[1]吴倩倩,张清华,马莉.人大肠癌线粒体DNA重组质粒的构建[J].南方医科大学学报,2005,(08):1016-1019.
 WU Qian-qian,ZHANG Qing-hua,MA Li.Construction of recombinant eukaryotic expression plasmid pcDNA3.1(+)-mtDNA of human colorectal carcinoma cells[J].Journal of Southern Medical University,2005,(08):1016-1019.
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人大肠癌线粒体DNA重组质粒的构建()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2005年08期
页码:
1016-1019
栏目:
出版日期:
2005-08-01

文章信息/Info

Title:
Construction of recombinant eukaryotic expression plasmid pcDNA3.1(+)-mtDNA of human colorectal carcinoma cells
作者:
吴倩倩1 张清华2 马莉3
1. 中国人民解放军军事体育学院门诊部, 广东, 广州, 510515;
2. 解放军第458医院, 广东, 广州, 510000;
3. 北京二炮干休所, 北京, 1000086
Author(s):
WU Qian-qian1 ZHANG Qing-hua2 MA Li3
1. Out-patient Department, Military College of Sports of PLA, Guangzhou 510515, China;
2. 458 Hospital of PLA, Guangzhou 510000, China;
3. Sanatorium for Retired Military Officer of Second Artillery Force of PLA, Beijing 100086, China
关键词:
大肠癌线粒体DNAD-环区突变质粒
Keywords:
mitochondriaDNAD-loopmutationplasmid
分类号:
R392.24;R735.34
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.I(+)-mtDNA真核表达重组体,并导入NIH3T3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细胞。结果 检测出大肠癌细胞株SW480、LoVo、HT29细胞mtDNAD-LOOP分别有10、9、8个突变位点。成功克隆1119bp的mtDNAD-LOOP区至表达质粒pcDNA3.1(+),并导入NIH3T3细胞中。结论 线粒体DNAD-LOOP区是一个具有高度多态性和突变性的区域,在大肠癌细胞株中突变率较高。
Abstract:
Objective To construct recombinant eukaryotic expression plasmid pcDNA3.1(+)-mtDNA for investigation of mutations in the D-loop region of mitochondrial DNA in human colorectal carcinoma. Methods The D-loop region of 3 colorectal carcinoma cell lines (SW480, LoVo, and HT29) were amplified by PCR and sequenced. The mtDNA fragment was recombined in the eukaryotic expression plasmid pcDNA3. 1(+), and the resultant pcDNA3.1(+)-mtDNA recombinant was used to infect murine fibroblast cell line NIH3T3. Results Among the 3 colorectal carcinoma cell lines (SW480, LoVo, HT29), 10, 9, 8 mutations, were identified, respectively. The 1119-bp fragment of mtDNA was successfully cloned. DNA sequencing analysis demonstrate total agreement of the sequence with that in GenBank. The mtDNA fragments were cloned into the multiple cloning sites of vector pcDNA3.1(+) properly and the recombinant was introduced into NIH3T3 cells. Conclusions The D-loop region of mitochondrial DNA is a highly polymorphoric and mutable region with high mutation rate in human colorectal carcinoma cells. The recombinant eukaryotic expression plasmid pcDNA3.1(+)-mtDNA is successfully constructed.

参考文献/References:

[1] Jerry WS, Harold W. Are mitochondrial DNA mutations involved in the carcinogenic process[J] ? Mutat Res, 1987, 186(10): 149-60.
[2] Luciane RC, Bertrand CL. Mutagenesis, tumorigenicity and apoptosis: are the mitochondria involved[J] ? Mutat Res, 1998, 398:19-26.
[3] Millar CB, Guy J, Sansom OJ, et al. Enhanced CpG mutability and tumorigenesis in MBD4-deficient mice[J]. Science, 2002, 297(5):403-5.
[4] Matsuda T, Vande Berg BJ, Bebenek K, et al. The base substitution fidelity of DNA polymerase beta-dependent single nucleotide base excision repair[J]. J Biol Chem, 2003, 278(5): 25947-51.
[5] Servant L, Bieth A, Hayakawa H, et al. Involvement of DNA polymerase beta in DNA replication and mutagenic consequences[J]. J Mol Biol, 2002, 315(10): 1039-47.
[6] Takeuchi H, Fujimoto A, Hoon DS. Detection of mitochondrial DNA alterations in plasma of malignant melanoma patients[J]. Ann N Y Acad Sci, 2004, 1022(8): 50-4.
[7] Tong BC, Ha PK, Dhir K, et al. Mitochondrial DNA alterations in thyroid cancer[J]. J Surg Oncol, 2003, 82(1): 170-3.
[8] Zhou S, Kachhap S, Singh KK. Mitochondrial impairment in p53-deficient human cancer cells[J]. Mutagenesis, 2003, 18 (10):287-92.
[9] Hansel DE, Kern SE, Hruban RH. Molecular pat hogenesis of pancreatic cancer[J]. Annu Rev Genomics Hum Genet, 2003, 4 (10):237-56.
[10] Wong LJ, Tan DJ, Bai RK, et al. Molecular alterations in mitochondrial DNA ofhepatocellular carcinomas: is there a correlation with clinicopathological profile[J] ? J Med Genet, 2004, 41 (12): 59-65.
[11] Solano A, Roig M, Vives-Bauza C, et al. Bilateral striatal necrosis associated with a novel mutation in the mitochondrial ND6 gene[J].Ann Neurol, 2003, 54(1): 527-30.
[12] Liu CY, Lee CF, Hong CH, et al. Mitochondrial DNA mutation and depletion increase the susceptibility of human cells to apoptosis[J].Ann N Y Acad Sci, 2004, 101(6): 133-45.
[13] Abnet CC, Huppi K, Carrera A, et al. Control region mutations and the ’common deletion’ are frequent in the mitochondrial DNA of patients with esophageal squamous cell carcinoma[J]. BMC Cancer, 2004, 4(2): 27-30.
[14] Beretta S, Mattavelli L, Sala G, et al. Leber hereditary optic neuropathy mtDNA mutations disrupt glutamate transport in cybrid cell lines[J]. Brain, 2004, 127(10): 2183-92.
[15] Makiko SF, Henning U, Otavia LC, et al. Facile detection of mitochondrial DNA mutations in tumor and bodily fluids[J]. Science,2000, 287(6): 2017-9.
[16] Habano W, Sugai T, Nakamra SI, et al. Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma[J]. Gastroenterology, 2000, 118(11): 835-41.
[17] 王平忠,蒙世杰,刘佩,等.大鼠乳腺肿瘤线粒体基因突变研究[J].中国病理生理杂志,2001,17(1):297-301.
[18] Habano W, Sugai T, Nakamura SI, et al. Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma[J]. Gastroenterology, 2000, 118(5): 835-41.

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备注/Memo

备注/Memo:
收稿日期:2005-1-18。
作者简介:吴倩倩(1978-),女,住院医师,电话:020-61640381,E-mail:swb535@126.com
更新日期/Last Update: 1900-01-01