JPH01143900A - Hepatic reparative factor - Google Patents

Abstract

NEW MATERIAL:A hepatic reparative factor, fractionated from a homogenate of an intestine mucous membrane of an animal and consisting of a hepatocyte growth control factors consisting of a complex of thermostable glycoprotein, having 6,000-50,000 molecular weight. USE:A cultivation promoter for hepatocytes or liver function promoter. PREPARATION:For example small intestines of cattle, pig, etc., is finely cut and washed with cold water to remove the contents. Only an intestinal epithelial mucous membrane part is then excoriated and ablated from the muscular and sheath membrane layers. A phosphoric acid buffer solution is subsequently added to homogenize the separated epithelial mucous membrane. The produced homogenate is ultracentrifuged to separate the supernatant liquid, which is then heat-treated at 100 deg.C for 3-5min., centrifuged and dialyzed against the phosphoric acid buffer solution. Furthermore, this dialyzate is concentrated and subjected to gel filtration to collect a fraction having 6,000-50,000 molecular weight. Thereby the aimed hepatic reparative factor is obtained.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to liver repair factors, and in particular, the present invention relates to liver repair factors that have the effect of proliferating hepatocytes as well as promoting differentiation and tissue formation of hepatocytes. Regarding factors.

[Conventional technology]

The inventors have discovered that intestinal mucosal homogenates from warm-blooded animals have a hepatocyte proliferation effect. In particular, we discovered a factor with a high specific activity and a hepatocyte proliferation effect in the fraction with a molecular weight of about 20,000, and this hepatocyte growth factor was first disclosed (Japanese Unexamined Patent Application Publication No. 1983-1998-1).
No. 243019).

However, in order to proliferate hepatocytes and form liver tissue, simply proliferating hepatocytes is insufficient; it is necessary to promote hepatocyte differentiation and tissue formation. The previously disclosed hepatocyte growth factor has not been found to have any activity with respect to this kind of action.

[Problem that the invention seeks to solve]

In view of the above circumstances, an object of the present invention is to provide a liver repair factor that has higher hepatocyte proliferation activity and also has the effect of promoting hepatocyte differentiation and tissue formation.

[Means for solving problems]

As a result of further research into intestinal mucosal homogenates of warm-blooded animals regarding the above-mentioned problem, the inventor found that a fraction completely different from the one proposed earlier not only had high hepatocyte proliferation activity;
The present invention has been based on the discovery that this method promotes hepatocyte differentiation and tissue formation.

That is, the liver repair factor according to the present invention is fractionated from an animal intestinal mucosal homogenate and has a molecular weight of about 6,000 to about 50.
It is characterized by consisting of a growth-promoting factor and a growth-regulating factor, which are heat-stable sugar-protein complexes of 1,000,000.

The above-mentioned hepatocyte growth factor can be obtained as follows.

First, the intestinal mucosa of the source animal is isolated.

Examples of source animals include cows, pigs, sheep,
Examples include rabbits and chickens. Types of intestinal mucosa of these animals include the epithelial mucosa of the small intestine, cecum, duodenum, jejunum, ileum, or large intestine. There is no difference in the effectiveness of the extracted and separated hepatocyte growth factors even if the intestinal compartment from which the mucous membrane was collected differs.

Note that the separation operation of the epithelial mucosa is performed at low temperature (0 to 5°C).

Next, a saline solution substantially free of calcium and preferably substantially free of magnesium, such as phosphate buffered saline (hereinafter referred to as PBS), is added to the collected intestinal mucosa, and this is homogenized to dissolve the tissue. A homogenate is prepared by finely grinding and mixing.

Next, a supernatant is obtained from the above homogenate by means such as centrifugation. When using centrifugation, it is usually 8,000~
12.000G for several minutes or more.

The obtained supernatant is heat-treated, for example, at 60 to 100°C for 1 minute or more. As a result, the denatured high molecular weight protein is precipitated and removed. Subsequently, the supernatant is dialyzed against the above saline solution to remove low molecular weight components.

Next, the supernatant subjected to the above dialysis treatment (in the case of a supernatant obtained by centrifugation, the above heating and/or dialysis treatment does not need to be performed) was mixed with Sephadex G 50 (Sephadex
phadex G-50).

