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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。
OBJECTIVE: To establish a cell model of renal fibrosis in vitro that can be used for high connotation analysis and lay the foundation for the screening of anti-renal fibrosis compounds and their mechanisms. Methods In this study, normal rat renal fibroblasts NRK49F were induced by TGF-β1, α-smooth muscle actin (α-SMA) was used as a detection index, and α-SMA was detected by high content imaging analysis system Intensity of detection and analysis, and cell density, induction time, induction medium concentration and other conditions are optimized to establish a stable model of renal fibrosis in vitro. On this basis, the model was validated by TGF-β1 blockers SB525334 and curcumin and emodin. And to further explore whether pirfenidone has anti-renal fibrosis. Results The establishment of an in vitro model of NRK49F renal fibrosis that could be used for high content analysis was established by optimizing the cell density, induction time, and serum concentration in the medium. Both SB525334, curcumin, emodin and pirfenidone could reduce TGF-β1-induced α-SMA overexpression and inhibit the activation of NRK49F. Conclusion The experiment established an in vitro model of renal fibrosis that could be used for high content screening and could screen the drug with anti-renal fibrosis relatively stably. Pirfenidone inhibits the transformation of fibroblasts to myofibroblasts in this model, suggesting that it may have anti-renal fibrosis, providing a new idea for the selection of anti-fibrotic drugs.