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CN-122005516-A - Construction method and application of animal model of self-onset systemic lupus erythematosus

CN122005516ACN 122005516 ACN122005516 ACN 122005516ACN-122005516-A

Abstract

The invention discloses a construction method of a spontaneous systemic lupus erythematosus animal model, which is characterized in that an EP300 (E1A Binding Protein P) in keratinocytes is knocked out to enable mice to spontaneously develop a systemic lupus erythematosus phenotype, including local skin unhairing, continuous skin damage, continuous proteinuria, serum autoantibodies, histological examination shows systemic lupus erythematosus-like changes such as dermal immune cell infiltration, glomerular IgG deposition and the like, and clinical symptoms of the systemic lupus erythematosus are simulated, so that an important tool is provided for related researches of the systemic lupus erythematosus.

Inventors

  • LU QIANJIN
  • TIAN JINGRU
  • SHI LIQING

Assignees

  • 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所)

Dates

Publication Date
20260512
Application Date
20260211

Claims (10)

  1. 1. The method for constructing the spontaneous systemic lupus erythematosus animal model is characterized by comprising the following steps of: (1) Constructing a keratinocyte-conditional EP300 knockout mouse, the keratinocyte-conditional EP300 knockout mouse comprising C57BL/6 mice with genotypes EP300 flox/flox Krt5-CreERT2/+ and EP300 flox/+ Krt5-CreERT 2/+; (2) The EP300 gene in keratinocytes is knocked out using a gene knockout activator to mediate disease phenotype development.
  2. 2. The method of claim 1, wherein the knock-out activator is a compound that is metabolized to bind to the estrogen receptor mutant ERT to allow CreERT2 to exert Cre recombinase activity for gene knockout.
  3. 3. The method of claim 2, wherein the gene knockout activator is tamoxifen or 4-hydroxy tamoxifen.
  4. 4. The method according to claim 3, wherein tamoxifen is administered by intraperitoneal injection with or without needle, and wherein 4-hydroxy tamoxifen is administered by any of topical application, skin application, subcutaneous injection with or without needle, and intradermal injection.
  5. 5. The construction method according to claim 3, wherein the tamoxifen solution is continuously injected intraperitoneally into the diagonally-forming cell conditioned EP300 knockout mice for 1 to 7 days once a day when tamoxifen is used as a gene knockout activator, and wherein the 4-hydroxy tamoxifen solution is continuously applied to the skin on the ventral and dorsal sides of the skin ears of the diagonally-forming cell conditioned EP300 knockout mice without hair or after shaving for 1 to 7 days once a day when 4-hydroxy tamoxifen is used as a gene knockout activator.
  6. 6. The method according to claim 5, wherein the 4-hydroxy tamoxifen solution is prepared in a proportion of 50mg of 4-hydroxy tamoxifen, 1mlDMSO and 9ml of corn oil, and the dose of the 4-hydroxy tamoxifen solution to be applied externally to the mice is 10 to 80. Mu.L.
  7. 7. The method of claim 5, wherein the mice develop disease phenotype 7 days after initial intraperitoneal injection of tamoxifen or 7 days after topical application of 4-hydroxy tamoxifen.
  8. 8. The method of claim 1, wherein the keratinocyte conditional EP300 knockout mouse is constructed by: Crossing a mouse with the genotype of EP300 flox/flox and a Krt5-CreERT2/+ mouse to obtain a filial generation, reserving a heterozygote mouse with the genotype of EP300 flox/+ Krt5-CreERT2/+ and crossing the heterozygote mice with each other to obtain a mouse with the genotype of EP300 flox/flox Krt5-CreERT 2/+.
  9. 9. Use of the spontaneous systemic lupus erythematosus animal model constructed by the construction method of any one of claims 1-8 in research of SLE disease mechanism.
  10. 10. Use of the spontaneous systemic lupus erythematosus animal model constructed by the construction method of any one of claims 1-8 in screening drugs for targeted treatment of lupus skin lesions.