When the absorbance of each fraction is determined by 71Ilj,
Four fractions corresponding to absorbance peaks are obtained. The liver repair factor according to the present invention is obtained as a fraction corresponding to the fourth peak.

Examples of the physicochemical properties of the liver repair factor of the present invention obtained as described above are as follows.

・Molecular weight is 6. The range is QQQ to 5Q, OQO.

・It is stable against heat as it withstands heat treatment during the extraction process from intestinal mucosal substances.

- Its chemical entity is a glycoprotein, which is an aggregate of several types of glycoproteins with different molecular weights (herein, this is referred to as a glycoprotein complex).

[Effect]

The liver repair factor according to the present invention exhibits a highly active hepatocyte proliferation effect.

It also has the effect of promoting hepatocyte differentiation and tissue formation. Therefore, liver tissue can be artificially induced from a single hepatocyte. Such an effect does not exist in conventional hepatocyte growth factors.

Furthermore, the liver repair factor according to the present invention has a remarkable effect of improving liver function.

〔Example〕

Hereinafter, the present invention will be explained in further detail based on specific examples.

However, the following specific examples are merely illustrative and do not limit the present invention.

Preparation of small intestinal epithelial mucosal homogenate Rat (male), 2-3 months old, weighing 200-300 g
were used as supply animals. These rats had free access to both food and water and were raised in an environment kept at around 23°C.

The rat was lightly anesthetized with ether, the jugular vein was cut to expose the release surface, and the entire small intestine was removed through laparotomy. After removing the contents of the excised small intestine, a vertical incision was made using ophthalmic scissors.
Washed with PBS. Next, the mucous membrane was scraped and collected using tweezers.

Alternatively, a product prepared in the following manner using a large animal such as a cow or pig as a source may also be used. That is, the small intestine is removed from the carcass, cut into pieces of 20 to 30 cI11 length, and washed with cooling water (ice water) to remove the contents. Next, using a scalpel, scissors, or a bottle set, only the intestinal epithelial mucosal part is scraped and peeled off from the muscle layer and the sheath layer. After washing with cooled distilled water, it was sealed in a plastic container and stored frozen in a refrigerator at -20°C.

Approximately twice the amount by weight of PBS, which is substantially free of calcium and magnesium, was added to the separated epithelial mucosa and homogenized. A Waring blender was used for the homogenization operation at its maximum speed for 1 minute.

Treatment of homogenate The homogenate obtained above was ultracentrifuged for 30 minutes at lO, ooOG to obtain a supernatant. Dispense this supernatant into large test tubes and
After heat treatment at 00°C for 3 to 5 minutes, ultracentrifugation was performed again at 10.000G for 30 minutes. The separated supernatant is sealed in a dialysis membrane, and while cooling with water, PB that is substantially free of Ca and Mg is extracted.
Dialyze against S for 24 hours.

This was appropriately concentrated, dialyzed again against distilled water for several hours, and then lyophilized and stored at -20 to 0°C.

Obtaining effective fractions 0.2 g of the freeze-dried product stored above was dissolved in 10 m of buffer solution, and separated into each fraction by gel filtration under the following conditions.

Separation conditions>Column; diameter 3.53. Length 4ocII gel; Sephadex G50 buffer; 25mM ammonium formate flow rate; 0.5-1 rril/win The results of the gel filtration described above are shown in FIG. In the figure, the horizontal axis shows the fraction number, and the vertical axis shows the absorbance for light with a wavelength of 280 nm. As shown in the results, it was separated into four fractions corresponding to absorbance peaks. Among these, the fraction corresponding to the fourth peak (Fr 4) is the object of the present invention.

The molecular weight of this fraction ranges from approximately 6,000 to 50,000.

Experiments conducted to confirm the effect of the liver repair factor (Fr 4) obtained as described above will be described below as an example.

