PURPOSE:The titled substance, obtained by treating an intestinal mucous membrane of a warm-blooded vertebrate with a salt solution containing no calcium, centrifugung the resultant homogenate, and dialyzing the resultant supernatant liquid, and useful as a hepatic functin promotor, etc. CONSTITUTION:The intestinal mucous membrane of a warm-blooded vertebrate, e.g. a rat, mouse or domestic fowl, is collected, and treated with a salt solution, e.g. Tyrode's solution, containing substantially no calcium to give a homogenate, which is then centrifuged. The resultant supernatant liquid after the centrifugation is heated at 60-100 deg.C and dialyzed to afford the aimed substance. The resultant substance is nondialytic and eluted by the chromatography with ''ConA- Sepharose '' without exhibiting the property of polysaccharide or glycoprotein. The compound has multiplication promoting action on the hepatic cell, and the hepatic parenchymal cell can be cultivated by adding the substance even to a culture medium containing no blood serum. The substance is expected to be applied as a hepatic function promotor, etc.

Description

[Detailed description of the invention] Original F! A relates to a hepatocyte growth promoting substance.

The present inventor has arrived at the present invention as a result of various studies aimed at obtaining a substance that promotes the proliferation of intestinal mucosa, liver cells, and cells in warm-blooded animals. That is, the method of the present invention is to collect intestinal mucosa from warm-blooded animals.

Add this to salt 1Illii which does not substantially contain calcium.
a non-dialyzable substance, obtained by treatment with a treatment solution to obtain a homogenate, followed by heating of the kernel supernatant and further dialysis);
The present invention is a hepatocyte proliferation-promoting substance, which is eluted by Madgutuffy and has a hepatocyte proliferation-promoting effect.

The present invention will be described in detail below.

Warm-blooded animals in the present invention include rats, mice, chickens, goats, cows, rabbits, pigs, and horses.

The polar types of intestinal mucosa of these animals include the small intestine, Tw&,
Examples include the intestine, jejunum, stent, and large intestine, but usually, the intestine and cecum of cows, pigs, and chickens are preferably used.

The BAK-promoting substance of the present invention can be obtained, for example, by the following method.

First, the intestinal mucosa of the above animal is collected. Intestinal epithelial mucosal tissue is usually collected by a scraping method.

This is treated with a saline solution that is qualitatively free of calcium to obtain a homogenate. ζStraw process is usually O--℃
It is carried out at a certain temperature. 2 Examples of the above salt solution include Tyrode's solution (Tyrode's solution).
ion) (hereinafter referred to as “OMFJ.

) is used. The composition of this solution is as follows.

Ha□tr, 00 Ha, HPO4/, /jK
OJ! o,,zo in,po,
o,,2゜(g/l) In other words, salt? As the Ili solution, one containing X in Na+Na% is suitable.

Calcium is i! If the #1 liquid contained in the present invention is used, the intended purpose of the present invention cannot be achieved, and a salt flIr liquid which does not substantially contain KY magnesium is most preferred. In other words, it is best to use one that does not contain calcium or magnesium.

Next, a supernatant is obtained from the homogenate by centrifugation, etc., and when centrifuged, the supernatant is usually 100 ρ~/co, ooo
The supernatant obtained is heated to precipitate and remove denatured high molecular weight proteins. Heating is 6
0~100℃sF! The supernatant is then dialyzed against the above saline solution, such as GMF, to remove low molecular weight components.

The stone material thus obtained has the following properties.

b) Shows good thermal stability and is non-dialyzed.

o) Concanavalin A - eluted by Sepharose gelatinography.

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

2) The electropherogram obtained from the BD8 polyacrylic acid gel has a band with a molecular weight of about 4t/,000-/l, 000KJ.

→ It has the effect of promoting the growth of hepatocytes in animals (does not exhibit QIA properties), 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.

Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples unless it exceeds the gist thereof.

Example/ In addition, to observe its proliferation, 90 (1) Collection and extraction method of small intestine mucosa A rat was lightly anesthetized with ether, the carotid artery was cut, and after exsanguination,
Open the abdomen, remove the entire small intestine, squeeze out the contents of the small intestine,
Washed with cold OMF solution. Cut this open vertically with surgical scissors.

Scrape the small intestine mucosa with a bottle set and collect approximately /θ times the volume of OM.
After homogenization with F solution, i-,oo.

XW, centrifuged for 30 minutes, and the supernatant was used as a small intestine mucosal extract. (2) Heating and dialysis treatment The obtained small intestine mucosal extract was heated at 100°C for - minutes, and then heated i2. oooxt, ao min-related heart isolation and supernatant, 'O
Dialyzed with MF solution for 2y hours, Regenerative Liver Surgery (Note 1) or Regenerative Liver II!
Small intestinal mucosal extract (/, j W pro
tein/7)/Q% addition E.

