A DGKα - / - ζ - / - mouse model, a method for constructing the same, and an application thereof in simulating the progression of clinical autoimmune hepatitis

Abstract

The present invention relates to a DGKα<supgt;‑ / ‑< / supgt;ζ<supgt;‑ / ‑< / supgt; mouse model, in which both the DGKα subtype and the DGKζ subtype are deleted; the present invention also relates to a method for constructing a DGKα<supgt;‑ / ‑< / supgt;ζ<supgt;‑ / ‑< / supgt> mouse model; the present invention also relates to the application of a DGKα<supgt;‑ / ‑< / supgt;ζ<supgt;‑ / ‑< / supgt> mouse model in simulating the progression of clinical autoimmune hepatitis. The research team of the present invention independently constructed DGKαζ double‑deficient mice. Without the induction of stimulating factors, these mice can spontaneously present symptoms of hepatocyte inflammatory injury such as persistently high levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin, positive autoantibodies against nuclear antigens, and progress to liver fibrosis and cirrhosis, which is extremely similar to the clinical manifestations and evolution process of AIH patients.

Description

Technical Field

[0001] The present invention relates to a mouse model, specifically a DGKα - / - ζ - / - mouse model and its construction method and application in simulating the progression of clinical autoimmune hepatitis. Background Art

[0002] Autoimmune hepatitis (AIH) is liver parenchymal cell damage mediated by abnormal autoimmune reactions, causing acute or chronic liver inflammation, with clinical features such as elevated serum transaminases, IgG globulins, formation of autoantibodies, and interface hepatitis visible in liver pathological tissue biopsy. The pathogenesis of AIH is unclear and may be related to environmental factors, genetic susceptibility, etc. After the liver immune tolerance is broken, an autoimmune response occurs, causing damage to the corresponding target tissues, apoptosis and necrosis of target cells. Currently, the treatment methods for AIH are limited. Mainly, symptomatic treatment is carried out using ursodeoxycholic acid and immunosuppressants, but the curative effect is poor. Advanced patients usually can only undergo liver transplantation. In recent years, the global incidence of AIH has gradually increased, and the risk of patients suffering from liver cancer and other malignant tumors has also increased significantly. Therefore, clarifying the pathogenesis of AIH, finding the key regulatory nodes in the disease occurrence and development, and revealing its molecular regulatory network will surely promote the discovery of new strategies for its clinical diagnosis, treatment and effective prevention.

[0003] Currently, there are about 7 models for studying AIH, including liver homogenate immunization method, alternative antigen (Con A / α-GalCer) immunization method, antigen / TCR transgenic method, antigen-loaded DC cell immunization method, virus infection method using molecular similarity, specific gene knockout method, AIH antigen immunization method, etc. However, the existing methods can either only simulate acute immune liver injury (lacking AIH specificity), or the excessive inflammatory damage is lethal and cannot simulate the whole process of clinical AIH occurrence and development (that is, these mouse models only have liver autoimmune inflammatory damage, but the excessive inflammatory damage causes the mice to die in infancy and cannot present characteristic lesions in the middle and late stages of clinical AIH such as liver fibrosis). Therefore, the lack of a good animal model for simulating clinical AIH seriously hinders the in-depth study of this disease. Summary of the Invention

[0004] An object of the present invention: In order to overcome the defects of the prior art, the present invention provides a DGKα - / - ζ - / - mouse model.

[0005] A technical solution of the present invention: A DGKα - / - ζ - / - mouse model, in which the mouse lacks both DGKα subtype and DGKζ subtype simultaneously.

[0006] Another object of the present invention: In order to overcome the defects of the prior art, the present invention provides a method for constructing a DGKα - / - ζ - / - mouse model.

[0007] Technical solution of the present invention: A method for constructing a DGKα - / - ζ - / - mouse model, which has steps of constructing DGKα - / - mice, constructing DGKζ - / - mice and constructing double gene knockout mice,

[0008] wherein,

[0009] Steps for constructing DGKα - / - mice: Knock out the exon 184-328 segment in the catalytic domain part encoded by DGKα to obtain DGKα - / - mice with lost DGKα enzyme activity;

[0010] Steps for constructing DGKζ - / - mice: Knock out the exon 11-15 segment in the catalytic domain part encoded by DGKζ to obtain DGKζ - / - mice with lost DGKζ enzyme activity;

[0011] Steps for constructing double gene knockout mice: Cross DGKα - / - mice and DGKζ - / - mice to obtain offspring DGKα - / - ζ - / - mice.

