CN103749388A - Method for breeding IL-12p40 (-/-) IL-2R alpha (-/-) mice used as hepatic fibrosis and primary biliary cirrhosis animal models - Google Patents
Method for breeding IL-12p40 (-/-) IL-2R alpha (-/-) mice used as hepatic fibrosis and primary biliary cirrhosis animal models Download PDFInfo
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Abstract
The invention discloses a method for breeding IL-12p40 (-/-) IL-2R alpha (-/-) mice used as novel hepatic fibrosis and primary biliary cirrhosis animal models. The method includes steps of (1), performing cross breeding on IL-2R alpha (+/-) mice and IL-12p40(-/-) mice to obtain IL-12p40(+/-) IL-2R alpha (+/-) mice by means of breeding; (2), performing cross breeding on IL-12p40(-/-) mice and the IL-12p40(+/-) IL-2R alpha (+/-) mice obtained in the step (1) to obtain IL-12p40(-/-) IL-2R alpha (+/-) mice by means of breeding; (3), performing inbreeding on the IL-12p40(-/-) IL-2R alpha (+/-) mice obtained in the step (2) and obtaining the identified IL-12p40(-/-) IL-2R alpha (-/-) mice. The method has the advantages that IL-12p40 is an important cell factor in the immune system of a patient, and functions and differentiation of CD4+T and CD8+T cells can be affected by the IL-12p40; IL-2R alpha is an alpha chain of an IL-2 receptor and plays a key role in keeping the balance of the immune system of the patient.
Description
Technical field
The invention belongs to medical biotechnology field, specifically relate to the method for a kind of cultivation as the IL-12p40 of liver fibrosis and primary biliary cirrhosis of liver animal model (/-) IL-2R α (/-) mouse.The IL-12p40 that obtains by method of the present invention (/-) IL-2R α (/-) mouse, can be as new animal model for screening the medicine for the treatment of liver fibrosis and primary biliary cirrhosis of liver.
Background technology
Liver fibrosis is the especially pathologic process of the excessive accumulation of collagen of extracellular matrix protein causing due to chronic liver injury.Liver fibrosis is not independently disease of one, and multiple chronic hepatic diseases all can cause liver fibrosis, comprises that virus hepatitis infects, alcohol drinks too much, nonalcoholic steatohepatitis, oneself immunity hepatitis etc.In liver fibrosis process, liver astrocyte is topmost effector cell, the synthetic a large amount of collagen of liver astrocyte of activation.Liver fibrosis has multiple mouse model, comprise carbon tetrachloride model, bile duct ligation model, alcohol feed model, thioacetamide model etc., but these mouse models are the Liver Fibrosis Model causing due to environmental stimuli substantially, and it is not the Liver Fibrosis Model that autoimmune liver disease causes.And the IL-12p40 that we find (/-) IL-2R α (/-) mouse is spontaneous Liver Fibrosis Model, and this liver fibrosis is caused by autoimmunity disease.
Primary biliary cirrhosis of liver is a kind of chronic intrahepatic cholestasis type disease, is mainly in female middle-aged, and normal and other autoimmunity diseases coexist.In histopathology, primary biliary cirrhosis of liver shows the specific Damage of immunocyte to little bile duct in liver.AMA (AMA) in serum is the specific index of primary biliary cirrhosis of liver, and this mitochondrial antibody is mainly in a series of mitochondria antigen, as PDC-E2.Primary biliary cirrhosis of liver can develop into cirrhosis even by liver fibrosis, and primary biliary cirrhosis of liver is difficult for treatment, is urso (UDCA) at present for the unique effective medicine of primary biliary cirrhosis of liver.Therefore need suitable animal model to carry out medicament research and development.
IL-2R α (/-) mouse is owing to lacking IL-2R alpha molecule, growth and the function of regulatory T cells have been subject to impact, therefore IL-2R α (/-) mouse has serious general autoimmunity disease [Willerford, D.M., et al. (1995) .Immunity 3 (4): 521-530].Work before finds that IL-2R α (/-) mouse has portal vein inflammation and bile duct destruction, and in serum, can detect AMA (AMA), the content of multiple inflammatory cytokine rising [Wakabayashi in serum, K., et al. (2006) .Hepatology 44 (5): 1240-1249].Because IL-2R α (/-) mouse has these and be similar to the symptom of mankind's primary biliary cirrhosis of liver, therefore can be used as the animal model of primary biliary cirrhosis of liver.IL-2R α (/-) mouse has serious colitis equally, and the colitis of finding IL-2R α (/-) mouse is mainly by CD4
+t cell causes, and autoimmune cholangitis is mainly by CD8
+t cell causes [Hsu, W., et al. (2009) .Hepatology 49 (1): 133-140].The autoimmune cholangitis of but IL-2R is α (/-) mouse is not very serious, and the level that portal vein inflammation and bile duct destroy is lower, does not also develop into the degree of liver fibrosis.And IL-2R α (/-) mouse exists colitis simultaneously, and colitis is not present in mankind's patients with primary biliary cirrhosis conventionally.These have limited the application of this animal model, therefore need a more suitable primary biliary cirrhosis of liver animal model.
