中文摘要：

[目的]研究显示人线粒体转录终止因子3（human mitochondrial transcription termination factor 3,h MTERF3）基因表达异常在肿瘤发生发展中具有重要意义。该研究旨在构建h MTERF3基因慢病毒表达载体,建立稳定过表达h MTERF3基因的人宫颈癌He La细胞株。[方法]采用RT-PCR技术从HEK293细胞中克隆出h MTERF3基因c DNA开放阅读框（open reading frame,ORF）序列,使目的基因连接入载体质粒中,构建成重组慢病毒载体质粒。对重组慢病毒载体质粒进行PCR鉴定、酶切鉴定和DNA测序鉴定后,制备包装病毒病感染人宫颈癌He La细胞株,通过药物筛选及维持稳定过表达h MTERF3基因的He La细胞株。倒置荧光显微镜下观察转染后各组细胞中绿色荧光蛋白（green fluorescence protein,GFP）的表达情况,RT-q PCR检测转染后各组细胞中h MTERF3基因的表达水平。[结果]成功构建了含有h MTERF3基因c DNA ORF序列的载体质粒p Lenti6.3-h MTERF3-IRES2-EGFP,DNA测序结果完全正确,经慢病毒包装后所得病毒上清液的滴度为1.1×10^9TU/m L。稳转He La细胞株在荧光显微镜下可见GFP表达,RTq PCR实验证实稳定转染重组慢病毒后He La细胞中h MTERF3 mRNA水平比未转染细胞极显著增高（P〈0.01）。[结论]成功构建了适用于h MTERF3基因研究的慢病毒表达系统,构建了稳定过表达h MTERF3基因的宫颈癌He La细胞株,从而为研究宫颈癌的基因诊治提供了一定的实验依据。

英文摘要：

[ Objective ] Studies show that the abnormal expression of human mitochondrial transcription termination factor 3 （ hMTERF3 ） plays an important role in the development and progression of cancer. The present study aims to provide some experimental evidence for the gene therapy of cancer by constructing a lentiviral expression vector carrying the hMTERF3 gene and further establishing colon cancer cell lines with stable overexpression of hMTERF3. [ Methods ] hMTERF3 gene cloned from pGEM - hMTERF3 plasmid was double restriction digested as well as lentiviral vector plasmid. Connection of target gene and vector plasmid was followed to generate the lentiviral expression vector plasmid. After confirmed by sequencing, the vector pLenti6.3 - hMTERF3 - IRES2 - EGFP and its helper vectors were mixed and co - transfected into 293T cells to obtain recom- binant virus containing the hMTERF3 gene. The lentiviral titer was detected and the resulting recombinant lentiviruses carrying hMTERF3 or control virues only carrying green fluorescence protein （GFP） were used to infect the human HeLa cell lines. The expression of GFP was determined under the inverted fluorescence microscope and the level of hMTERF3 mRNA in the infected cells deteetd by qPCR. [ Results ] The lentiviral expression vector pLenti6.3 - hMTERF3 - IRES2 - EGFP carrying correct hMTERF3 gene sequence was successfully constructed. The titer of the recombinant hMTERF3 lentiviral supernatant Lenti6.3 - hMTERF3 was 1 ×10^9 TU/mL. The expression of GFP was observed in the transduced cells under the fluorescence microscope, and that of hMTERF3 mRNA in the transfected HeLa cells was significantly up - regulated as compared with the control and blank groups. [ Conclusion] hMTERF3 gene lentiviral expression vector was successfully constructed, and so were HeLa cancer cell lines stably overexpressing hMTERF3, which may shed light on the lentivirus - mediated genetic theraphy for human cervical cancer.