突变的大肠癌线粒体DNA转染NIH3T3及LST细胞的研究

日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，并导人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人真核表达质粒pcDNA3．1（＋）。并用脂质体法导人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和HT29细胞mtDNAD-LOOP分别有10、9和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和4个突变点，并相应有3、4、3和2个多态性变化。结论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；通过转染后突变的外源性的mtDNA可以整合到核基因组内；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；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。

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

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 into murine fibroblast cells. Methods] The D-loop region of the three colorectal carcinoma cell lines (SW480, LOVO, HT29) was amplified by PCR and sequenced. The fragment of mtDNA was recombined in the eukaryotic cell expression plasmid pcDNA3.1(+), the pcDNA3.1(+)-mtDNA recombinant was used to i...