大鼠心肌成纤维细胞组织金属蛋白酶抑制剂mRNA水平检测(英文)

Detection of tissue inhibitors of metalloproteinases in rat cardiac fibroblasts

目的通过建立多重反转录聚合酶链反应(RT-PCR)方法,旨在测定大鼠心肌成纤维细胞组织金属蛋白酶抑制剂-1(TIMP-1)、组织金属蛋白酶抑制剂-2(TIMP-2)的mRNA水平。方法采用胶原酶-胰蛋白酶消化法培养成年雌性SD大鼠的心肌成纤维细胞,提取细胞总RNA并进行反转录反应。自行设计大鼠TIMP-1,TIMP-2和内参照GAPDH的PCR引物序列,进行多重PCR反应,PCR产物经20g/L琼脂糖凝胶电泳分析目的基因片段。结果20g/L琼脂糖凝胶电泳在同一条泳道显示TIMP-1,TIMP-2和GAPDH3条目的DNA条带,表明成功地同时扩增了TIMP-1,TIMP-2和GAPDH基因。结论采用多重RT-PCR方法,测定了大鼠心肌成纤维细胞TIMP-1,TIMP-2mRNA水平,此方法为研究高血压病心肌纤维化提供了可靠手段。

Aim To investigate the mRNA levels of tissue inhibitor of metalloprot einase-1(TIMP-1),tissue inhibitor of metalloproteinase-2 (TIMP-2) in rat car diac fibroblasts by multiplex reverse transcription-polymerase chain reaction ( RT-PCR). Methods Cardiac fibroblasts of adult female Sprague-Dawley rats were isolated. Total RNA was extracted and then reverse transcription was performed. Multiplex RT-PCR was carried out and PCR products were analyzed by agarose gel electrophoresis. Results 2%agarose gel electrophoresis showed that there were t hree targeted PCR products in a single lane which matched rat TIMP-1, TIMP-2 a nd GAPDH respectively. Conclusion We establish a feasible, simple and sensitive approach to measure mRNA of rat TIMP-1, TIMP-2 by multiplex RT-PCR, which may contribute to the research on cardiac fibrosis associated with hypertension.