目的 建立可用于高内涵分析的肾纤维化体外的细胞模型,为抗肾纤维化中药化合物的筛选和机制研究奠定基础.方法 本研究通过TGF-β1诱导正常大鼠肾成纤维细胞NRK49F,以肌成纤维细胞标志 α-平滑肌肌动蛋白(α-SMA)作为检测指标,通过高内涵成像分析系统对α-SMA荧光表达强度进行检测分析,并对细胞种板密度、诱导时间、诱导培养基中血清浓度等各种条件进行优化,建立稳定的肾纤维化体外模型.在此基础上以TGF-β1受体阻滞剂SB525334和姜黄素、大黄素对模型进行验证.并进一步探讨吡非尼酮是否具有抗肾纤维化功效.结果 实验通过对细胞密度、诱导时间、培养基中血清浓度进行优化,确立了可用于高内涵分析的NRK49F肾纤维化体外模型的建立条件.SB525334、姜黄素、大黄素和吡非尼酮均能减少TGF-β1诱导的α-SMA过表达,抑制NRK49F的活化.结论 实验建立了可用于高内涵筛选技术的肾纤维化体外模型,并能相对稳定地筛选具有抗肾纤维化作用的药物.吡非尼酮在本模型中抑制了成纤维化细胞向肌成纤维细胞转化,提示其可能具有抗肾纤维化作用,为抗肾纤维化药物初筛对象的选择提供了思路.%Aim Toestablishacellularmodelofrenal fibrosis in vitro,which can be used for the high-content analysis,and lay a good foundation for the screening and mechanism study of traditional Chinese medicine compoundswithanti-fibroticeffect.Methods Inthis study,normal rat kidney fibroblasts(NRK49F)were induced by TGF-β1 .As the maker ofmyofibroblast,al-pha smooth muscle actin (α-SMA)was detected andanalyzed by high content imaging analysis system,and the cell plate density,induction time,the concentra-tion of serum in medium were optimized,to establish a stable model of renal fibrosis in vitro.Then the model was validated by TGF-β1 receptor blocker SB525334, curcumin and emodin.On this basis,whether pirfeni-done had the effect of anti-renal fibrosis was explored. Results Byoptimizingthecelldensity,inductiontime and serum concentration in culture medium,the conditions of NRK49 F renal fibrosis model in vitro for high content analysis were established. SB525334, curcumin and emodin and pirfenidone reduced the ex-pression of α-SMA in NRK49 F induced by TGF-β1 . Conclusions Aninvitromodelofrenalfibrosis, which can be used for high content screening technolo-gy,is established and can be used to screen drugs withanti-renal fibrosis effects relatively stably.Pirfenidone in this model inhibits the transformation of NRK49 F in-to myofibroblasts,suggesting that it may be able to in-hibit renal fibrosis,providing a way for the selection of objects for anti-renal fibrosis drug screening.
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