The results are shown in Table 2.The method for culturing neonatal rat hepatocytes and the method for measuring the number of cells is as follows.

a) Neonatal rat hepatocyte culture method Neonatal rats)/~10w74 were cut off from electricity, exsanguinated, and sterilized with 2θ% ethanol, then placed in a sterile box.
Thereafter, all operations except centrifugation were performed in a cage-free box. Make an incision in the abdomen with scissors, remove the whole liver, and clean it with CMF solution.
It was washed and cut into pieces of approximately /H square size using a force-ζ sled blade.

Shredded liver at 0. O7% collagenase (Hawaks)
The composition of the mosquito solution is shown in Table 1) and incubate for 1 hour at 32°C, then filtered through double nylon gauze, washed with oMy solution, and filtered again through 21 nylon gauze. It was filtered with □. The filtered crude cell suspension was centrifuged at low speed for dθ x t, - minutes to collect hepatic parenchymal cells, the supernatant was removed, OMν solution was added again, pipetted gently, centrifuged in the same way, and the same The centrifugation operation was repeated once to thoroughly wash the cells.

The washed cells were placed in the target medium, and the cells were sown in Silastic Petri dishes (3jX/jlIJI) K/jCC each, and incubated at 32°C, 2J96air-jX00. Gas phase culture t-*.

b) Method for measuring cell number After culturing, remove the petri dish on the day of measurement.

Remove the medium and 0. −2j% trypsin (CMνflI
The cells were incubated for 30 minutes at 37° C. and then peeled off from the petri dish. 10% of the cell suspension
0Xf for 10 min, isolated the relevant hearts, collected the cells, and stained them with a well-known CM1 solution, KMI, and Turin Blue.
Only living cells in the liver parenchyma were measured using a hematometer (
Cells with no nuclear staining were considered viable cells by Trivan blue staining). Note that measurements were taken for each of three Petri dishes, and the average value was taken as the cell number.

Note Regenerative liver surgery method Rat liver regenerative liver surgery involves removing lead in the liver. Hlgginll & Munderson
The method was used. That is, after the animal was lightly anesthetized with ether and fixed in the supine position on a fixation table, the surgical field was disinfected with a brush and 70% ethanol, Jm (5 incisions were made on the linea alba up to the xiphoid process, and the anterior thorax was made with a finger. The middle lobe and left lobe were exposed by applying pressure, sutures were placed on the back side of the exposed liver lobe, the liver lobe was tightly ligated, and the liver lobe was excised.It was sterilized with a resection bottle (surgery was always performed at 10:30 a.m. From / It took place for an hour.)
Note-2) Regenerative liver sham surgery method: The abdomen was incised in the same manner as in regenerative liver surgery, and the liver lobe was extracted outside the peritoneal cavity for 5 minutes, disinfected with 2θ% ethanol, and returned into the axillary cavity.

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

Table - Micro MI number of neonatal rat hepatocytes (ml) Addition of heat-dialyzed small intestine mucosal extract from rats that underwent sham surgery (10%) (**) Addition of heat-dialyzed small intestine mucosa extract from rats that underwent regenerative liver surgery (io%) That is, with the addition of the above-mentioned small intestine mucosal extract, **proliferation of cultured hepatocytes of neonatal rats was observed.

Composition of J Hanks solution pH 7,44 *Example Small intestine mucosal extract was added to hepatocyte culture of adult rats, and cell adhesion rate, proliferation, and liver function were observed (basic medium: lag
The animals to be tested were 8-month-old 8-month-old male rats.

a) Measurement of cell number Cell number was measured in the same manner as in Example 9. Heat-dialyzed small intestine mucosal extract of normal rats (0.3W
prots+tn 7wt) was added at 7θ% and compared with the case where normal rat serum was added at 70%. Table 4 shows the results.
tK is shown.

Table D Number of adult rat hepatocytes (x10@) (*3) Addition of normal rat serum (70%) (*4) Addition of heat-dialyzed small intestine mucosal extract of normal rat (10'X) Above small intestine mucosa extract It can be seen that the number of rat cells is maintained for 7 days in the medium supplemented with 100% rat serum, but gradually decreases in the medium supplemented with normal rat serum.

b) Cell adhesion rate Heat-dialyzed small intestinal mucosal extract of normal rats θ, t w
10% g protein/ml and 0.0 g protein/ml. JMf prot・1m7@l was added at 7θ%. As a control, normal rat serum was ioX
Nine additives and one without additives were used. Show your results!
The method for measuring the adhesion rate of cell numbers is as follows.

