反义人端粒酶RNA组分基因对胃癌细胞端粒酶活性的影响

Influence of antisense hTR eukaryotic expression vector on telomerase activity in gastric cancer cells

目的 克隆人胃癌细胞端粒酶RNA组分（hTR）基因片段并构建其正义和反义真核表达载体,研究反义端粒酶RNA对人胃癌细胞株MKN-45端粒酶活性的影响。方法 采用RT-PCH方法从人胃癌细胞株MKN-45中扩增出人hTR部分cDNA序列,将该片段分别正向和反向插入PEF6/V5-His-TOPO载体后构建人端粒酶RNA组分（hTR）基因正、反义真核表达载体,随后采用脂质体转染法将该正、反义载体转染入人胃癌细胞株MKN-45,用TRAP法观察后者端粒酶活性的改变。结果 所克隆的基因片段其碱基序列与文献报导完全一致,且插入载体的方向完全正确。与正常对照、转染正义载体、空载体者比较,转染反义载体者端粒酶活性显著下降。结论 本实验已成功克隆了人端粒酶RNA组分（hTR）基因的部分序列并成功构建hTR正反义真核表达载体,而且反义端粒酶RNA能有效降低人胃癌细胞株MKN-45端粒酶活性。

Objective To clone human telomerase RNA components (hTR) gene from human gastric cancer cells MKN - 45, construct its eukaryotic sense and antisense expression vector and study the effect of antisense telomerase UNA on telomerase activity in human gastric cancer cells by antisense ribonucleotide technique. Methods The hTR cD-NA was cloned from human gastric cancer cell line (MKN - 45) by RT - PCR and subcloned into eukaryotic expression vector (PEF6/V5 - His - TOPO vector) in cis - direction or trans - direction by DNA recombinant methods. The recombinant vector was then transfected into MKN - 45 cells by lipofectin - mediated method and the telomerase activity of MKN-45 was detected by TRAP. Results The cloned hTR cDNA was sequenced and confirmed by comparison with the database of the Genbank. The constructed hTR sense and antisense eukaryotic expression vector (pEF - hTR and pEF - ahTR) was proved to be the same as original design by restriction endonuclease analysis and sequencing. The telomerase activity of MKN - 45 was significiantly inhibited after antisense vecter transfection when compared with the control cell lines. Conclusions We succeeded cloning hTR cDNA was cloned and its sense and antisense eukaryotic expression vector were constructed successfully. The antisense RNA of hTR can greatly inhibit the telomerase activity of MKN - 45 cell line.