应用荧光定量RT-PCR法检测mdr-1基因的表达

发布时间：2018-11-07 20:58

【摘要】：【目的】建立用实时荧光定量RT—PCR法在mRNA水平上检测肿瘤细胞中多药耐药基因(mdr-1基因)表达的方法，实现对肿瘤细胞中mdr-1 mRNA快速，准确的检测，对临床治疗和肿瘤预后判断提供辅助手段。【材料与方法】 1．细胞培养：用含10％小牛血清的RPML1640培养基对乳腺癌耐阿霉素细胞株MCF-7／ADR于孵箱内单层连续传代培养，用0.25％的胰酶及0.25％的EDTA用直接吹打法传代。 2．细胞和组织总RNA提取：用Biozol试剂分别提取乳腺癌耐阿霉素细胞株MCF-7／ADR细胞和人乳腺癌肿瘤组织中的总RNA。 3．cDNA的合成：以所提取的乳腺癌耐阿霉素细胞株MCF-7／ADR及人乳腺癌肿瘤组织所提取的RNA为原料分别进行逆转录反应，获得cDNA。 4．PCR反应及重组质粒的构建：以乳腺癌耐阿霉素细胞株MCF-7／ADR逆转录的cDNA为模板，进行普通PCR扩增。扩增后的PCR产物用琼脂糖凝胶回收法进行纯化。用PGEM—T Easy质粒载体连接PCR纯化产物，构建重组质粒。将重组质粒转化大肠杆菌JM109感受态细胞，，蓝白斑筛选重组质粒。 5．荧光定量PCR标准曲线的建立：优化荧光定量PCR体系和条件，并以重组质粒为标准品建立检测mdr-1 mRNA的标准曲线。 6．用所建立的荧光定量PCR方法对58例乳腺癌标本中mdr-1 mRNA的含量进行测定，同时用免疫组化的方法检测标本中P-gP的表达，两种方法进行比较，采用X~2检验对数据进行统计学分析。【结果】构建了mdr-1基因的重组质粒，并且成功转化大肠杆菌JM109感受态细胞，经蓝白斑筛选提取重组质粒，对质粒进行梯度稀释，以稀释的质粒为模板优化荧光定量PCR体系和条件，建立最佳荧光定量PCR体系，
[Abstract]:[objective] to establish a real-time fluorescent quantitative RT-PCR method for the detection of multidrug resistance gene (mdr-1 gene) in tumor cells at mRNA level, and to detect mdr-1 mRNA in tumor cells quickly and accurately. To provide the auxiliary means for clinical treatment and prognosis judgment of tumor. [materials and methods] 1. Cell culture: using RPML1640 medium containing 10% calf serum, the MCF-7/ADR cell line of breast cancer resistant to doxorubicin was cultured continuously in the incubator. 0.25% trypsin and 0.25% EDTA were subcultured by direct blowing. 2. Total RNA extraction from cells and tissues: total RNA. was extracted from MCF-7/ADR cells and human breast cancer tumor tissues by Biozol reagent, respectively. Synthesis of 3.cDNA: cDNA. was obtained by reverse transcription reaction of MCF-7/ADR, a breast cancer cell line resistant to doxorubicin, and RNA extracted from human breast cancer tissue, respectively. 4.PCR reaction and construction of recombinant plasmid: using MCF-7/ADR reverse transcription cDNA of breast cancer adriamycin resistant cell line as template, general PCR amplification was carried out. The amplified PCR products were purified by agarose gel recovery method. The recombinant plasmid was constructed by ligating the purified PCR product with PGEM-T Easy plasmid vector. The recombinant plasmid was transformed into E. coli JM109 competent cells, and the recombinant plasmid was screened by blue and white spot. 5. Establishment of standard curve of fluorescence quantitative PCR: optimize the system and condition of fluorescent quantitative PCR, and establish standard curve of mdr-1 mRNA with recombinant plasmid as standard. 6. The content of mdr-1 mRNA in 58 cases of breast cancer was determined by fluorescence quantitative PCR method, and the expression of P-gP in 58 cases of breast cancer was detected by immunohistochemical method. The data were analyzed statistically by X2 test. [results] Recombinant plasmid of mdr-1 gene was constructed and transformed into E. coli JM109 receptive cells successfully. The recombinant plasmid was extracted by blue and white spot screening, and the plasmid was diluted by gradient dilution. The system and conditions of fluorescent quantitative PCR were optimized by using diluted plasmid as template, and the optimal fluorescent quantitative PCR system was established.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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