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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 investigate the effect of cystatin C (CysC) on the proliferation of rat cardiac fibroblasts (CFs). Methods Ten (10 ~ 15) Sperague-Dawley (SD) clean-grade rats from 1 to 3 days old were used to culture neonatal rat cardiac fibroblasts with differential adhesion method. The recombinant adenovirus vector was constructed by Gateway (Ad-CysC) and transfected CFs in vitro. The logarithmic growth phase CFs were randomly divided into normal group (PBS group), adenovirus group (GFP group) and Ad-GFP group And CysC adenovirus expression group (Ad-CysC group). The expression of CysC protein in CFs was detected by Western blotting (WB). The effect of CysC overexpression on the proliferation of CFs was detected by BrdU incorporation assay. Results After DNA sequencing and WB analysis, the CysC adenovirus expression vector was constructed successfully. The transfection efficiency of Ad-CysC was over 90% after transfected with CFs for 24 hours (MOI = 200). After transfection of CFs with recombinant CysC adenovirus, CysC protein was up-regulated at 24h (P <0.05). Compared with PBS group and Ad-GFP group, the BrdU uptake rate of CFs transfected with Ad-CysC was significantly increased at 72h (P <0.05). Conclusion The high expression of CysC in rats can promote the proliferation of CFs and accelerate the process of myocardial fibrosis.