[0012] Preferably, the steps for constructing DGKα - / - mice: Use the targeting technology to target and insert two Loxp sites at both ends of exon 184 and 328 of the mouse DGKα gene to obtain Loxp-DGKα mice;

[0013] Then mate the Loxp-DGKα mice with Cre tool mice to obtain offspring DGKα - / - mice with the exon 184-328 segment of the DGKα gene knocked out.

[0014] Preferably, the steps for constructing DGKζ - / - mice: Use the targeting technology to target and insert two Loxp sites at both ends of exon 11 and 15 of the mouse DGKζ gene to obtain Loxp-DGKζ mice;

[0015] Then mate the Loxp-DGKζ mice with Cre tool mice to obtain offspring DGKζ - / - mice with the exon 11-15 segment of the DGKζ gene knocked out.

[0016] Another object of the present invention: In order to overcome the defects of the prior art, the present invention provides a DGKα - / - ζ - / - mouse model for use in simulating the progression of clinical autoimmune hepatitis.

[0017] Numerous studies have shown that diacylglycerol kinases (DGKs) can promote the conversion of diacylglycerol (DAG) into phosphatidic acid (PA) through phosphorylation, and are important regulatory molecules for peripheral immune regulation, especially for maintaining peripheral immune tolerance. Among the 10 subtypes of DGKs, DGKα and ζ subtypes are expressed in T cells and are crucial for the development and tolerance of T cells.

[0018] Furthermore, using this mouse, we found that DGKα and DGKζ in it coordinately regulate the diacylglycerol (DAG) metabolism of T cells (CD4, CD8, NKT, and Treg) in the liver, and play a decisive role in liver immune tolerance by maintaining T cell quiescence, inducing T cell anergy, and promoting Treg function.

[0019] Therefore, the research team of the present invention independently constructed DGKαζ double knockout mice. Without the induction of stimulating factors, these mice can spontaneously present symptoms of hepatocyte inflammatory injury such as persistently high levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin, positive autoantibodies against nuclear antigens, and progress to liver fibrosis and cirrhosis - which is extremely similar to the manifestations of clinical AIH patients. Further, using these mice, we found that DGKα and DGKζ coordinately regulate the diacylglycerol (DAG) metabolism of T cells (CD4, CD8, NKT, and Treg) in the liver, and play a decisive role in liver immune tolerance by maintaining T cell quiescence, inducing T cell anergy, and promoting Treg function. Therefore, the present invention constructs a mouse model that can accurately simulate the occurrence and development process of clinical autoimmune hepatitis, providing a good animal model for exploring the pathogenesis of AIH and intervention research. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram showing that double knockout of DGKα and ζ (DGKα - / - ζ - / - , namely DKO) leads to spontaneous activation of T cells. Note: n.s. no significant statistical difference, *p < 0.05, **p < 0.01;

[0021] Figure 2 Schematic diagram showing the occurrence of spontaneous autoimmune hepatitis in DKO mice. Note: ***p < 0.001;

[0022] Figure 3Schematic diagram of chronic hepatitis and liver fibrosis in DKO aged mice, Note: ns no significant statistical difference, **p<0.001, ***p<0.001;

[0023] Figure 4 Adoptive transfer of T cells from DKO mice to TCRβ - / - δ - / - Schematic diagram of AIH induced in (T cell-deficient) mice, Note: *p<0.05, **p<0.01, ***p<0.001;

[0024] Figure 5 Schematic diagram showing that adoptive transfer of wild-type Tregs to DKO mice can inhibit the occurrence of AIH. Note: ns, no statistical difference, ***p<0.001. DETAILED DESCRIPTION

[0025] The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The embodiments provided are only illustrative of the method of the present invention and do not limit the rest of the content disclosed by the present invention in any way.