Example 1 (Influence on hepatocyte proliferation in adult rats) Using the medium supplemented with the liver repair factor (Fr 4) obtained above, hepatic parenchymal cells (Rat parenchymal cells) obtained from adult rats of the S D (Spraque Dawley) strain were cultured.
A culture experiment was conducted on the yma+)1epatoeyte). When the number of cells was examined on the 1st, 4th, and 7th day after culture, the results indicated by the 0 mark in FIG. 2 were obtained. The results are shown as the average value and standard error of 6 dates. The medium used is WE medium (νilliams'
E Med1ua+) plus 5% normal repair serum (Norm
al Bovfne Calf'Serlum; hereinafter abbreviated as NBC3) and 10''6IIM insulin were used.Furthermore, as a liver repair factor, 5% F r4 (211tSF Protein) was used.
/M) was used.

Also, for comparison, 10% NBC3 was added to WE medium.
and 10″6 IOM of insulin only, F
A culture experiment similar to that described above was conducted using a medium to which r4 was not added. This result is also shown in Figure 2 with .

As is clear from the results in FIG. 2, addition of the liver repair factor Fr4 resulted in a 3-fold proliferation promoting effect within 7 days.

Example 2 (Influence on hepatocyte proliferation in neonatal rats) Using the medium supplemented with the liver repair factor (Fr 4) obtained above, hepatic parenchymal cells (Rat Parentchymal Hepato
A culture experiment was conducted on the following. During one week after culturing,
When the number of cells was examined every two days, the results shown by O in FIG. 3 were obtained. This result is shown as an average value over 6 dates. In addition, as a medium, WE medium (Vi
5% inactivated normal rat serum and 5% F r 4 (2119
Protein/1! The one to which Ll) was added was used.

For comparison, 10% inactivated portal blood serum obtained from normal rats was added to WE medium.
A culture experiment similar to that described above was conducted using a medium to which only Serium (Serium) was added and Fr4 was not added. This result is also shown in Figure 3 with .

As is clear from the results in FIG. 4, the addition of the liver repair factor Fr4 resulted in a 7- to 8-fold growth promoting effect.

Example 3 (Influence on hepatocyte tissue formation and differentiation) Liver parenchymal cells obtained from SD adult rats (Rat Pa
renchyLIlal Hepatocytes),
Cultured for 24 hours in WE medium supplemented with 5% NBCS and 10''6 mM insulin.
5% F r 4 (219Prot
ein/IIj? ) was added, and culture was continued for 90 days.

As a result, remarkable proliferation of hepatocytes was observed, and as shown in the micrographs in Figures 4 to 8, tissue formation and differentiation (fat,
cholesterol, bilirubin, bile pigment production, etc.) were induced.

That is, FIG. 4 shows the state after 15 days of culture, where formation of tubes (arrows) and production of bilirubin are observed. Fifth
The figure shows the state after 20 days of culture, and the production of neutral fat, cholesterol, or bilirubin is observed as indicated by the white dots in the figure. Figure 6 shows the state after 30 days of culture.
Bilirubin production (arrow) is observed. FIG. 7 shows the state after 50 days of culture, and dense arrays of cells (arrows) are observed. Figure 8 shows the state after 90 days of culture.
The formation of a tissue that could be called a micro-artificial liver was observed.

Example 4 (Suppressive effect on the development of liver damage by administration of CC4) A 1:1 mixture of CCl4 and olive oil was administered by subcutaneous injection in the neck to Wistar rats, 7 weeks old and weighing 180 g. - The dose per 100g body weight is 0.
2 mi, and was administered twice a week for 6 consecutive weeks. Also,
24 hours after administering CCl4, the liver repair factor F
r4 is 1d per 100g of body weight (2ηProtein/
rxi) was administered by intraperitoneal injection.

As a comparative example, instead of administering liver repair factor Fr4, body weight 10
1 g of physiological saline was administered per 0 g, and otherwise the same procedure as above was performed.

As a control example, body ffi was 0.211 per 100g.
Ll olive oil alone was administered twice a week.

Six weeks after the above continuous administration, blood was collected from the jugular vein of each rat and serum was separated. GOT (glutamic oxal acetic transaminase) in each serum sample,
When the respective activities of GPT (glutamic birvic transaminase), ALP (alkaline phosphatase) and catebusin D were measured, the results were shown in Figures 9 to 1.
The results shown in Figure 2 were obtained.

