荧光定量RT-PCR检测有机阴离子转运多肽OATP-E基因表达的方法学研究

发布时间：2018-11-02 10:11

【摘要】：【目的】探讨人类有机阴离子转运多肽超家族成员OATP-E mRNA在乳腺癌细胞株MCF-7和对阿霉素耐药的乳腺癌细胞株MCF-7/ADR中的表达;建立荧光定量RT-PCR检测肿瘤细胞OATP-E基因表达的方法并绘制出外标准曲线,为基础研究、临床治疗和药物筛选提供针对人类有机阴离子转运多肽超家族成员OATP-E方面研究的有效手段。【材料与方法】 1.细胞培养:以含有10%胎牛血清的RPMI1640培养基常规培养乳腺癌细胞株MCF-7和对阿霉素耐药的乳腺癌细胞株MCF-7/ADR,传代待其生长稳定后分别提取细胞总RNA待用。 2.细胞总RNA的提取:用Trizol试剂分别提取乳腺癌细胞株MCF-7和对阿霉素耐药的乳腺癌细胞株MCF-7/ADR的细胞总RNA。 3.RT-PCR反应:以所提取的乳腺癌细胞株MCF-7的细胞总RNA为原料,逆转录反应获得编码人类有机阴离子转运多肽超家族成员OATP-E的cDNA并进行普通PCR扩增。 4.构建克隆载体pGEM-OATP-E标准重组质粒:提取并纯化PCR产物,与pGEM-T Easy Vector质粒载体相连接,构建克隆载体pGEM-OATP-E重组质粒并转化大肠杆菌菌株JM109感受态细胞。 5.外标准曲线的建立:采用蓝白斑染色方法筛选并提取pGEM-OATP-E重组质粒,并将该pGEM-OATP-E重组质粒定量后作为标准品,绘制实时荧光定量RT-PCR方法检测OATP-E mRNA的外标准曲线。 6.采用本实验建立的实时荧光定量RT-PCR方法及其外标准曲线检测OATP-E mRNA在对阿霉素耐药的乳腺癌细胞株MCF-7/ADR中的表
[Abstract]:[objective] to investigate the expression of OATP-E mRNA, a member of the human organic anion transport polypeptide superfamily, in breast cancer cell line MCF-7 and adriamycin resistant breast cancer cell line MCF-7/ADR. To establish a fluorescence quantitative RT-PCR method for the detection of OATP-E gene expression in tumor cells and draw the standard curve for the study. Clinical treatment and drug screening provide an effective method for the study of OATP-E members of the human organic anion transporter polypeptide superfamily. [materials and methods] 1. Cell culture: breast cancer cell line MCF-7 and adriamycin resistant breast cancer cell line MCF-7/ADR, were routinely cultured on RPMI1640 medium containing 10% fetal bovine serum. The total cell RNA was extracted after stable growth. 2. Extraction of total cell RNA: total RNA. of breast cancer cell line MCF-7 and adriamycin resistant breast cancer cell line MCF-7/ADR were extracted by Trizol reagent 3.RT-PCR reaction: the cDNA encoding human organic anion transporter polypeptide superfamily OATP-E was obtained by reverse transcription reaction from the total RNA of the extracted breast cancer cell line MCF-7 and was amplified by ordinary PCR. 4. The standard recombinant plasmid of clone vector pGEM-OATP-E was constructed. The PCR product was extracted and purified, and then ligated with pGEM-T Easy Vector plasmid vector. The recombinant plasmid of clone vector pGEM-OATP-E was constructed and transformed into Escherichia coli JM109 receptive cells. 5. The establishment of external standard curve: the recombinant plasmid of pGEM-OATP-E was screened and extracted by blue and white spot staining, and the recombinant plasmid of pGEM-OATP-E was used as standard after quantification. Draw real-time fluorescence quantitative RT-PCR method to detect the external standard curve of OATP-E mRNA. 6. Detection of OATP-E mRNA in adriamycin resistant breast cancer cell line MCF-7/ADR by real-time fluorescent quantitative RT-PCR and its external standard curve established in this study
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R361.3

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