中文摘要：

目的：在细胞水平探讨西罗莫司[sirolimus,又名雷帕霉素（rapamycin）]的抗肾纤维化作用。方法：采用原代培养的大鼠肾皮质肌成纤维细胞（myofibroblast,MyoF）。实验各时间点分别设对照组和用西罗莫司40mg/L处理组。将细胞培养12h,24h,48h后分别收集细胞和细胞培养上清液,用Western blot方法检测细胞内α-平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）、纤维连接蛋白（fibronectin,FN）、Ⅰ型胶原（typeⅠcollagen,ColⅠ-）和上清液中FN,ColⅠ-蛋白水平的表达;用实时荧光定量PCR（real-time quantitative PCR）法检测细胞培养1h,2h,4h,6h各时间点前胶原Ⅰ（procollagenⅠ-）mRNA表达的变化;用明胶酶谱法检测细胞培养上清液中基质金属蛋白酶-2（matrix metalloproteinase-2,MMP-2）和基质金属蛋白酶-9（matrix metalloproteinase-9,MMP-9）的活性。实验结果分别以各时间点对照组作为基线1,计算与对照组的比值。结果：（1）西罗莫司于各个时间点对细胞内α-SMA的表达均无影响。（2）用西罗莫司处理后,于24h对细胞内ColⅠ-蛋白表达量明显少于对照组,并持续至48h,分别为各时间点对照组的（0.58±0.05）倍和（0.63±0.18）倍,差异有统计学意义（P〈0.05）。（3）与各时间点对照组相比,细胞内procollagen-ⅠmRNA在1h,2h时表达均减少,分别为（0.38±0.05）倍和（0.55±0.16）倍,差异有统计学意义（P〈0.05）,于4h恢复到基础水平。（4）培养细胞上清液中12h即有ColⅠ-基础表达,经西罗莫司处理24h其表达量较对照组明显减少,为对照组的（0.59±0.25）倍,差异有统计学意义（P〈0.05）,持续至48h仍呈减少趋势,为对照组的（0.52±0.21）倍,P〈0.05。（5）细胞内FN蛋白表达趋势与上清液中一致,24h其表达量较对照组明显减少,分别为对照组的（0.44±0.09）倍和（0.40±0.15）倍,差异均具有统计学意义（P〈0.05）,48h西罗莫司对FN的抑制作用消失。?

英文摘要：

Objective： To investigate the anti-fibrotic effect of sirolimus （rapamycin） at the cell level. Methods： The primary cultured rat renal cortical myofibroblasts were divided into two groups, control group and sirolimus 40 mg/L group at each time point. The protein levels of α-SMA, Col-Ⅰ , fibronectin （FN） were analyzed by Western blot in both the whole cell lysates and supernatant culture media 12 h , 24 h and 48 h after incubation, respectively. Real-time quantitative PCR was carried out to measure the levels of procollagen-Ⅰ mRNA 1 h, 2 h, 4 h, and 6 h after cell incubation. The activities of gelatinase MMP-2 and MMP-9 in the supernatant from the cultured cell media were assayed by gelatin zymography. Results： （1） Sirolimus had no effect on the expression of α-SMA of myofibroblasts at differnet time points. （2） The expression of Col-Ⅰ in the whole cell lysates both reduced at the end of 24 h and 48 h in sirolimus group significantly [（0.58±0.05） and （0.63±0.18 ）,P〈0.05] compared with control group at each time point, respectively. （3） The levels of procollagen-I mRNA reduced significantly at the end of 1 h and 2 h compared with control group at each time point [（0.38±0. 05） and （0.55±0.16）, P 〈0.05], but increased to basic level at the end of 4 h. （4） The myofibroblasts had basic expression of Col-Ⅰ early at the end of 12 h, its expression in supernatant culture medium reduced obviously both at 24 h and 48 h in sirolimus group compared with control group of each time point [（0.59±0.25）and （0.52±0.21）, P〈0.05]. （5） The expression of FN in the whole cell lysates had the same trend as that in supernatant culture medium, which reduced obviously at the end of 24 h in sirolimus group compared with control group at each time point[（0.44±0.09） and （0.40±0.15）, P〈0.05], but the inhibitive effect of sirolimus on FN disappeared at the end of 48 h. （6） The activities of MMP-2 and MMP-9 in the supernatant culture media were not sig