癌细胞mtDNA限制性片段长度多态性分析

Restriction Fragment Length Polymorphism Analysis of Mitochondrial DNA from Carcinoma Cells

目的探讨人癌细胞ｍｔＤＮＡ与正常细胞ｍｔＤＮＡ一级结构的差异。方法采用一步法制备包括肺腺癌ＳＰＣ－Ａ－Ａ、ＰＬＡ－８０１Ｄ、Ａ５４９、Ａ５３１，肝细胞癌ＳＭＭＣ－７７２１，膀胱上皮癌ＥＪ共６个癌细胞系ｍｔＤＮＡ；用ＰｖｕⅡ、ＸｈｏⅠ、ＰｓｔⅠ、ＥｃｏＲⅠ、ＢｓｔＥⅡ、ＨｉｎｄⅢ、ＨｐａⅠ、Ｂｃ１Ⅰ、ＥｃｏＲⅤ、ＳｃａⅠ和ＸｂａⅠ共１１种限制性内切酶对所得ｍｔＤＮＡ进行ＲＦＬＰ分析；并用ＰＣＲ－ＲＦＬＰ方法分析了癌细胞ｍｔＤＮＡ非编码区结构变化。结果６个癌细胞系其ｍｔＤＮＡ基因编码区的３２个酶切位点均无变异，而有３个癌细胞系在其ｍｔＤＮＡ非编码区第１６２７６位核苷酸处出现了ＥｃｏＲⅤ新的酶切位点。结论提示癌细胞ｍｔＤＮＡ基因编码区一级结构相当稳定，而主要的核苷酸变异可能位于其ｍｔＤＮＡ非编码区。

Objective To understand the primary structure difference between human carcinoma and norml mitochondrial DNA(mtDNA).Methods SINGLE-STEP Method was used to isolate the mtDNA from 6 human carcinoma cell lines,including lung adenocarcinoma cell lines of SPC-A-1,PLA-801D,A531,A549;hepatocellular carcinoma cell line SMMC-7721 and bladder epithelioma cell line EJ.The mtDNA was analyzed by restriction fragment length polymorphism (RFLP) with 11 kinds of restriction endonuclease,which were PvuⅡ XhoⅠ,PstⅠ,EcoRⅠ,BstEⅡ,HindⅢ,HpaⅠ,BclⅠ,EcoRⅤ,ScaⅠ and XbaⅠ.The structure of noncoding region of mtDNA was analyzed by PCR-RFLP.Results In the coding region of mtDNA from 6 carcinoma cell lines,any variation at 32 restriction-sites failed to be detected.But a new site was found in three carcinoma cell lines (3/6) at nucleotide 16276(EcoRⅤ) within the noncoding region.Conclusions These results indicate that the primary structure of gene coding region of mtDNA isolated from carcinoma cells is highly conserved.While the major variation of nucleotide is probably located in the noncoding region.