突变的大肠癌线粒体DNA转染NIH3T3及LST细胞的研究  

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

作　　者：宋卫兵[1] 肖冰[1] 张振书[1] 毛丹 姬宏莉[1] 王新颖[1] 

机构地区：[1]南方医科大学南方医院消化病研究所,广东广州510515 [2]广州军区卫生员训练队,广东佛山51000

出　　处：《中国现代医学杂志》2008年第11期1496-1499,1502,共5页China Journal of Modern Medicine

基　　金：广东省自然科学基金(No:037049)

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mtDNAD-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。[Objective] To investigate mutations in the D-loop region of mitoehondrlal DNA in eolorectal carcinoma cell lines, construct eukaryotie cell expression recombinant peDNA3.1（＋）-mtDNA and transfeet peDNA3.1（＋）- mtDNA into murine fibroblast cells. [Methods] The D-loop region of the three eoloreetal carcinoma cell lines （SW480, LOVO, HT29） was amplified by PCR and sequenced. The fragment of mtDNA was reeombined in the eukaryotie cell expression plasmid peDNA3.1（＋）, the peDNA3.1（＋）-mtDNA recombinant was used to infect marine fibroblast cell NIH3T3. [Results] In the three eoloreetal carcinoma cell lines （SW480, LOVO, HT29）, there were 10, 9, 8 mutations identified respectively. There were no obvious differences in apoptosis between the groups after transfeetion. Target gene and Neo gene could be amplified in nuclear genome of transfeeted cells. There were 9, 11, 8, 4 mutations identified respectively in the four transfected NIH3T3 cell lines. And there were respectively 3, 4, 3, 2 polymorphism mutation points. [Conclusions] After transfected with the mutated mtDNA of colorectal carcinoma, the mtDNA D-loop region of the transfected cells displays new mutation points. The exogenous pieces of the mutated mtDNA can integrate into nuclear genome after transfection. There are no differences in apoptosis of transfected cells after transfection of the mutated mtDNA into NIH3T3 and LST cells. The mutated mtDNA may affect the mechanism of occurrence and development of colorectal carcinoma through affecting its mtDNA mutation or integrating exogenous mtDNA to its nuclei which may cause the abnormal expression of oncogene or tumor suppressor gene.

关 键 词：线粒体DNA D-环区 突变 质粒pcDNA3.1(+) 转染 

分 类 号：Q754[生物学—分子生物学] R574.6[医药卫生—消化系统]