CHRNA9基因绝对定量PCR标准曲线的建立

Standard curve generation of CHRNA9 gene for absolute quantification using Real-time PCR

[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。

[Objective] This study explored the expression of the α9 nicotine acetylcholine receptor（ nAChRs） subunits mRNA between human breast cancer cells and human normal mammary epithelial cells. [Methods] Total RNA was isolated from human cells lines,and reverse transcribed to cDNA. A 171 bp characteristic segment of α9 nAChRs gene was cloned and inserted into the pMD18 vector,and sequenced. Plasmid standard curve generation of CHRNA9 gene for absolute quantification by qRT-PCR. Then quantitative analyzed the original copy number of CHRNA9 gene in MCF-10 A and MCF-7 cell lines. [Results] Using the standard curve,the mRNA expression of alpha 9 nAChR subunit gene between MCF-10 A and MCF-7 cell lines,that is,the reverse transcription of cDNA copy number was detected successfully. The copy number of alpha 9 nAChR subunit gene were 2236. 55 and 712. 61. 31,respectively. Compared with human normal mammary epithelial cells MCF-10 A,the mRNA expression level higher in human breast cancer cells MCF-7,with fearfully significant（ p〈 0. 01）. [Conclusion]The standard curve generation of CHRNA9 gene can be used for detecting the expression of CHRNA9 gene in different breast cancer cell line. In addition,α9 nAChRs as an ideal specific target,with extremely important value in new drug research and development in their treatment of nAChRs related diseases.