应用RNAi抑制食管癌细胞端粒酶活性实验探究.docVIP

应用RNAi抑制食管癌细胞端粒酶活性实验探究 　 作者：彭彦辉 王亮 陈卫云 郝玉宾 邢邯英 【摘要】 目的 研究载体介导RNA干扰技术(RNAi)对人食管癌细胞株ECA109端粒酶逆转录酶(human telomerase reverse transcriptase，hTERT)表达及端粒酶活性的抑制作用。方法 设计并构建3条hTERT基因的siRNA 序列干扰质粒Ⅰ、Ⅱ、Ⅲ及对照质粒，并将其分别转染ECA109食管癌细胞,应用RTPCR法观察各转染细胞hTERTmRNA表达水平的改变，末端重复片段扩增酶联免疫吸附(TRAPELISA)方法测定细胞端粒酶活性。结果 与对照质粒比较，转染24 h后，干扰质粒Ⅰ、Ⅱ、Ⅲ组细胞端粒酶活性均显著下降(Plt;0.01);转染48 h后,各干扰质粒的抑制率依次为49%、79%、53%。干扰质粒转染ECA109细胞中hTERTmRNA的表达较对照质粒转染ECA109细胞中hTERTmRNA的表达量显著下降，仅为对照组的55.4 %。结论 成功构建载体介导的hTERT靶向RNA干扰重组体，能有效抑制ECA109细胞端粒酶活性及hTERTmRNA的表达。 【关键词】 RNA干扰;食管癌;人端粒酶逆转录酶;端粒酶 　　【Abstract】 Objective To investigate the effect of RNA interference(RNAi) on the expression of human telomerase reverse transcriptase (hTERT) and the activity of telomerase. Methods According to the nucleotide sequence of hTERT in gene bank and referring to the design strategies of siRNA, mRNA interfering doublestranded DNA vector Ⅰ，Ⅱ，Ⅲ and the control vector were constructed respectively and then were transfected into the ECA109 cells. The expressions of hTERT of the transfected cells were determined by RTPCR and the activity of telomerase was determined by telomeric repeat amplificationELISA(TRAPELISA). Results Compared with the control vector group, the telomerase activation of ECA109 cells in interfering vector Ⅰ，Ⅱ，Ⅲ group were lower after transfected for 24 and 48 h (Plt;0.01) .TRAPELISA results showed that the inhibition rate of interfering vectorⅠ，Ⅱ，Ⅲ on the telomerase activation were 49%，79%，53%respectively and the maximum inhibition rate was interfering vector Ⅱ. Interfering vector Ⅱ also declined the mRNA expression level of hTERT gene in ECA109 cells as compared with the control group. Conclusions The siRNA expression plasmid was successfully constructed and could inhibit the expression of hTERTmRNA and telomerase activity ,which may be beneficial in searching for new gene therapy of the tumors. 　　【Key words】 RNA interference; Esophageal carcinoma; Human telomerase reverse transcriptase; Telomerase 　 近年研究证实肿瘤组织中端粒酶阳性率高达85