Summary of the invention
The present invention is the weak point for having avoided original mouse model, on the basis of IL-2R α (/-) mouse, knock out IL-12p40, the colitis of model mouse is alleviated greatly, and autoimmune cholangitis increases the weight of greatly, and has occurred liver fibrosis.
The invention provides the method for a kind of cultivation as the IL-12p40 of a kind of new liver fibrosis and primary biliary cirrhosis of liver animal model (/-) IL-2R α (/-) mouse, described method comprises the steps:
(1) IL-2R α (+/-) mouse and IL-12p40 (/-) mouse is hybridized, cultivates and obtain IL-12p40 (+/-) IL-2R α (+/-) mouse;
(2) step (1) is cultivated to IL-12p40 (+/-) IL-2R α (+/-) mouse that obtains and IL-12p40 (/-) mouse and hybridize, cultivate and obtain IL-12p40 (/-) IL-2R α (+/-) mouse; With
(3) step (2) is cultivated to the IL-12p40 that obtains (/-) IL-2R α (+/-) mouse and carry out selfing cultivation, identify and obtain IL-12p40 (/-) IL-2R α (/-) mouse.
Wherein the IL-2R α of step (1) C57BL/6J background used (/-) mouse (B6.129S4-I12ratm1Dw) and IL-12p40 (/-) mouse (B6.129S1-I112btm1Jm) is purchased from U.S. Jackson laboratory.
The method of wherein identifying mouse IL-12p40 gene is to identify with methods of genotyping the wild type gene that whether has IL-12p40 in mouse offspring; The method of identifying mouse IL-2R α gene is the average fluorescent strength with the CD25 of CD4 positive cell in Flow cytometry peripheral blood.IL-2R α (/-) the CD4 positive cell of mouse does not comprise the cell of the CD25 positive; And the average fluorescent strength of the CD25 of IL-2R α (+/-) mouse CD4 positive cell is the half of IL-2R α (+/-) mouse, can identify accordingly the IL-2R α gene of mouse.
The invention still further relates to the IL-12p40 that obtains by breeding method of the present invention (/-) IL-2R α (/-) mouse, further experimental results show that this mouse can screen and treat liver fibrosis and primary biliary cirrhosis of liver medicine as new liver fibrosis and primary biliary cirrhosis of liver animal model.
In the present invention, the result of hepatic pathology proves that the portal vein inflammation of IL-12p40 (/-) IL-2R α (/-) mouse will obviously be better than IL-2R α (/-) mouse, and the degree that bile duct destroys is higher (but not having significant difference).IL-2R α (/-) mouse shows the enteritis symptoms such as prolapse of the anus, diarrhoea, and IL-12p40 (/-) IL-2R α (/-) mouse does not almost have such symptom.The result of colon pathology also proves that IL-2R α (/-) mouse rather than IL-12p40 of the present invention (/-) IL-2R α (/-) mouse exists comparatively serious colitis.
The spleen weight of IL-12p40 in the present invention (/-) IL-2R α (/-) mouse will be apparently higher than IL-2R α (/-) mouse, in IL-12p40 (/-) IL-2R α (/-) mouse, about 60% mouse spleen quality is more than 0.3000 gram, and its ratio is far above IL-2R α (/-) mouse.Splenomegaly can be found in patients with primary biliary cirrhosis, it is generally acknowledged relevant to portal hypertension.We find that liver mononuclearcell number and the spleen weight of IL-12p40 (/-) IL-2R α (/-) mouse is proportionate, and this shows that inflammation and spleen have close relationship.