[0026] Specific solutions of the present invention

[0027] A DGKα - / - ζ - / - Mouse model that lacks both the DGKα and DGKζ subunits.

[0028] A DGKα - / - ζ - / - Methods for constructing mouse models with DGKα - / - Mouse constructs, DGKζ - / - Mouse construction and double knockout mouse construction steps,

[0029] Among them,

[0030] DGKα - / - Mouse construction steps:

[0031] First, construct the targeting vector of the target gene, clone two Loxp sites in the same direction into the two ends of the target gene DGKα exon 184 and 328, respectively, and then use CRISPR / Cas fertilized egg injection or TurboKnockout blastocyst injection technology to establish the target gene Loxp-DGKα mice;

[0032] Then, the Loxp-DGKα mice were mated with Cre tool mice that specifically expressed certain cells / tissues. Cre was able to cut the region in the middle of Loxp and remove the exon 184-328 segment encoding the DGKα catalytic domain, causing the DGKα enzyme activity to be lost. At the same time, Cre also removed the ATP binding site of the activation domain in the exon 184-328 segment to prevent the DGKα enzyme from being reactivated. The above method was used to obtain the offspring DGKα - / - Mice;

[0033] DGK - / - Mouse construction steps:

[0034] First, construct the targeting vector of the target gene, clone two Loxp sites in the same direction into the two ends of exon 11 and 15 of the target gene DGKζ, and then use CRISPR / Cas fertilized egg injection or TurboKnockout blastocyst injection technology to establish the target gene Loxp-DGKζ mice;

[0035] The Loxp-DGKζ mice are then mated with Cre tool mice that specifically express a certain cell / tissue. Cre can cut the region in the middle of Loxp and remove the exons 11-15 that encode the DGKζ catalytic domain, causing the DGKζ enzyme to lose activity. At the same time, Cre also removes the ATP binding site of the activation domain in exons 11-15 to prevent the DGKζ enzyme from being reactivated. The offspring DGKζ obtained by the above method - / - Mice;

[0036] Steps for constructing double knockout mice, DGKα - / - Mice and DGKζ - / - Mice are crossed and the offspring mice are genetically identified to obtain DGKα - / - ζ - / - Mouse.

[0037] A DGKα - / - ζ - / - Mouse models are used to mimic the progression of clinical autoimmune hepatitis.

[0038] Verification Example

[0039] Verification Example 1: DGKα of the present invention - / - ζ - / - T cells spontaneously activate in the DKO mouse model.

[0040] DKO mice showed obvious spleen and lymph node enlargement at 2-3 months of age ( Figure 1 -A), with increased cellularity in the spleen and lymph nodes ( Figure 1-B).

[0041] Figure 1 Flow cytometry (FCM) results in -C showed that the peripheral T cells of DKO mice were reduced.

[0042] In the spleen, the proportions of CD4 + and CD8 + T cells were both decreased, especially the absolute number of CD8 + T cells was significantly reduced ( Figure 1 -D), reflecting a partial block in the development and maturation of thymocytes (from the CD4CD8 double-positive stage to the single-positive stage) in these mice.

[0043] The expression of CD69 was upregulated in the T cells of DKO mice ( Figure 1 -E), and most of the phenotypes were CD62L low CD44 hi memory / effector T cells ( Figure 1 -F); among them, a considerable proportion of CD4 + and CD8 + T cells in DKO mice could produce IFNγ after being stimulated with PMA and ionomycin in vitro for 4 hours ( Figure 1 -G).

[0044] As Figure 1 shown in -H, when a part of CD4 + and CD8 + T cells from DKO mice were cultured in vitro for 72 hours, they could divide without TCR stimulation, but CD4 + or CD8 + T cells from wild-type mice did not divide; when stimulated with anti-CD3 antibody, the division rate of T cells from DKO mice was significantly faster than that of T cells from wild-type mice, and the T cells could still divide even when the CD28 co-stimulatory signal was blocked by CTLA4-Ig; similarly, before and after stimulation with anti-CD3 antibody, the phosphorylation level of Erk1 / 2 in DKO T cells was enhanced, indicating that these T cells were indeed spontaneously activated and were more easily activated ( Figure 1 -I).

