【摘要】 目的 了解大肠癌细胞株(SW480，LOVO，HT29)线粒体DNA的突变，克隆突变的大肠癌线粒体DNA(mtDNA)基因，构建pcDNA3.1(+)-mtDNA真核表达重组体，并导入NIH3T3及LST细胞，以探讨线粒体基因突变与肿瘤发生的关系。方法 提取大肠癌细胞株(SW480，LOVO，HT29)mtDNA,扩增D-LOOP区，产物用DNA自动测序法进行序列分析。利用DNA重组技术将其定向插入真核表达质粒pcDNA3.1(+),并用脂质体法导入NIH3T3及LST细胞。用MitoCapture Mitochondrial Apoptosis Detection Kit试剂盒染色后用流式细胞仪及荧光显微镜检测转染细胞的凋亡情况。扩增并测序分析转染细胞的D-LOOP区突变特点。结果 检测出大肠癌细胞株SW480，LOVO，HT29细胞mtDNA D-LOOP分别有10，9，8个突变位点。转染前后，各组间细胞凋亡无明显变化。转染细胞的核基因组可扩增出目的基因及Neo基因。4株NIH3T3转染细胞mtDNA D-环区分别检测到9，11，8，4个突变点，并相应有3，4，3，2个多态性变化。结论 (1)转染突变的大肠癌细胞mtDNA后转染细胞的mtDNA均可发生多处的突变位点。(2)通过转染后突变的外源性的mtDNA可以整合到核基因组内。(3)突变的mtDNA转染LST细胞及NIH3T3细胞后，不影响转染细胞的凋亡改变。(4)mtDNA的突变可能通过影响体细胞mtDNA的突变和通过外源性mtDNA在核内的整合从而影响癌基因或抑癌基因的表达异常，从而参与肿瘤的发生发展。

    【关键词】 线粒体DNA；D—环区；突变；质粒；pcDNA3.1(+);转染

    The research of transfecting mutated mtDNA of colorectal carcinoma cell line into NIH3T3 cell line and LST cell line

    SONG Wei-bing,XIAO Bing,ZHANG Zhen-shu,et al.Institute for Digestive Disease of Nanfang Hospital,

    The Nanfang Medical University,Guangzhou 510515,China

    【Abstract】 Objective To investigate mutations in the D-LOOP region of mitochondrial DNA in colorectal carcinoma cell lines,construct eukaryotic cell expression recombinant pcDNA3.1(+)-mtDNA and transfect pcDNA3.1(+)-mtDNA in murine fibroblast cells.Methods The D-LOOP region of the three colorectal carcinoma cell line (SW480,LOVO,HT29) were amplified by PCR and sequenced.The fragment of mtDNA were recombined in the eukaryotic cell expression plasmid pcDNA3.1(+),the pcDNA3.1(+)-mtDNA recombinant were used to infect marine fibroblast cell NIH3T3.Results Among the three colorectal carcinoma cell line (SW480,LOVO,HT29),there were respectively 10,9,8 mutations were identified.There's no obvious differences in apoptosis between groups after transfection.Target gene and neo gene can be amplified in nuclear genome of transfected cells.There were respectively 9,11,8,4 mutations identified in the four transfected NIH3T3 cell lines.And there were respectively 3,4,3,2 polymorphism mutation points.Conclusion 1,After transfected the mutated mtDNA of colorectal carcinoma,the mtDNA D-loop region of the transfected cells displays new mutation points.2,The external source pieces of the mutated mtDNA can integrate to nuclear genome after transfection.3,There's no differences in apoptosis between combinations after transfected the mutation of mtDNA in NIH3T3 and LST cells.4,The mutated mtDNA may affect the action mechanism of occurrence and development in colorectal carcinoma through affecting its mtDNA mutation or integrating exogenetic mtDNA to its nuclear which may cause the abnormal expression of oncogene or anti-oncogene. ......