In the present invention, we find to occur that the frequency of liver fibrosis is higher in IL-12p40 (/-) IL-2R α (/-) mouse, and IL-2R α (/-) mouse there will not be liver fibrosis.By kinds of experiments methods such as Azan dyeing, SABC, Liver hydroxyproline content and RT-PCR, we prove that IL-12p40 (/-) IL-2R α (/-) mouse has liver fibrosis, IL-2R α (/-) mouse without.
In the present invention, we have compared the number of IL-12p40 (/-) IL-2R α (/-) mouse and IL-2R α (/-) liver of mouse and the various cell subsets of spleen, find that the cell mainly increasing is T cell, comprise CD4
+t cell and CD8
+t cell, and B cell, NK cell and NKT cell there was no significant difference.We are to liver CD4
+t cell and CD8
+the subgroup of T cell is analyzed, and finds two kinds of mouse CD4
+t cell is all effect memory cell (effector memory cells) substantially, and CD8
+t cell in IL-12p40 (/-) IL-2R α (/-) mouse take effect memory cell as main, in IL-2R α (/-) mouse take maincenter memory cell (central memory cells) as main.
In the present invention, the result of dyeing proves in cell factor born of the same parents, liver CD4 in IL-12p40 (/-) IL-2R α (/-) mouse
+mouse is high than IL-2R α (/-) for the ratio of the IFN-γ positive of T cell, and the ratio of the IL-17A positive is lower.Liver CD8
+in T cell, the ratio of the IFN-γ positive is also IL-12p40 (/-) IL-2R α (/-) mouse higher than IL-2R α (/-) mouse.The result of liver RT-PCR also shows, the IFN-γ expression of IL-12p40 (/-) IL-2R α (/-) mouse is higher, and the expression of IL-17A is lower.
In sum, the invention provides following technical proposals:
1. a method of cultivating IL-12p40 as new liver fibrosis and primary biliary cirrhosis of liver animal model (/-) IL-2R α (/-) mouse, described method comprises the steps:
(1) IL-2R α (+/-) mouse and IL-12p40 (/-) mouse is hybridized, cultivates and obtain IL-12p40 (+/-) IL-2R α (+/-) mouse;
(2) step (1) is cultivated to IL-12p40 (+/-) IL-2R α (+/-) mouse that obtains and IL-12p40 (/-) mouse and hybridize, cultivate and obtain IL-12p40 (/-) IL-2R α (+/-) mouse; With
(3) step (2) is cultivated to the IL-12p40 that obtains (/-) IL-2R α (+/-) mouse and carry out selfing cultivation, identify and obtain IL-12p40 (/-) IL-2R α (/-) mouse.
2. according to the method described in the 1st, wherein step (1) IL-2R α used (/-) mouse and IL-12p40 (/-) mouse is purchased from U.S. Jackson laboratory.
3. according to the method described in the 1st, the method for wherein identifying mouse IL-12p40 gene is to identify with methods of genotyping the wild type gene that whether has IL-12p40 in mouse offspring; The method of identifying mouse IL-2R α gene is the average fluorescent strength with the CD25 of CD4 positive cell in Flow cytometry peripheral blood.
Advantage of the present invention is: the specificity of the animal model of current primary biliary cirrhosis of liver is not high, and course advancement is less than the degree of liver fibrosis.IL-12p40 (/-) IL-2R α (/-) mouse not only has stronger portal vein inflammation and bile duct destroys, generation liver fibrosis that can also be spontaneous, and the degree of colitis is very weak.Therefore, IL-12p40 (/-) IL-2R α (/-) mouse can be used as the animal model of a better liver fibrosis and primary biliary cirrhosis of liver.
Accompanying drawing explanation
In detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
HE dyeing (A, B) and the liver portal vein inflammation (C) of Fig. 1 .IL-2R α (/-) and IL-12p40 (/-) IL-2R α (/-) mouse contrast with bile duct injury (D).
HE dyeing (A, B) and the colon Pathology (C, pathological score, D, colon weight) of Fig. 2 .IL-2R α (/-) and IL-12p40 (/-) IL-2R α (/-) mouse.
Megalosplenia (the A of Fig. 3 .IL-12p40 (/-) IL-2R α (/-) mouse, rightmost), spleen is (B) heavily, the mononuclearcell number (C) of liver, and liver mononuclearcell number and spleen correlation (D) heavily.
The liver fibrosis of Fig. 4 .IL-12p40 (/-) IL-2R α (/-) mouse, A, liver fibrosis scoring, B, Azan dyeing, C, the RT-PCR result of liver.
Fig. 5. the number (A) of liver T cell and ratio (B).
