JP4107838B2 - Oncostatin M receptor gene-deficient animals and uses thereof - Google Patents

Description

【００３１】

【００３２】

【図面の簡単な説明】
【図１】図１のａは野生型OSMRβ遺伝子の制限酵素地図、翻訳開始コドンの位置、サザンブロット法に用いたプローブの位置、並びにＰＣＲに用いたプライマー及び増幅領域の位置と増幅領域のサイズを示す図である。図１のbは、構築したターゲティング・ベクターの構造を示す図である。図１のcは、図1のbに示すターゲティング・ベクターと相同組換えが起きた、破壊されたOSMRβ遺伝子の構造並びにＰＣＲに用いたプライマー及び増幅領域の位置と増幅領域のサイズを示す図である。図１のdは、サザンブロットの結果を遺伝子型と共に示す図である。図１のeは、ＰＣＲ産物を電気泳動した結果を遺伝子型と共に示す図である。
【図２】四塩化炭素を投与した野生型マウス及びノックアウトマウス(OSMR-/-)における、四塩化炭素投与後の時間と血中GPT活性との関係を示す図である。
【図３】四塩化炭素を投与した野生型マウス及びノックアウトマウス(OSMR-/-)における、四塩化炭素投与後の時間と血中GOT活性との関係を示す図である。
【図４】四塩化炭素を投与した野生型マウス及びノックアウトマウス(OSMR-/-)における、四塩化炭素投与後の時間と増殖細胞核抗原陽性細胞数との関係を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an oncostatin M receptor gene-deficient animal and its use for producing a liver injury model animal.
[0002]
[Prior art]
Conventionally, hepatic toxins such as carbon tetrachloride and D-galactosamine were administered to normal mice and rats as liver disease model animals for the search for therapeutic agents for liver diseases and the evaluation of hepatocyte or stem cell transplantation. Things are widely used.
[0003]
[Problems to be solved by the invention]
However, the liver is an extremely recoverable organ, and the liver of an animal once administered with liver toxin also recovers quickly and returns to its original state. For example, when carbon tetrachloride is administered to mice, blood GPT activity, which shows central necrosis and cell destruction, peaks at 48 hours after administration and then rapidly recovers, and blood GPT after 72 hours from administration. Activity is restored to normal levels. Therefore, when such a liver injury model animal is used, it is difficult to search for and evaluate a drug that takes time to exert its medicinal effect, and the liver does not need to be administered a drug. Since it recovers rapidly, it is not suitable for accurate evaluation of drug efficacy. If there is an animal whose liver recovery after administration of hepatotoxin is delayed compared to normal animals, it is more preferable as a liver disease model for searching for liver disease therapeutic agents and for evaluation of hepatocyte or hepatic stem cell transplantation. .
[0004]
Accordingly, it is an object of the present invention to provide an animal whose liver recovery after administration of liver toxin is delayed compared to normal animals.
[0005]
[Means for Solving the Problems]
As a result of intensive studies, the inventors of the present invention have a broader and more persistent liver damage and delayed recovery than normal mice when hepatotoxin is administered to mice in which the oncostatin M receptor gene is disrupted. Further, the inventors have conceived that such a mouse is more advantageous as a liver disease model than the conventional liver disease model, and completed the present invention.
[0006]
That is, the present invention induces liver injury by administering liver toxin to an animal for producing a liver injury model animal comprising an oncostatin M receptor gene-deficient animal (excluding humans) in which the oncostatin M receptor gene is disrupted. A liver injury model animal is provided.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
As described above, the animal of the present invention has an oncostatin M receptor (hereinafter sometimes referred to as “OSMR”) gene disrupted. The OSMR gene to be destroyed may be the OSMRα gene or the receptor β gene. In addition, the animal species is not limited and may be any species (except humans), but mice are preferred from the viewpoint of the availability of embryonic stem cells (ES) cells. Also preferred are animals in which the disrupted OSMR gene is homozygous. Hereinafter, an animal in which the disrupted OSMR gene is homozygous may be referred to as “OSMR-/-”. Here, “−” means that there is no normal gene, and when it has a normal gene, it is represented by “+”. Therefore, the wild type is `` OSMR + / + '', the heterozygous of the normal OSMR gene and the disrupted OSMR gene is `` OSMR
+/- ".
[0008]
In animals in which the interleukin-6 (hereinafter sometimes referred to as “IL-6”) gene is disrupted in addition to the OSMR gene, the recovery of the liver after inducing liver disease due to liver toxin is further delayed. More preferred as a liver disease model. Regarding the IL-6 gene, an animal in which the disrupted IL-6 gene is homozygous is preferable (similar to the OSMR gene, sometimes expressed as “IL-6 − / −”).
[0009]
The OSMR gene itself is well known and its base sequence has also been determined (GenBank Accession No. AB015978). In addition, since a method for producing a knockout mouse using a gene targeting method is well known and established, the production of an OSMR gene-deficient mouse itself can be performed based on a conventional method. For example, as will be described in detail in the following examples, it can be produced as follows. That is, a targeting vector in which the OSMR gene is disrupted by inserting an exogenous LacZ and neomycin resistance gene into the gene portion encoding the OSMR protein on the genome is prepared. This is introduced into mouse ES cells by electroporation or the like, and ES cells that have undergone homologous recombination are cloned. The ES cells are microinjected into a mouse blastocyst and then returned to the womb of a foster parent to give birth to a chimeric mouse. This chimeric mouse is bred with a wild-type mouse to obtain a mouse in which one of the pair of chromosomes has its OSMR gene disrupted. By crossing these mice, a knockout mouse (OSMR − / −) in which the OSMR gene is completely deleted on the chromosome can be produced.