Description

Construction method and application of animal model of self-onset systemic lupus erythematosus Technical Field The invention belongs to the technical field of disease animal model construction methods, and particularly relates to construction of an animal model of systemic lupus erythematosus. Background Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease of high heterogeneity, whose central features are the sustained activation of the type I interferon signaling pathway and the widespread production of autoantibodies. The occurrence of this disease is the result of complex interactions of genetic, environmental and endocrine factors, the specific mechanisms of which have not yet been fully elucidated. Epidemiological data shows that global SLE prevalence is in a continuously growing state, and has created a significant disease burden. Under the background, the construction of an animal model which can accurately simulate key clinical characteristics and has good operability and economy has become a key premise for promoting the research and treatment innovation of the disease mechanism. SLE animal models commonly used at present are divided into two main types, namely spontaneous type and induced type, according to the construction mode. The spontaneous model such as NZBWF, BXSB/MpJ and MRL/lpr strain mice has the characteristics of clear genetic background and spontaneous occurrence of diseases, and is an important tool for researching genetic factors and immune disorders. The induced model is induced by exogenous antigens (such as chromatin and campylobacter jejuni) or immunostimulants (such as pristane), and has certain advantages in cost and period. However, both models have obvious defects that spontaneous models are high in price, different in attack time and obvious in individual difference, genetic basis of the spontaneous models is basically different from human SLE, induced models are short in period but often lack genetic susceptibility basis, the correlation of disease processes and clinical manifestations of the disease is weak, and modeling processes are strong in dependence on technical operation. More notably, the existing models are mostly difficult to reproduce the typical skin pathological changes of SLE, and the differences between the genetic mechanism and the disease evolution level and the human beings greatly restrict the application efficacy of the SLE in pathogenesis exploration, pathological phenotype analysis and treatment strategy evaluation. Thus, the development of new spontaneous models based on the true genetic background of patients has become an urgent need to deepen SLE cognition. Skin is not only a common target organ for SLE, but its local immune microenvironment (composed of keratinocytes, resident immune cells and cytokine networks) may play a role beyond the past cognition in the initiation and maintenance of systemic autoimmunity. Clinical studies have shown that there is often significant inflammatory infiltration and interferon signaling activation in SLE patient skin, and that part of cutaneous lupus erythematosus cases can eventually progress to systemic lesions. Although the direct causal chain between keratinocyte dysfunction and systemic onset of SLE is not yet fully established, the sustained type I interferon microenvironment can drive immune cell aberrant activation and autoantibody production. Together, these evidence suggest that disruption of local immune homeostasis in the skin may be an important link in triggering or amplifying systemic autoimmune responses, providing a new perspective for understanding SLE pathogenesis. EP300 (p 300) is a histone acetyl transferase and transcriptional coactivator, and is a key regulatory molecule for various signaling pathways (e.g., CREB, NF- κ B, STAT, etc.). The method plays an important role in the processes of cell differentiation, immune response, cell cycle, apoptosis and the like by the participation of acetylated histones and non-histones in gene expression regulation. The functional abnormality of EP300 is closely related to autoimmune diseases, tumorigenesis, and the like. At present, no report related to the construction of an animal model of self-induced SLE by using keratinocyte conditional EP300 knockout mice is known. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a new spontaneous SLE animal model for research and development of pathogenesis, pathological features and treatment methods of SLE. In order to achieve the technical aim, the invention discloses a method for constructing a spontaneous systemic lupus erythematosus animal model, which comprises the following steps: (1) Constructing a keratinocyte-conditional EP300 knockout mouse, the keratinocyte-conditional EP300 knockout mouse comprising C57BL/6 mice with genotypes EP300 flox/flox Krt5-CreERT2/+ and EP300 flox/+ Krt5-CreERT 2/+; (2) The EP300 gene in keratinocytes is knocked out using