[0045] Figure 1 -C showed that at 4 months of age, we observed that the proportion of peripheral CD4 + / - ζ - / - T cells in DGKα + mice was decreased, and the proportions of CD8 + / - ζ - / - and DGKα - / - ζ + / - in CD8 +The proportions of T cells all decreased. Although not as significant as in DGKα - / - ζ - / - T cells, in DGKα + / - ζ - / - T cells, there was a significantly increased expression of CD69 and mainly CD62L low CD44 hi effector / memory T cell phenotype; while in DGKα - / - ζ + / - mice, only CD8 + T cells showed a weak activation phenotype, suggesting that DGKζ has a stronger effect on T cell activation than DGKα.

[0046] In summary, the above results indicate that DGKα and ζ coordinately regulate the homeostasis of normal T cells, and a decrease in the activities of both DGKα and ζ can lead to spontaneous activation of T cells in vivo. T cells in DGKαζ double-deficient mice can be spontaneously activated, accompanied by over-activation of Erk1 / 2; DGKαζ double-deficient mice spontaneously develop AIH (but do not have juvenile lethality) and progress chronically.

[0047] Verification Example 2: The mouse model of the present invention develops spontaneous autoimmune hepatitis.

[0048] Liver tissues were taken from WT and DKO mice (3 months old), and it was found that DKO mice had obvious hepatomegaly ( Figure 2 -A).

[0049] Liver tissues were fixed with 10% formaldehyde, dehydrated with alcohol, and embedded in paraffin; thin sections were prepared and stained with hematoxylin-eosin (H&E), and it was found that there were obvious mononuclear cell infiltration, piecemeal necrosis, and regeneration of hepatocytes in the liver of DKO mice ( Figure 2 -B), indicating that obvious inflammatory reactions occurred in the livers of DKO mice.

[0050] Detection with a hepatocyte injury marker AST / ALT diagnostic kit showed that the levels of ALT and AST in the sera of DKO mice (3 months old or above) were significantly increased ( Figure 2 -C).

[0051] Figure 2 -D's FCM results showed that there were a large number of T cells (mainly CD4 + and CD8 + cell infiltration increased) in the livers of DKO mice.

[0052] Verification Example 3: The mouse model of the present invention develops chronic hepatitis and liver fibrosis as it ages.

[0053] Compared with wild-type mice, elevated serum ALT could be detected in DKO mice as early as 6 weeks of age, and most mice had elevated serum ALT at 12 weeks of age and maintained it throughout their lives ( Figure 3 -A).

[0054] Collagen staining of the liver with Sirius red revealed a significant increase in the content of intraliver collagen fibers in DKO mice at 4 months of age and older, especially around the portal vein and in the periportal area, indicating liver fibrosis. Notably, the degree of liver fibrosis in 6-month-old DKO mice was significantly more severe than that in 4-month-old DKO mice ( Figure 3 -B).

[0055] The lifespan of DKO mice was generally 6 to 12 months, and eventually severe jaundice occurred, accompanied by elevated serum total bilirubin and direct bilirubin ( Figure 2 -C, D).

[0056] ELISA kits were used to detect the levels of serum autoantibodies IgG and IgM in DKO mice (5 - 11 weeks old or 19 - 25 weeks old), and it was found that only IgM was significantly elevated ( Figure 4 -E).

[0057] Verification Example 4: Adoptively transferring the peripheral cells of the mouse model of the present invention to TCRβ - / - δ - / - mice would induce AIH.

[0058] 2 - 3×10 6 purified peripheral T cells from DKO mice or wild-type (WT) mice were intravenously injected into TCRβ - / - δ - / - (T cell-deficient) mice.

