摘要
目的 利用 CRISPR技术敲除 SIRT3 胶原来诱导建立类风湿关节炎大鼠模型,分析对其免疫紊乱和软骨损伤的影响.方法 实验分成 WT组(野生型)、KO 组(CRISPR 技术敲除 SIRT3)、WT-RA 组(野生型类风湿关节炎大鼠模型)、KO-RA组(CRISPR技术敲除 SIRT3 类风湿关节炎大鼠模型).检测大鼠胸腺指数和脾指数,Western blot方法检测 SIRT3、MMP-2、MMP-3、MMP-9 蛋白表达,流式细胞术分析 CD4+/T淋巴细胞和 CD8+/T淋巴细胞百分比,ELISA法检测血清中 Th1(TNF-α、IFN-γ)和 Th2(IL-4、IL-10)细胞因子.结果 与 WT 组比较,WT-RA 组 SIRT3、CD4+、CD4+/CD8+、胸腺指数、脾指数、TNF-α、IFN-γ、MMP-2、MMP-3、MMP-9 水平显著升高,CD8+、IL-4、IL-10 水平显著降低(P<0.05).与 KO组比较,KO-RA 组 CD4+、CD4+/CD8+、胸腺指数、脾指数、TNF-α、IFN-γ、MMP-2、MMP-3、MMP-9 水平显著升高,CD8+、IL-4、IL-10 水平显著降低(P<0.05).与 WT-RA 组比较,KO-RA 组 SIRT3、CD4+、CD4+/CD8+、胸腺指数、脾指数、TNF-α、IFN-γ、MMP-2、MMP-3、MMP-9 水平显著降低,CD8+、IL-4、IL-10 水平显著升高(P<0.05).结论 利用 CRISPR技术敲除 SIRT3 缓解胶原诱导的类风湿关节炎大鼠造成的免疫系统的紊乱和软骨组织的损伤.
Abstract
Objective The rat model of rheumatoid arthritis was established by using CRISPR technology to knockout SIRT3,and the effects on immune disorder and cartilage damage were analyzed.Method The experiment was divided into WT group(wild type),KO group(SIRT3 was knockout by CRISPR technology),WT-RA group(wild type rat model of rheumatoid arthritis),KO-RA group(a rat model of rheumatoid arthritis in which SIRT3 was knocked out by CRISPR).The thymus index and spleen of rats were detected.Protein expression of SIRT3,MMP-2,MMP-3,and MMP-9 were detected by Western blot,and the percentage of CD4+/T lymphocytes and CD8+/T lymphocytes was analyzed by flow cytometry.The serum cytokines of Th1(TNF-α,IFN-γ)and Th2(IL-4,IL-10)were detected by ELISA.Result Compared with WT group,the levels of SIRT3,CD4+,CD4+/CD8+,thymus index,spleen index,TNF-α,IFN-γ,MMP-2,MMP-3 and MMP-9 in WT-RA group were significantly increased,while the levels of CD8+,IL-4 and IL-10 were significantly decreased(P<0.05).Compared with KO group,the levels of CD4+,CD4+/CD8+,thymus index,spleen index,TNF-α,IFN-γ,MMP-2,MMP-3 and MMP-9 in KO-RA group were significantly increased,while the levels of CD8+,IL-4 and IL-10 were significantly decreased(P<0.05).Compared with WT-RA group,the levels of SIRT3,CD4+,CD4+/CD8+,thymus index,spleen index,TNF-α,IFN-γ,MMP-2,MMP-3 and MMP-9 in KO-RA group were significantly decreased,while the levels of CD8+,IL-4 and IL-10 were significantly increased(P<0.05).Conclusion Using CRISPR technology to knock out SIRT3 alleviated the immune disorder and cartilage damage in rheumatoid arthritis rats induced by collagen.
维普
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