[0010]
In addition, knockout mice (IL-6 − / −) in which the IL-6 gene is disrupted are well known (eg, Cressman, DE et al. (1996) Nature 274: 1379-1383; Kovalovich, K. et al. , (2000) Hepatology 31: 149-159), so that by crossing a well-known IL-6 knockout mouse with an OSMR knockout mouse produced by the above method, a mouse in which both genes are disrupted is produced. Can do.
[0011]
As specifically shown in the examples below, OMSR gene-deficient animals have liver damage induced by administration of hepatotoxins such as carbon tetrachloride for a longer period of time compared to normal animals. However, obstacles are also widespread. Therefore, OMSR gene-deficient animals are suitable as animals for producing liver injury model animals.
[0012]
A liver injury model animal can be easily obtained by administering a liver toxin that induces liver injury to the above animal, as in the conventional art. As the liver toxin for inducing liver damage, any of the conventionally known liver toxins such as carbon tetrachloride and D-galactosamine can be used, and the dose may be the same as before. . When carbon tetrachloride is used, for example, a liver disorder can be effectively induced by administering about 4 to 8 μL / g body weight of a carbon tetrachloride solution having a concentration of about 20 to 30%.
[0013]
In the following examples, as specifically shown, in the wild-type mice after carbon tetrachloride administration, central necrosis and serum GOT and GPT activities became maximal at 48 hours after carbon tetrachloride administration, and thereafter, recovery toward recovery was observed. It was shown that. On the other hand, in the OSMR − / − mice, the range of necrosis was very wide and was not improved even at 72 hours, and persistent liver damage was observed. In addition, the disorder of extracellular matrix remodeling due to the marked inhibition of TIMP expression induction was also observed. These results suggest that OSM has the activity to reduce liver damage and promote tissue repair in vivo. OSMR-/-mice and IL-6 / OSMR-/-mice, which are expected to induce chronic and persistent liver damage more easily than wild-type mice, are normal mice and rats. It is more suitable as a system for evaluating the drug efficacy than the liver injury model animal using. It also means that anti-hepatitis drug targets are included in the OSMR signal downstream, that is, in genes induced by OSMR. By using this mouse, identification of new target genes and expression of these targets And screening for drugs that control activity may be possible.
[0014]
Moreover, when considering the construction of an artificial liver, and hepatocyte transplantation and hepatic stem cell transplantation, which are expected as a treatment alternative to liver transplantation, it is necessary to increase the collected mature hepatocytes and hepatic stem cells in vitro. As can be seen from the phenomenon of liver regeneration, hepatocytes are highly proliferating cells in vivo, and HGF and EGF are known as growth factors for hepatocytes. Unfortunately, at the present stage, even with these growth factors, hepatocytes cannot be expanded in vitro. This is because the proliferation mechanism of hepatocytes in liver regeneration is not fully understood. According to the present invention, it was found that OSMR − / − mice and IL-6 / OSMR − / − mice were remarkably inhibited in the proliferation of residual hepatocytes after liver injury. These mice can be used to search for hepatocyte growth mechanisms and hepatocyte growth-promoting factors, and the results obtained from them will be useful for the above clinical applications.
[0015]
Furthermore, hepatocyte transplantation and hepatic stem cell transplantation are expected as next-generation medical care that replaces liver transplantation with severe donor shortage. For evaluation of these cell transplants, transplantation experiments into individuals are essential. The adult liver is a very regenerative organ, and in normal liver resection and liver damage caused by liver toxins such as carbon tetrachloride, the average growth rate of the liver due to the rapid proliferation of mature hepatocytes. It is said that it will return to its original volume after 1.7 divisions, and there is no capacity for the transplanted donor cells to engraft and proliferate sufficiently. Conventionally, in rats, an alkylating agent such as retrocin is administered and pretreatment is performed to inhibit the growth of recipient hepatocytes. This pretreatment takes about one month. Was necessary and inconvenient. According to the present invention, it was revealed that OSMR − / − mice and IL-6 / OSMR − / − mice have markedly inhibited proliferation of hepatocytes after injury. From these results, it is considered that these mice can be used as recipient mice for cell transplantation without performing the pretreatment as described above.
