RNAi研究TrxR1在肝癌细胞增殖中的作用及不同中医治法的调节

RNA Interference Studying the Role of TrxR1 Gene in Proliferation of Hepatocarcinoma Cells and Adjustment of Several TCM Therapeutical Principles

目的观察不同中医治法对大鼠肝癌TrxR1表达的调控差异,以及TrxR1在人肝癌细胞增殖中的作用。方法将84只雄性Wistar大鼠随机分为正常组和模型组以及实验组,除正常组外均采用DEN诱发大鼠肝癌,实验组分别经中药健脾益气、清热解毒、活血化瘀等治疗,检测各组大鼠肝癌组织(正常组取肝组织)TrxR1表达的差异;设计2个人TrxR1基因siRNA靶点,将重组质粒通过脂质体转染入SMMC-7721人肝癌细胞株,MTT法检测转染前后细胞生长增殖情况、Real-time-PCR检测该基因的表达差异。结果芯片结果显示,TrxR1基因在大鼠肝癌形成后表达显著增加,益气健脾治法明显下调其表达;采用MTT法筛选到一个TrxR1基因RNA干扰有效靶序列,脂质体转染144 h后细胞的生长增殖得到了明显的抑制;实时荧光定量PCR检测表明,转染72h后该基因的表达量明显降低。结论肝癌细胞恶性增殖有赖于TrxR1基因的高表达,益气健脾方药能显著下调该基因表达。

Objective To study the different effects of several common TCM formula therapies on the expression of TrxR1 gene in rat liver cancer and its role in growth and proliferation of human hepatocarcinoma cells. Methods 1） Rat hepatocellular carcinoma（HCC） models were induced by diethylnitrosamine（DEN）,the rats were treated by different therapeutic methods including nourishing spleen and enriching ＂qi＂、clearing away heat and toxic material、activating blood circulation.Then we used gene chips measured the different expression of TrxR1 gene.2） Two siRNA targets toward TrxR1 gene were designed,recombinant plasmid was transfected to hepatocellular carcinoma cell line SMMC-7721,the gene expression of TrxR1 were determined using real-time quantitative PCR,and the cell viability were determined by MTT assay. Results According to the gene chip results,the expression of TrxR1 increased significantly after formation of Hepatocellular Carcinoma,TCM therapeutic methods of nourishing spleen and enriching ＂qi＂ obviously down-regulated its expression;2） an effective siRNA target sequence in TrxR1 was got by MTT assay,the cell growth and proliferation were inhibited obviously when interferenced target effectivly was transfected 144 hours.3）Real-time quantitative PCR result displayed the gene expression decreased greatly at the 72 hour after transfected. Conclusion Malignant proliferation of hepatocarcinoma cells have relationship with high expression of TrxR1 gene.