胱抑素C对大鼠心肌成纤维细胞增殖的影响

Effects of CysC on the proliferation of cultured rat cardiac fibroblasts

目的探讨胱抑素C（CysC）高表达对大鼠心肌成纤维细胞（CFs）增殖能力的影响。方法取出生1~3d的Sperague-Dawley（SD）清洁级大鼠10只,体质量10~15g,差速贴壁法培养乳鼠心肌成纤维细胞;采用Gateway技术构建重组CysC腺病毒高效表达载体（Ad-CysC）并体外转染大鼠CFs;将对数生长期的大鼠CFs随机分成正常细胞组（PBS组）,携有绿色荧光蛋白（GFP）的腺病毒组（Ad-GFP组）及CysC腺病毒高表达组（Ad-CysC组）。采用蛋白免疫印迹技术（WB）检测CysC蛋白在CFs中的表达情况,通过5-溴脱氧尿嘧啶核苷（BrdU）掺入法检测CysC高表达对CFs增殖能力的影响。结果经DNA测序及WB结果分析,CysC腺病毒高效表达载体构建成功,转染CFs24h后（MOI=200）,Ad-CysC的转染效率高达90%以上;用重组CysC腺病毒转染CFs后,CysC蛋白在24h即有明显的表达上调（P〈0.05）;与PBS组及Ad-GFP组相比,转染Ad-CysC的CFs在72h的BrdU摄取率显著提高（P〈0.05）。结论大鼠CysC腺病毒高效表达能够促进CFs增殖,可能加速心肌纤维化进程。

Objective To explore the effects of cystatin（CysC）on the proliferation of rat cardiac fibroblasts.Methods Cardiac fibroblasts from 1～3 days old Sprague-Dawley rats（clean grade,weight 10～15 g） were cultured by the anchorage velocity-dependent separation method.The recombinant adenovirus vector for CysC gene（Ad-CysC）was constructed with the GatewayTM technique and transfected into rat cardiac fibroblasts in vitro.Rat cardiac fibroblasts were randomly divided into the normal control group,the Ad-GFP group and the Ad-CysC group.Expression of CysC at the protein level was examined by means of Western-blot.Cell proliferation rate was measured by bromodeoxyuridine（BrdU）. Results DNA sequencing results and Western blot analysis indicated that recombinant Ad-CysC was successfully constructed.The efficiency of Ad-CysC infecting rat cardiac fibroblasts was above 90% when multiplicity of infection（MOI） was 200.Furthermore,protein expression of CysC was significantly increased at 24 h after transfection（P0.05）.Compared to the normal control group and the Ad-GFP group,the BrdU incorporation rate of the Ad-CysC group was much higher at 72 h after transfection（P0.05）.Conclusions High expression of CysC can stimulate the proliferation of cardiac fibroblasts,which could promote the myocardial fibrosis.