应用RNAi抑制食管癌细胞端粒酶活性的实验研究.docVIP

应用RNAi抑制食管癌细胞端粒酶活性的实验研究 目录 TOC \o 1-9 \h \z \u 目录 1 正文 1 文1：应用RNAi抑制食管癌细胞端粒酶活性的实验研究 1 1 材料与方法 3 2 结 果 4 3 讨 论 5 文2：RNAi抑制胰腺癌细胞株VEGF表达的实验研究 7 1材料与方法 7 参考文摘引言： 11 原创性声明（模板） 12 文章致谢（模板） 13 正文 应用RNAi抑制食管癌细胞端粒酶活性的实验研究 文1：应用RNAi抑制食管癌细胞端粒酶活性的实验研究 【Abstract】 Objective To investigate the effect of RNA interference(RNAi) on the expression of human telomerase revee tracriptase (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 cotructed respectively and then were trafected into the ECA109 cells. The expressio of hTERT of the trafected 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 trafected for 24 and 48 h (P) .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. Conclusio The siRNA expression plasmid was successfully cotructed and could inhibit the expression of hTERTmRNA and telomerase activity ,which may be beneficial in searching for new gene therapy of the tumo. 【Key words】 RNA interference; Esophageal carcinoma; Human telomerase revee tracriptase; Telomerase 近年研究证实肿瘤组织中端粒酶阳性率高达85%以上,人端粒酶逆转录酶(human telomerase revee tracriptase，hTERT)是端粒酶活化和细胞癌变的限速步骤〔1〕。抑制其表达可有效地抑制肿瘤细胞的生长和诱导凋亡，目前已成为肿瘤基因 治疗 的理想靶标。RNA干扰(RNAi)作为一种简单有效的代替基因敲除的工具正在功能基因组学领域掀起一场真正的革命。本研究拟选择hTERT基因作为研究靶点，运用体外转录法合成多对小干扰RNA(siRNA),转染人食管癌细胞，检测其对端粒酶活性的抑制，筛选出高效抑制端粒酶活性的siRNA，为开发抗肿瘤新药和基因治疗提供实验和理论依据。 1 材料与方法 材料 人食管癌细胞株ECA109购自