摘要
目的探讨mTOR信号通路在肾间质成纤维细胞增生活化过程中的调控作用，并研究其抑制剂在抗肾纤维化治疗中的可行性。方法用8周龄雌性C57BL／6小鼠构建单侧输尿管结扎（UUO）肾间质纤维化动物模型（n=30），按数字随机法分为雷帕霉素组（n=15）及UUO组（n=15）。雷帕霉素组术前1d开始腹腔注射雷帕霉素（2mg·kg^-1·d^-1）至实验结束；UUO组注射生理盐水。分别于术后1、3、7、14d处死小鼠（n=3），留肾组织进行相关检测。同时，体外实验评估雷帕霉素对TGF—β诱导鼠成纤维细胞株（NIH3T3细胞）活化的干预作用。结果UUO小鼠肾组织中活化的肌成纤维细胞[α肌动蛋白（α-SMA）阳性】高表达mTOR通路下游效应因子pS6K。雷帕霉素显著抑制pS6K表达及肾间质中肌成纤维细胞的活化，改善肾小管间质损伤及纤维化程度。实时荧光定量PCR结果提示雷帕霉素组小鼠肾皮质组织中成纤维细胞特异蛋白1（FSPl）、转化生长因子B（TGF-β）、结缔组织因子（CTGF）及Ⅳ型胶原蛋白基因仅l（Co14A1）的mRNA水平显著下降。体外实验结果示TGF-β诱导小鼠成纤维细胞株（NIH3T3）的mTOR通路显著活化，并大量合成α—SMA。雷帕霉素能够明显抑制mTOR通路活性，降低细胞的纤维化活性。结论肾间质纤维化过程中成纤维细胞内的mTOR信号通路高度活化。抑制mTOR通路能够显著降低成纤维细胞的活性，改善肾间质纤维化程度。
Objective To evaluate the regulatory role of roTOR signaling in activation of renal interstitial fibroblasts and the potential effect on interstitial fibrosis. Methods 8-week old female C57BL/6 mice （n=30） underwent unilateral ureteral obstruction （UUO） to induce renal interstitial fibrosis. Animals were randomly divided into rapamycin （2 mg·kg^-1· d^-1） group and UUO group （vehicle-treated） （n=15 each group）. Daily intraperitoneal injection of rapamycin or saline was applied to animals from day 1 before operation to the end of experiment. Three mice were sacrificed at day l, 3, 7, 14 respectively and kidneys were harvested for further analysis. NIH3T3 cells were stimulated by TGF-β for 12 hours with the presence or bsence of rapamycin （100 nmol/L）. Results Immunofluorescent co-staining revealed that active fibroblasts highly expressed pS6K and α-SMA in kidney interstitium. Administation of rapamycin significantly inhibited activation of roTOR signaling in fibroblasts and ameliorated interstitial fibrosis of obstructed kidneys. Real-time PCR confirmed that rapamycin decreased the mRNAexpression of FSP1, TGF-β, CTGF and Co14A1 in fibrotic kidneys. In vitro experiment revealed that TGF-β induced highly expression of pS6K and α-SMA cultured NIH3T3 cells, which could be markedly inhibited by rapamycin. Conclusions mTOR signaling highly activates in interstitial fibroblasts during kidney fibrosis. Inhibition of mTOR signaling by rapamycin decreases the activation of fibroblasts and ameliorates interstitial fibrosis.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2012年第3期226-231,共6页
Chinese Journal of Nephrology
基金
国家自然科学基金（30871169）
教育部新教师项目资助课题（200805331032）
中南大学青年教师助推专项资助项目（2011QNZT165）