[0016]
Thus, an animal in which liver toxicity is induced by administering liver toxin to an OSMR gene-deficient animal is useful as a liver injury model animal, and in particular, drug evaluation for liver injury, search for a factor for promoting liver regeneration, or hepatocyte transplantation. Useful for evaluation.
[0017]
【Example】
Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
[0018]
Materials and methods
(1) Generation of OSMR, IL-6 / OSMR knockout mice Immediately under the starting methionine of the gene encoding OSMRβ protein on the genome (denoted as “ATG” in a in FIG. 1), LacZ (GenBank Accession No V00296) and a neomycin resistance gene (Neo) (GenBank Accession No. E00425) were inserted to prepare a targeting vector in which the OSMR gene was disrupted. The targeting vector has the structure shown in FIG. 1b. The HindIII / BglII fragment of the wild type OSMRβ gene was cleaved with ApaI and SacII, and LacZ and Neo were inserted into this portion. This targeting vector has regions homologous to mouse genomic sequences of 1.5 kb and 6 kb, respectively, upstream and downstream of LacZ / Neo.
[0019]
This targeting vector was introduced into mouse ES cells by electroporation based on a conventional method, and a claw having a neomycin resistance gene was first selected in a medium supplemented with G418. Next, genomic DNA is prepared from the obtained clones, digested with Hind III or Eco RV restriction enzymes, and the resulting fragment changes are analyzed by Southern blotting and PCR to perform homologous recombination at the target position. The resulting ES cells were cloned. The position of the probe used for the Southern blot is indicated by a white square in FIG. The result of Southern blotting is shown in FIG. In addition, two kinds of primer pairs were used for PCR. The positions of the primer pairs used are indicated by black arrows in FIG. 1a and FIG. 1c, respectively. In the normal gene, a 364 bp region is amplified as shown in FIG. 1a, and in the disrupted gene, a 750 bp region is amplified as shown in FIG. 1c. The results of electrophoresis of Southern blots and PCR products are shown in d and e of FIG.
[0020]
These ES cells were microinjected into the blast cysts of mice by the blast cyst injection method, and then returned to the womb of the foster parent, thereby giving birth to a chimeric mouse in which somatic cells were partially derived from the introduced ES cells. . Subsequently, this chimeric mouse was bred with a wild-type mouse to obtain a mouse in which one OSMR gene of two pairs of chromosomes was disrupted in whole cells. By crossing these mice, a knockout mouse in which the OSMR gene was completely deleted on the chromosome was prepared. In addition, by repeating the mating of this OSMR knockout mouse and IL-6 knockout mouse, a mouse in which both genes were disrupted was prepared. The disruption of the OSMR gene or OSMR / IL-6 gene of the obtained mouse was confirmed by the Southern blot method and the Northern blot method in the newborn lung as described above. In addition, the probe used for the Northern blotting used what amplified 1218 bp contained in the coding region of OSMR gene by PCR (5'-primer: atccaaaggctccgcaggac; 3'-primer: agcgtgcatctggagttgtg).
[0021]
(2) Wild-type mice (C57 / BL6), OSMR-/-, and IL-6 / OSMR-/-males aged 10 to 12 weeks were used for the acute liver injury experiment with carbon tetrachloride. A 25% carbon tetrachloride solution (diluted with olive oil) was intraperitoneally administered at 6 μL / g (body weight). Two to three mice in each group were bled from the abdominal vena cava under Nembutal anesthesia after 0, 0.5, 1, 2, 4, 8, 24, 48, 60, 72, 96, 120 hours after administration of carbon tetrachloride. Later, the cervical vertebrae were slaughtered. The liver was then removed and used for histochemistry, immunohistochemistry, and gene expression analysis.
[0022]
(3) Measurement of serum liver injury marker GOT activity and GTP activity were measured using GOT-UV test Wako and GPT-UV test Wako (Wako Pure Chemical Industries) according to the protocol attached to the kit.
[0023]
(4) Using a histochemistry and immunohistochemistry cryostat, 7 μm frozen liver sections were prepared and fixed with 4% paraformaldehyde or acetone. Histochemistry is stained with hematoxylin and eosin, and immunostaining for proliferating cell nuclear antigen (PCNA) is performed by reacting anti-PCNA antibody (rat IgG), biotinylated anti-rat IgG and streptavidin HRP, respectively, and using DAB as a substrate. Dyeed [0024]
(5) Gene expression analysis by Northern blot RNA was extracted from the collected liver using Trizol reagent (Nippon Gene). Ten micrograms of total RNA per lane was electrophoresed on a formaldehyde-denaturing gel, transferred to a nylon membrane, and then hybridized using a DIG-labeled cDNA probe. Bands were detected by chemiluminescence using CDP-star as a substrate.
[0025]
(6) Gelatin zymography analysis