Fig. 6. the subgroup of liver T cell naivety (naive) and Memorability (memory).
Fig. 7. dyeing in liver cell factor born of the same parents.
Embodiment
Below with reference to specific embodiment, further describe the present invention, but it should be appreciated by those skilled in the art that the present invention is not limited to these specific embodiments.
It should be appreciated by those skilled in the art that if not otherwise specified, in following embodiment, chemical reagent used is the reagent of the pure rank of commercially available analysis.Following zoopery is carried out in accordance with China Science & Technology University's Experimental Animal Center laboratory animal nursing and guide for use.
The foundation of embodiment 1.IL-12p40 (/-) IL-2R α (/-) mouse
The IL-2R α of C57BL/6J background (/-) mouse (B6.129S4-I12ratm1Dw) [Willerford, D.M., et al. (1995) .Immunity 3 (4): 521-530] and IL-12p40 (/-) mouse (B6.129S1-I112btm1Jm) [Magram, J., et al. (1996) .Immunity 4 (5): 471-481] purchased from U.S. Jackson laboratory (The Jackson Laboratory, maine state of u.s.a, http://www.jax.org/), raise in the Experimental Animal Center of China Science & Technology University without special pathogen (SPF) environment in.
We hybridize IL-2R α (+/-) mouse and IL-12p40 (/-) mouse, obtain IL-12p40 (+/-) IL-2R α (+/-) mouse; Hybridize with IL-12p40 (/-) mouse again, obtain IL-12p40 (/-) IL-2R α (+/-) mouse.The IL-2R α that uses in experiment (/-) mouse is bred by IL-2R α (+/-) mouse, and IL-12p40 (/-) IL-2R α (/-) mouse is bred by IL-12p40 (/-) IL-2R α (+/-) mouse.Because the mutator gene of IL-12p40 and IL-2R α all comprises a neo gene, so identify that the method for mouse IL-12p40 gene is to identify the wild type gene of IL-12p40 by the method for Genotyping (genotyping), the method for identifying mouse IL-2R α gene is the average fluorescent strength with the CD25 of CD4 positive cell in Flow cytometry peripheral blood.Wherein IL-2R α (/-) the CD4 positive cell of mouse does not comprise the cell of the CD25 positive; And the average fluorescent strength of the CD25 of IL-2R α (+/-) mouse CD4 positive cell is the half of IL-2R α (+/-) mouse, can identify thus mouse IL-2R α gene.All mouse were all used for experiment 12 to 18 week age, and experiment meets treatment and the guide for use of China Science & Technology University's Experimental Animal Center.
The liver of mouse and colon's piece are fixed in 4% neutral formalin after 1～2 day through dewatering and being embedded in the middle of paraffin.Paraffin-embedded liver organization is cut into the thin slice of 4 μ m, and the thin slice of 6 μ m is cut in colon.After all thin slice dewaxings, carry out H & E dyeing.By Pathology Doctors ', liver portal vein inflammation, bile duct destruction, fibrillatable, colitis are marked.Liver organization is by the Azan display fibers that dyes.
The results are shown in Figure 1, Fig. 2 and Fig. 4.The degree that can see portal vein lymphocytic infiltration in the liver of IL-12p40 (/-) IL-2R α (/-) mouse from the HE colored graph of Fig. 1 and Fig. 2 will be apparently higher than IL-2R α (/-) mouse, and the degree of colitis will be starkly lower than IL-2R α (/-) mouse.Pathology comments the result of marking identical with it.The Azan dyeing of Fig. 4 demonstrates the collagen deposition degree of IL-12p40 (/-) IL-2R α (/-) mouse will be higher than IL-2R α (/-) mouse and IL-2R α (+/-) mouse.
The separation of the each internal organs mononuclearcell of embodiment 3.
Take out the liver of mouse, use containing the PBS of 0.2%BSA and grind, the centrifugal 1min of 650rpm after steel wire filters, gets supernatant.Spleen and lymphonodi mesenterici grind with two slides and PBS/0.2%BSA, through nylon net filter.Mononuclearcell from the cell suspension of resuspended liver and spleen carries out density gradient centrifugation 20min by 40% with 70%Percoll (GE Healthcare company), collects intermediate layer cell.By hemacytometer, carry out under the microscope cell counting.
The results are shown in Figure 3.The liver mononuclearcell number of IL-12p40 (/-) IL-2R α (/-) mouse wants significance higher than IL-2R α (/-) mouse and IL-2R α (+/-) mouse.
