摘要
目的:观察不同中医治法对大鼠肝癌CK18表达的调控差异,以及CK18在人肝癌细胞增殖中的作用。方法:(1)采用DEN诱发大鼠肝癌,分别经中药健脾益气、清热解毒、活血化瘀等治疗后,检测肝癌组织CK18表达的差异;(2)设计2个人CK18基因siRNA靶点,将重组质粒电转入SMMC-7721人肝癌细胞株,MTT法检测细胞生长增殖情况、Realtime-PCR检测该基因的表达差异。结果:(1)芯片结果显示,CK18基因在大鼠肝癌形成后表达显著增加,不同中医治法对其具有不同程度的轻度调控作用,其中健脾益气治法下调作用较明显;(2)采用MTT法筛选到一个CK18基因RNA干扰有效靶序列,转染144h后细胞的生长增殖得到了明显的抑制;(3)实时荧光定量PCR检测表明,转染72h后该基因的表达已明显降低。结论:肝癌细胞恶性增殖有赖于CK18基因的高表达,目前常用治法对该基因的调控作用有限,因而该基因可望作为肝癌疗效筛选评价指标。
Objective：To study the different effects of several common TCM formula therapies on the expression of CK18 gene in rat liver cancer and its role in growth and proliferation of humane hepatocarcinoma cells.Methods：1）Rat hepatocellular carcinoma（HCC）models were induced by diethylnitrosamine（DEN）,the rats were treated by different therapeutical 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 CK18 gene.2）Two siRNA targets toward CK18 gene were designed,recombinant plasmid was transfected to hepatocellular carcinoma cell line SMMC-7721,the gene expression of CK18 were determined using real-time quantitative PCR,and the cell viability were determined by MTT assay.Results：1）According to the gene chip results,the expression of CK18 increased significantly after formation of Hepatocellular Carcinoma,several TCM therapeutical methods had slight effections,and down-regulating effect of nourishing spleen and enriching qi prescription was obivious;2）an effective siRNA target sequence in CK18 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 CK18 gene.Now the adjustment and control of common TCM formula therapies is limited to this gene,so it maybe act as a target for screening and evaluation the curative effect to hepatocarcinoma.
出处
《辽宁中医杂志》
CAS
北大核心
2010年第4期748-751,共4页
Liaoning Journal of Traditional Chinese Medicine
基金
国家自然基金项目(30672574
30271576)
关键词
肝癌
CK18
基因芯片
RNA干扰
治法
hepatocellular carcinoma
CK18
RNA interference
gene chip
therapeutical principles and methods