[0059] After 10 weeks, it was found that adoptive transfer of DKO mouse peripheral T cells led to a significant increase in the serum ALT level of recipient mice ( Figure 4 -A), and a large infiltration of lymphoplasmacyte cells ( Figure 4 -B) and CD8 + T cells in the liver tissue ( Figure 4 -C); further, 1 - 1.5×10 6 peripheral CD4 + and CD8 + T cells were obtained by density gradient centrifugation, MACS magnetic bead rough sorting, and flow cytometry sorting from DKO mice or WT mice, and were respectively adoptively transferred into TCRβ - / - δ - / - mice and tamoxifen was injected. It was found that adoptive transfer of only DKO peripheral CD8 + T cells or CD8 + T cells combined with CD4 +T cell adoptive transfer can lead to a significant increase in the serum ALT level of recipient mice ( Figure 4 -D); FACS analysis found that the proportions of CD4 + and CD8 + T cells in the spleens and livers of recipient mice after adoptive transfer were significantly increased ( Figure 4 -E); H&E staining showed that adoptive transfer of only CD8 + T cells from DKO mice was sufficient to cause severe liver injury, with a large infiltration of inflammatory cells in the liver tissue. This indicates that the T cells of DKO mainly induce AIH in recipient mice via CD8 + T cells.

[0060] Verification Example 5: Adoptive transfer of wild-type Tregs to the model mice of the present invention can inhibit the occurrence of AIH.

[0061] To verify the hypothesis that "DKO Tregs may have lost the ability to inhibit liver-specific effector T cells", wild-type Tregs were intravenously injected into 3-4-week-old DKO mice. At 3-4 months of age, the DKO mice injected with PBS all developed fulminant hepatitis as expected; however, the DKO mice injected with wild-type Tregs did not show obvious hepatitis, such as no increase in serum ALT, no infiltration of liver monocytes, or no hepatocyte necrosis ( Figure 5 ) - suggesting that wild-type Tregs can inhibit the autoimmune response in the livers of DKO mice. These research results indicate that DKO Tregs are dysfunctional through an intrinsic mechanism, while wild-type Tregs can inhibit the occurrence of liver inflammation in the DKO environment.

[0062] Note: "- / -" indicates that this gene has been knocked out, i.e., it does not exist; "+", on the other hand, represents expression. Taking the "C" gene knockout mice as an example, the normal wild-type mice of the "C" gene are usually represented by "+ / +"; the homozygous mice with the "C" gene knocked out are represented by "- / -"; the heterozygous mice with the "C" gene not completely knocked out are represented by "+ / -".

[0063] Such as "DGKα - / - ζ - / - " indicates that both the α and ζ subtypes have been completely knocked out.

Claims

1. A method for constructing a DGKα - / - ζ - / - mouse model, which has DGKα - / - mouse construction, DGKζ - / - mouse construction, and double - gene knockout mouse construction steps, wherein, DGKα - / - Mouse construction steps: Knock out the exon segments 184 - 328 in the catalytic domain - encoding part of DGKα to obtain a DGKα - / - mouse with lost DGKα enzyme activity; DGKζ - / - Mouse construction steps: Knock out the exon segments 11 - 15 in the catalytic domain - encoding part of DGKζ to obtain a DGKζ - / - mouse with lost DGKζ enzyme activity; Double - gene knockout mouse construction steps: Cross the DGKα - / - mouse and the DGKζ - / - mouse to obtain the offspring DGKα - / - ζ - / - mouse.

2. The method for constructing a DGKα - / - ζ - / - mouse model according to claim 1, wherein the DGKα - / - mouse construction steps: Targetedly insert two Loxp sites at both ends of exon 184 and exon 328 of the mouse DGKα gene to obtain Loxp - DGKα mice; Then cross the Loxp - DGKα mice with Cre tool mice to obtain the offspring DGKα - / - mouse with exon segments 184 - 328 of the DGKα gene knocked out.

3. The method for constructing a DGKα - / - ζ - / - mouse model according to claim 1, wherein the DGKζ - / - mouse construction steps: Targetedly insert two Loxp sites at both ends of exon 11 and exon 15 of the mouse DGKζ gene to obtain Loxp - DGKζ mice; Then cross the Loxp - DGKζ mice with Cre tool mice to obtain the offspring DGKζ - / - mouse with exon segments 11 - 15 of the DGKζ gene knocked out.

4. A DGKα - / - ζ - / - mouse model, which is constructed by the method of any one of claims 1 - 3 above, and the mouse lacks both the DGKα subtype and the DGKζ subtype.

5. The DGKα of claim 4 - / - ζ - / - Application of a mouse model in simulating the progression of clinical autoimmune hepatitis

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