Get 1 × 10
6individual separated liver mononuclearcell, seal with purifying CD16/32 antibody (Biolegend company), then the anti-CD3 of mark, CD4,, the fluorescence antibody of NK1.1, CD44 (Biolegend company), CD8 α (BD company), CD62L (eBioscience company).For dyeing in cell factor born of the same parents, cell is resuspended by the RPMI-1640 medium that contains 10% hyclone, uses the cytositimulation cocktail (purchased from eBioscience company) that contains albumen transport inhibitors to stimulate, and cultivates 4 hours for 37 ℃.Cell seals with purifying CD16/32 antibody, then uses the fluorescence antibody (Biolegend company) of anti-CD3, CD4, CD8 β, NK1.1 to carry out mark, then uses fixer (Biolegend company) to be fixed.Cell after fixing is worn film with wearing film liquid (Biolegend company), then uses the fluorescence antibody (Biolegend company) of anti-IFN-γ and IL-17A to carry out mark.
The results are shown in Figure 5, Fig. 6 and Fig. 7.Fig. 5 shows compared with IL-2R α (/-) mouse, and the cell increasing in the liver mononuclearcell of IL-12p40 (/-) IL-2R α (/-) mouse is mainly T cell, comprises CD4
+with CD8
+t cell.Fig. 6 is presented in liver, the CD4 of three kinds of mouse
+with CD8
+naivety (the naive) (CD44 of T cell
locD62L
hi), maincenter memory (central memory) (CD44
hicD62L
hi) remember (effector memory) (CD44 with effect
hicD62L
lo) three subgroups.Fig. 7 demonstrates the CD4 of IL-12p40 (/-) IL-2R α (/-) mouse
+with CD8
+in T cell, the ratio of the IFN-γ positive rises, and CD4
+in T cell, the ratio of the IL-17A positive declines.
The RNA of liver organization is used Trizol (Invitrogen company) to extract, then carries out reverse transcription by M-MLV transcriptase (Invitrogen company).What real-time fluorescence quantitative PCR was used is Premix Ex Taq kit (Takara company).The expression of genes of interest is by the expression standardization of house-keeping gene GAPDH.
The results are shown in Figure 4.The result of RT-PCR shows, several gene expression relevant to liver fibrosis raises in IL-12p40 (/-) IL-2R α (/-) mouse.
Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and described, but will be understood by those skilled in the art that, under the condition not deviating from by the appended defined the spirit and scope of the present invention of claim, the variation of various forms and details can be carried out therein, any combination of various embodiments can be carried out.
Claims (3)
1. a method of cultivating IL-12p40 as new liver fibrosis and primary biliary cirrhosis of liver animal model (/-) IL-2R α (/-) mouse, described method comprises the steps:
(1) IL-2R α (+/-) mouse and IL-12p40 (/-) mouse is hybridized, cultivates and obtain IL-12p40 (+/-) IL-2R α (+/-) mouse;
(2) step (1) is cultivated to IL-12p40 (+/-) IL-2R α (+/-) mouse that obtains and IL-12p40 (/-) mouse and hybridize, cultivate and obtain IL-12p40 (/-) IL-2R α (+/-) mouse; With
(3) step (2) is cultivated to the IL-12p40 that obtains (/-) IL-2R α (+/-) mouse and carry out selfing cultivation, identify and obtain IL-12p40 (/-) IL-2R α (/-) mouse.
2. method according to claim 1, wherein step (1) IL-2R α used (/-) mouse and IL-12p40 (/-) mouse is purchased from U.S. Jackson laboratory.
3. method according to claim 1, the method for wherein identifying mouse IL-12p40 gene is to identify with methods of genotyping the wild type gene that whether has IL-12p40 in mouse offspring; The method of identifying mouse IL-2R α gene is the average fluorescent strength with the CD25 of CD4 positive cell in Flow cytometry peripheral blood.
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| CN106834449A (en) * | 2017-01-10 | 2017-06-13 | 东南大学 | The IL-21 acceptor associated with primary biliary cholangitis and its application |
| CN107233579A (en) * | 2017-07-04 | 2017-10-10 | 华南理工大学 | The application of IL 12p40 (/) IL 2R α (/) mouse model |
| CN110777167A (en) * | 2019-11-06 | 2020-02-11 | 广州市妇女儿童医疗中心 | Method for constructing mouse model with movement dysfunction phenotype GTPCH enzyme deficiency disease |
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