80 micrograms of liver extract per lane was separated on 8% SDS-PAGE containing non-reduced, 0.1% gelatin, and the gel was washed with 2.5% Triton X-100® (30 minutes × 2) After reacting in the reaction solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 10 mM CaCl ₂ ) for 24 hours at 37 ° C., CBB staining was performed, followed by decolorization treatment (Methanol 30 / Acetic acid 10 / Water 60). In situ gelatin zymography was performed by pasting fresh frozen sections of liver on a polyester film-coated polyester film (Fuji Film), reacting at 37 ° C. for 3 hours, and then staining with Biebrich Scarlet. Observed with a microscope.
[0026]
result
(1) Deterioration of acute liver injury in OSMR-/-mice and IL-6 / OSMR-/-mice When a single dose of carbon tetrachloride is administered to mice, free radicals are generated from carbon tetrachloride by cytochrome P-450 in the liver. As a result, hepatocytes in the central part of the hepatic lobule are damaged, and the cell membrane permeability of the hepatocytes first increases, causing leakage of liver-specific enzymes such as GPT and GOT into the blood, followed by so-called central necrosis. (Recknagel, R. et al., (1989) Pharmacol. Thr. 43: 139-154). As a result of microscopic observation, extensive hepatocyte necrosis was observed after carbon tetrachloride administration in OSMR-/-mice, and persisted even 72 hours after administration. In addition, significant accumulation of lipid droplets was observed in the liver of OSMR-/-mice after carbon tetrachloride administration, suggesting that more severe damage occurred. Similarly, in IL-6 / OSMR − / − mice, significant hepatocyte necrosis was observed at 84 hours after administration. Serum GOT and GPT activities, which are markers of liver damage, showed similar changes in both wild type and OSMR-/-up to 48 hours after administration, but since then both GPT and GOT activities in wild type mice While both decayed rapidly, OSMR-/-mice showed a clear delay in recovery, and in particular, GOT activity maintained a peak value until 72 hours (Figs. 2 and 3). . In FIG. 2 and FIG. 3, “WT” indicates the results for the wild-type mouse, and “KO” indicates the results for the OSMR − / − mouse.
[0027]
(2) Delayed liver regeneration in OSMR-/-mice and IL-6 / OSMR-/-mice When liver parenchyma is lost due to liver injury, hepatocyte proliferation is switched on to repair the lost liver tissue Liver regeneration starts. As a result of examining the proliferation of hepatocytes at 48 hours after carbon tetrachloride administration by immunostaining with anti-proliferation nuclear antibody, in OSMR-/-mice, only about 20% of hepatocytes of wild-type mice had a proliferation signal. The liver regeneration was very strongly suppressed (FIG. 4). Induction of gene expression of cyclin-D, -A, -B, -E, which is a cell cycle control protein, was delayed in OSMR-/-mice, and the expression level was kept low. In IL-6 / OSMR-/-mice, the suppression was further enhanced, and the cyclin gene expression induction examined was below the detection limit.
[0028]
(3) Impairment of extracellular matrix reconstruction in OSMR-/-mice and IL-6 / OSMR-/-mice During liver regeneration, when the liver reconstructs its structure, along with cell proliferation, The extracellular matrix is reconstructed. In fact, it is known that the expression of matrix metalloproteases (MMPs) known as extracellular matrix degrading enzymes is induced in the early stage of liver regeneration, followed by the expression of inhibitor molecules (TIMPs). When the balance between these MMPs and TIMPs is lost, it is thought that the balance of the extracellular matrix reconstruction is lost, which leads to pathological conditions such as liver fibrosis. The expression of MMPs (especially gelatinase group) and TIMPs at the time of liver injury was analyzed by gelatin zymography and Northern blot. As a result, the expression of TIMP-1, 2 after liver injury was remarkably suppressed in OSMR-/-mice. Correspondingly, the activity of gelatinase (especially MMP9) was stronger and lasting than that of wild-type mice. In situ gelatin zymography also showed a wider range of tissue destruction in OSMR-/-mice. In IL-6 / OSMR-/-mice, it is not clear at this stage whether it is worse than OSMR-/-mice, but it clearly maintains high gelatinase activity compared to wild type. It was. It is expected that both mice in which the balance of the tissue repair process after liver injury is lost will shift to chronic hepatitis and cirrhosis more easily than wild type mice.
[0029]
【The invention's effect】
According to the present invention, a liver injury model animal produced from an animal for producing a liver injury model animal comprising OSMR gene-deficient animals is provided for the first time. When a liver injury model animal-producing animal induces liver injury by administering liver toxin, the recovery of the induced liver injury is significantly delayed from that of normal animals, and the injury is widespread and worse. Therefore, an animal in which liver injury is induced by administering a liver toxin to an animal for producing a liver injury model animal is useful as a liver injury model animal, and in particular, drug evaluation for liver injury, search for a factor for promoting liver regeneration, or hepatocyte It is considered that it is effective for transplantation evaluation.
[0030]
[Sequence Listing]

