CHRNA9基因绝对定量PCR标准曲线的建立

发布时间：2018-03-18 13:52

本文选题：乙酰胆碱受体α亚基基因　切入点：基因表达拷贝数　出处：《生物技术》2017年05期 　论文类型：期刊论文

【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(p0.01),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。
[Abstract]:[objective] to study the mRNA expression of the 伪 9 subunit of acetylcholine acetylcholine receptor nAChR9 in human breast cancer cell line MCF-7 and normal mammary epithelial cell line MCF-10A. The absolute quantitative standard curve of copy number of CHRNA9 gene transcripts was established. [methods] cells were cultured and collected, total RNAs were extracted, and cDNAs were synthesized by reverse transcription. Using cDNA as template, the 171bp characteristic fragment of CHRNA9 gene was amplified and subcloned into pMD-18T vector. Using gradient dilution plasmid as template, fluorescence quantitative PCR was used to establish the standard curve. The initial copy number of CHRNA9 in human breast epithelial cell line MCF-10A and the mRNA level of 伪 9 nAChR subunit gene in human breast cancer cell line MCF-7 were analyzed quantitatively. That is, the copy number of cDNA produced by reverse transcription was 712.61.31 and 2236.55 respectively. The difference of expression of CHRNA9 gene between the two groups reached a very significant level, that is, 伪 9 nAChR subunit gene in breast cancer cell MCF-7 was significantly higher than that in normal breast epithelial cell line MCF-10A. [conclusion] the absolute fluorescence quantitative standard curve of CHRNA9 gene obtained from the study was higher than that of normal breast epithelial cell line MCF-10A. It can be used to detect the expression of 伪 9 nAChR subunit gene in different breast cancer cell lines, and 伪 9 nAChR is an ideal and specific potential drug target. This provides the basis for the study of the pathogenesis of breast cancer and the development of targeted therapy drugs.
【作者单位】：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;海南大学热带农林学院;
【基金】：国家自然科学基金项目(“作用于α9α10乙酰胆碱受体的新颖芋螺毒素结构与功能的研究”,No.81420108028) 长江学者和创新团队发展计划(“热带特色海洋药物芋螺毒素资源的研究与利用”,No.IRT_15R15) 海南省普通高等学校研究生创新科研课题(“芋螺毒素抗乳腺癌活性及作用机制的初步研究”,No.Hyb2016-08)
【分类号】：R737.9

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