That is, take out the culture dish, remove the culture medium, and remove the culture medium. After washing with the solution, the cells were incubated with θ, COI% Trizucin at 32°C for Jθ minutes, the cells were scratched, and the number of cells was measured with Trivan blue. Percentage of cells attached to the bottom of the petri dish relative to the number of initially seeded Km cells. It was shown in

table! Adhesion rate: 0. JW protein/sl/B: 0
．． 11lvprotllln/d, that is, the above-mentioned small intestine mucosa? Add Il output IfM to l=[.

In the case of KFi, the engagement rate increased significantly.

C) Eagle MEM culture medium containing 1096 ml of ammonia in the culture medium, which was extracted from heat-dialyzed small viscosity of rats. After culturing J f3 i1, the culture medium was placed in a CMF bath, and 551111L of each medium of lcagl and MKM. ,6. fh cabinet.

The mixture was incubated at 32°C, and the amino acid, urine jk, and ammonia in each medium were added. &、According to the law.

-) Urea-form chloride: Based on a measurement reagent using the diacetyl monooxime method.

1it) Ammonia nitrogen: Parcelot (Be
rthslot) reaction was used.

Table 6 Changes in urea, ammonia, and amino acids in the medium (
urea) (7mmoni7) (agN/IIvcellular protein)
(acid) acid) 10 That is, the liver function of hepatocytes at St prime age m when the above-mentioned small intestine mucosal extract was added and cultured was Ml! Depending on the medium conditions of IM and OMF solutions, amino acids, urea,
Because the production of ammonia changes, sufficient liver 4!1
! It seems that he has retained his abilities.

Example 3 Heat-dialyzed micromucosal extracts of goats, rabbits, and chickens were added to the culture medium, and adhesion rate, #I was determined for rat hepatocytes.
I saw the breeding.

The goat is a female Saanen wing that is about 3 years old, and the rabbit is a New Sealant White male that is about 7 theta months old.

The chickens used were male white Leghorn chickens approximately -month old.

a) Adhesion rate of adult rat hepatocytes Heat-dialyzed small intestine mucosal extracts of goats, rabbits, and chickens were obtained by the method described in Examples, and this was carried out by adding 10% (θ, /sy protein /7
). Table 7 shows the adhesion rate of the mature rat hepatocytes after culture for λ days.

Table 7 Adhesion rate b) Number of neonatal rat hepatocytes 70% heat-dialyzed small intestinal mucosal extracts (θ, *19 protein/d) of goats, goats, and chickens were added. The results are shown in Table ♂.

Example a) Heat-dialyzed normal rat intestine mucosal extract tO
on Mu-Sefa p-chromatography) purification [
, i.e. 0.1 M-MILOJ! -10mM
After equilibration with Trim-110z buffer 7-pH 7j, the above extract was added.

Elute with the above buffer at a rate of /0-/hr,
The elution pattern is shown in the figure, and one mFi was eluted.

b) Concentrate this eluted fraction in a collodion bag, add it to the culture medium of iff newborn runt hepatocytes, and! The cells were cultured for one day, and the growth at that time represents the addition of the product (0.0 DaW/sf).

C) Eluted small intestinal mucosa extract! , -%BDB polyacrylic acid gel, pH 7,41,0,41M
) Electrophoresis was carried out in Lys-acetic acid buffer. Staining was carried out using Massie blue. Its electropherogram shows a molecular weight of approximately at
,oo.

/4,00OK It had 7 bands.

Example! A heat-dialyzed normal rat small intestine mucosal extract was separated by Siefadex chromatography, and the obtained fraction was collected using a fraction collector.
Absorption spectra were measured. The results are shown in Figure 3.

[Brief explanation of the drawing]

Figure 1-F shows the elution fraction of the heat-dialyzed small intestine and membrane extract of a normal rat by CONMU-CEPHOLOGY. Figure J shows the proliferation of neonatal rat liver mpii when the elution fraction is added;
The V absorption spectrum is shown. Applicant Mitsubishi Chemical Industries, Ltd. Agent Patent attorney Necessity - (7 people) ■ Mask of a (contents are not clear) 1956 Patent Application No. 161679 2 Name of the invention: Substance promoting proliferation of hepatocytes 3 Relationship to the case of the person making the amendment Applicant (596) 4th generation of Mitsubishi Chemical Industries, Ltd., Buried 2-chome Marunouchi, Chiyoda-ku, Tokyo 100 No. 5 No. 2 Mitsubishi Chemical Industries, Ltd. 5 Date of amendment order February 23, 1982 (shipment date)
6 Subject of amendment Application, specification, and drawings 7 Contents of amendment

Claims (1)

[Claims] (1) Collect the intestinal mucosa of a bloodletting animal, treat it with a saline solution that does not substantially contain calcium to obtain hosogenate, then heat the supernatant and further dialyze to obtain K. a non-dialyzable substance;
A hepatocyte proliferation-promoting substance, which is eluted by OOn Mucepharose chromatography and has a hepatocyte proliferation-promoting effect.