[0031]

[0032]

[Brief description of the drawings]
FIG. 1a shows a restriction map of a wild-type OSMRβ gene, the position of a translation initiation codon, the position of a probe used for Southern blotting, the position of primers and amplification regions used for PCR, and the size of the amplification region FIG. FIG. 1b shows the structure of the constructed targeting vector. FIG. 1c is a diagram showing the structure of the disrupted OSMRβ gene in which homologous recombination occurred with the targeting vector shown in FIG. 1b, the positions of primers and amplification regions used in PCR, and the size of the amplification region. is there. FIG. 1d shows the Southern blotting result together with the genotype. E of FIG. 1 is a figure which shows the result of having electrophoresed the PCR product with a genotype.
FIG. 2 is a graph showing the relationship between time after carbon tetrachloride administration and blood GPT activity in wild type mice and knockout mice (OSMR − / −) administered with carbon tetrachloride.
FIG. 3 is a graph showing the relationship between time after carbon tetrachloride administration and blood GOT activity in wild type mice and knockout mice (OSMR − / −) administered with carbon tetrachloride.
FIG. 4 is a graph showing the relationship between the time after administration of carbon tetrachloride and the number of proliferating cell nuclear antigen positive cells in wild type mice and knockout mice (OSMR − / −) administered with carbon tetrachloride.

Claims (6)

A liver injury model animal wherein liver injury was induced by administering liver toxin to an animal for producing a liver injury model animal comprising an oncostatin M receptor gene-deficient animal (excluding humans), wherein the oncostatin M receptor gene was disrupted.   The liver injury model animal according to claim 1, wherein the animal is a mouse.   The liver injury model animal according to claim 1 or 2, wherein the disrupted oncostatin M receptor gene is homozygous.   The liver injury model animal according to any one of claims 1 to 3, wherein the interleukin-6 gene is further disrupted.   The liver injury model animal according to claim 4, wherein the disrupted interleukin-6 gene is homozygous.   The liver injury model animal according to any one of claims 1 to 5, which is used for drug evaluation for liver injury, search for a factor for promoting liver regeneration, or liver cell transplantation evaluation.

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