CHRNA9基因绝对定量PCR标准曲线的建立

被引：1

作者：

孙志华 ^{[1
,2
]}

刘益巧 ^{[1
]}

钱江 ^{[1
]}

长孙东亭 ^{[1
]}

罗素兰 ^{[1
]}

机构：

[1] 海南大学热带生物资源教育部重点实验室、海口市海洋药物重点实验室

[2] 海南大学热带农林学院

来源：

生物技术 | 2017年 / 27卷 / 05期

关键词：

乙酰胆碱受体α9亚基基因; 基因表达拷贝数; 乳腺癌细胞系; 绝对荧光定量标准曲线;

D O I：

10.16519/j.cnki.1004-311x.2017.05.0073

中图分类号：

R737.9 [乳腺肿瘤];

学科分类号：

摘要：

[目的]为研究乙酰胆碱受体(nicotine acetylcholine receptor,nAChR)α9亚基基因(CHRNA9)的mRNA,在人乳腺癌细胞系MCF-7和人正常乳腺上皮细胞系MCF-10A中的差异表达情况,建立了CHRNA9基因转录本拷贝数的绝对定量标准曲线。[方法]培养并收集细胞,提取总RNA,反转录合成cDNA。以cDNA为模板,扩增CHRNA9基因171bp的特征片段,亚克隆到pMD-18T载体中,并进行测序鉴定。以梯度稀释的质粒为模板,进行荧光定量PCR来建立标准曲线,进一步对上述细胞系中CHRNA9的初始拷贝数进行定量分析。[结果]成功地对人正常乳腺上皮细胞系MCF-10A,人乳腺癌细胞系MCF-7中的α9 nAChR亚基基因的mRNA水平表达量,即反转录生成的cDNA拷贝数进行了定量,mRNA拷贝数分别为712.61.31和2 236.55个。经统计分析,二者之间CHRNA9基因的表达量差异达到了极显著水平(p<0.01),即α9 nAChR亚基基因在乳腺癌细胞MCF-7中显著高于乳腺正常上皮细胞MCF-10A。[结论]研究所获得的CHRNA9基因的绝对荧光定量标准曲线,可用于不同乳腺癌细胞系α9 nAChR亚基基因表达量的检测,而α9 nAChR作为一个理想的、特异性的潜在药物作用靶点,这为相应种类的乳腺癌发病机理研究和靶向治疗药物的研发提供基础。

引用

页码：460 / 465+483 +483

页数：7

共 11 条

[1] 乳腺癌靶向治疗的研究进展
孙志华
邴晖
刘益巧
钱江
长孙东亭
罗素兰
[J]. 生命科学研究, 2017, 21 (03) : 275 - 282
[2] Inhibition of α9α10 nicotinic acetylcholine receptors prevents chemotherapy-induced neuropathic pain
Romero, Haylie K.
Christensen, Sean B.
Mannelli, Lorenzo Di Cesare
Gajewiak, Joanna
Ramachandra, Renuka
Elmslie, Keith S.
Vetter, Douglas E.
Ghelardini, Carla
Iadonato, Shawn P.
Mercado, Jose L.
Olivera, Baldomera M.
McIntosh, J. Michael
[J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2017, 114 (10) : E1825 - E1832
[3] Anti-hypersensitive effect of intramuscular administration of αO-conotoxin GeXIVA[1,2] and GeXIVA[1,4] in rats of neuropathic pain[J] . Xiaodan Li,Yuanyan Hu,Yong Wu,Yi Huang,Shurun Yu,Qing Ding,Dongting Zhangsun,Sulan Luo. Progress in Neuropsychopharmacology & Biological . 2016
[4] Nicotinic acetylcholine receptors and cancer (Review)[J] . Ningning Dang,Xianguang Meng,Haiyan Song. Biomedical Reports . 2016 (5)
[5] The Oncogenic Functions of Nicotinic Acetylcholine Receptors[J] . Yue Zhao,Kalpesh Jani. Journal of Oncology . 2016
[6] The nicotinic acetylcholine receptor-mediated reciprocal effects of the tobacco nitrosamine NNK and SLURP-1 on human mammary epithelial cells
Kalantari-Dehaghi, Mina
Parnell, Erinn A.
Armand, Tara
Bernard, Hans-Ulrich
Grando, Sergei A.
[J]. INTERNATIONAL IMMUNOPHARMACOLOGY, 2015, 29 (01) : 99 - 104
[7] The enhancement of cancer stem cell properties of MCF-7 cells in 3D collagen scaffolds for modeling of cancer and anti-cancer drugs[J] . Lei Chen,Zhifeng Xiao,Yue Meng,Yannan Zhao,Jin Han,Guannan Su,Bing Chen,Jianwu Dai. Biomaterials . 2011 (5)
[8] Nitrosamines as nicotinic receptor ligands[J] . Hildegard M. Schuller. Life Sciences . 2007 (24-2)
[9] Nicotinic acetylcholine receptors in cancer: multiple roles in proliferation and inhibition of apoptosis[J] . Richard D. Egleton,Kathleen C. Brown,Piyali Dasgupta. Trends in Pharmacological Sciences . 2007 (3)
[10] Downregulation of Nicotinic Acetylcholine Receptors by Nerve Growth Factor in Human Small Cell Lung Carcinoma Cell Lines[J] . AGNESECODIGNOLA,CRISTINAMISSALE,PIERFRANCOSPANO,FRANCESCOCLEMENTI,EMANUELESHER. Annals of the New York Academy of Sciences . 2006 (1)

← 1 2 →