肺癌线粒体DNA诱导小鼠胚胎成纤维细胞NIH3T3恶性转化的研究

The research of mitochondrial DNA in lung cancer cell on inducing the mutation in NIH3T3 cells

目的观察转染了A549及H226肺癌细胞线粒体DNA(mtDNA)的小鼠胚胎成纤维细胞NIH3T3的生物学行为的变化。方法将重组载体pcDNA3.1(+)-A549 mtDNA、pcDNA3.1(+)-H226 mtDNA通过Lipofection2000TM转染NIH3T3细胞;荧光原位杂交(FISH)法观察mtDNA在核内的整合情况;分析转染前后细胞染色体核型;流式细胞仪(FCM)检测转染细胞的凋亡;四甲基偶氮唑盐微量酶反应比色法(MTT法)测定不同组别细胞的增殖率。结果转染A549-mtDNA、H226-mtDNA的NIH3T3细胞染色体出现了易位、断裂的结构畸形,其畸形的多倍体数目百分比(11.78±0.60)%、(11.33±1.05)%和染色体数目(3.55±0.57)%、(5.02±0.72)%高于未转染的NIH3T3细胞(1.38±0.17)%、(0.54±0.10)%。荧光显微镜观察到mtDNA能够整合在转染A549-mtDNA、H226-mtDNA的NIH3T3细胞染色体间期核中。同时,转染了A549-mtDNA、H226-mtDNA重组表达载体,A549-mtDNA组(5.20±0.20)%、H226-mtDNA组(7.75±0.11)%较NIH3T3组(19.04±0.08)%细胞的凋亡率明显下降(F=8381.21,P<0.001)。分别培养24 h、48 h、72 h,对MTT的吸光度,NIH3T3/A549-mtDNA组(0.58±0.06)、(0.96±0.08)、(1.45±0.06),NIH3T3/H226-mtDNA组(0.64±0.04)、(1.02±0.06)、(1.52±0.03)高于空NIH3T3组(0.26±0.04)、(0.42±0.03)、(0.81±0.04)、(1.18±0.09)。结论突变的肺癌mtDNA通过在核内的整合从而影响NIH3T3细胞的染色体核型及细胞增殖、凋亡等生物学行为。

Objective To observe the changes of biological behavior of NIH3T3 transfected with mitochondrial DNA(mtDNA)of A549 and H226 lung cancer cells.Methods The mtDNA eukaryotic expression vector of lung cancer cell was transfected into NIH3T3 cells by liposome method(Lipfection2000TM).The integration of mtDNA in nucleus was observed by fluorescence in situ hy⁃bridization(FISH).The pro-and post-transfection chromosomal nucleus cells were analyzed.The apoptosis of NIH3T3 in different group were detected by flow cytometry(FCM).The proliferation rate of different groups NIH3T3 are also determined by MTT method.Results After NIH3T3 cells transfected with A549 mtDNA and h226 mtDNA,the percentage of abnormal polyploid number(11.78±0.60),(11.33±1.05)and chromosome number(3.55±0.57),(5.02±0.72)were higher than those of untransfected NIH3T3 cells(1.38±0.17),(0.54±0.10).We found that mtDNA could be integrated into interphase nuclei of NIH3T3 cells transfected with A549 mtDNA and h226 mtDNA by Fluorescence microscopy.Moreover,The apoptotic rate in A549-mtDNA group(5.20±0.20)%and H226-mtDNA group(7.75±0.11)%was significantly lower than NIH3T3 group(19.04±0.08)%(F=8381.21,P<0.001).After the different group NIH3T3 were culture for 24 h,48 h,72 h,We found that the NIH3T3/A549-mtDNA group absorbance was(0.58±0.06),(0.96±0.08),(1.45±0.06),the NIH3T3/H-226 mtDNA group absorbance was(0.64±0.04),(1.02±0.06),(1.52±0.03),They were higher than in NIH3T3 group(0.42±0.03),(0.81±0.04),(1.18±0.09).Conclusion The mutant-lung cancer mtDNA can affect the karyotype,cell proliferation,apoptosis and other biological behaviors of NIH3T3 cells through nuclear integration.