As shown in these results, in the rats of the example in which the liver repair prisoner Fr4 was used in combination, each enzyme activity was close to the normal value (control example), and the effect of suppressing the induction of liver damage by CCl4 was observed.

Example 4 (Suppressive effect on the development of hepatitis by administration of D-galactosamine) 3 g of D-galactosamine hydrochloride (manufactured by Sigma) was added to 125 rIJ of physiological saline! and neutralized with IN NaOH. This solution was administered to Wistar rats, 7 weeks old and weighing 200 g, by intraperitoneal injection. The dose was 600n per 100g body weight. Immediately after administration of D-galactosamine hydrochloride, Fr4, a liver repair factor, was added to la'(2#Protein/if
) was administered by intraperitoneal injection.

Furthermore, liver repair factor Fr4 was similarly administered 24 hours and 48 hours later.

As a comparative example, the liver repair factor Fr4 was not administered and the above D-
Only galactosamine hydrochloride was administered.

As a control example, only physiological saline was injected.

Blood was collected from each of the rats mentioned above, serum was separated, and the activity of GOTSALP in each serum sample was measured.
The results shown in FIGS. 13 and 14 were obtained. In the figure, Δ indicates the results of the example, . indicates the comparative example, and 0 indicates the control example.

As is clear from these results, the activity of each enzyme was lower depending on the dose of liver repair factor Fr4, and it was observed that the onset of hepatitis was suppressed.

Finally, the results of a reference experiment in which the liver repair factor Fr4 was separated into smaller fractions will be described.

Reference Experiment 1 Samples of 20 fractions of liver repair factor Fr4 obtained as described above were freeze-dried and dissolved in 40 ml of redistilled water. lht/ was used as one batch and subjected to gel filtration again under the following conditions.

Separation conditions>Column; diameter 2art, length 45c111 gel; SP
-Sephadex C25 (SP-9ephadex C-25) buffer; 0.1M ammonium formate 140 0.1-0.3M ammonium formate 220! ! Li 013π0.5M ammonium formate 200 rnI! Flow rate: 0.33 m/min The above results are shown in FIG. 15. In the figure, the horizontal axis shows the fraction number and the type of eluent used, and the vertical axis shows the wavelength 2.
It shows the absorbance for light at 80 nm. As shown in the results, three active fractions A, B, and C were separated corresponding to the absorbance peaks.

Reference Experiment 2 Each of the three active fractions A, B, and C obtained above was subjected to gel filtration again under the following conditions.

Separation conditions>Column; diameter 3.5n, length 40CT1 gel; Sephadex G25 (5P-3ephadex C-25) buffer; IM
ammonium formate flow rate; natural fall The above results are shown in Figure 16. As shown in the figure,
Three active fractions A, B, and C were each obtained as a single fraction.

〔Effect of the invention〕

As detailed above, the liver repair factor according to the present invention is not only useful as a hepatocyte culture promoter, but is also expected to be used as a liver function promoter, and may also pave the way for the realization of an artificial liver. , has extremely high usefulness.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the results of gel filtration after separating the liver repair factor of the present invention from bovine small intestinal epithelial mucosal homogenate, and Figures 2 and 3 show the hepatocyte proliferation effect of the liver repair factor of the present invention. Figures 4 to 8 are micrographs showing hepatocyte differentiation and tissue formation effects by the liver repair factor of the present invention, and Figures 9 to 14 show liver function improvement by the liver repair factor of the present invention. Figures 15 and 16 showing the action are diagrams showing the results of further fractionation of the liver repair factor of the present invention by gel filtration. Applicant's representative Patent attorney Takehiko Suzue Fraction N□. Fig. 1 Figure 14

Claims (2)

[Claims] (1) Composed of hepatocyte growth-promoting factor and hepatocyte growth-regulating factor, which are fractionated from animal intestinal mucosal homogenates and are heat-stable glycoprotein complexes with a molecular weight of about 6,000 to about 50,000. Liver repair factor. (2) Claim (1) characterized by having the effect of proliferating hepatocytes, repairing damaged liver tissue, and forming artificial liver tissue by culturing from hepatic parenchymal cells.
Liver repair factors as described in section.