FABP7基因真核表达载体的构建及其对人乳腺癌细胞MCF-7增殖的影响

目的构建脑脂肪酸结合蛋白(BLBP/B-FABP,FABP7)基因的真核表达载体,并检测其对人乳腺癌细胞MCF-7增殖的影响。方法采用逆转录方法从星型细胞瘤组织中扩增FABP7基因,双酶切后插入线性化的pcDNA3.1载体真核启动子下游,构建重组真核表达载体,转染人乳腺癌细胞MCF-7后,采用半定量RT-PCR检测FABP7基因mRNA的表达,MTT法检测细胞的增殖活性、流式细胞术检测细胞的周期变化情况,并对细胞进行计数。结果FABP7基因重组真核表达质粒经双酶切及测序鉴定证明构建正确,转染MCF-7细胞后48、72和96h,pcDNA3.1-FABP7组的细胞数和细胞的A490值均比pcDNA3.1空质粒组明显降低,G1期细胞百分含量均明显升高。结论已成功构建了FABP7基因的真核表达载体,该载体可抑制人乳腺癌细胞MCF-7的增殖。

Construction of Eukaryotic Expression Vector for FABP7 Gene and Its Effect on Proliferation of Human Breast Cancer MCF-7 Cells

Objective To construct a eukaryotic expression vector for the gene encoding human brain fatty acid binding pro-tein 7(FABP7)and observe its effect on the proliferation of human breast cancer MCF-7 cells.Methods Amplify FABP7 gene from stellate cell tumor tissue by RT-PCR,digest with BamHⅠand Hind Ⅲ and insert downstream to the promoter of linearized pcDNA3.1 vector.Tranfect the constructed recombinant plasmid pcDNA3.1-FABP7 to human breast cancer MCF-7 cells and determine the ex-pression of FABP7 mRNA by semi-quantitative RT-PCR.Count the cells 48,72 and 96 h after transfection respectively,and deter-mine the cell proliferation activity by MTT method and the change of cell cycle by flow cytometry.Results Both restriction analysis and sequencing proved that recombinant plasmid pcDNA3.1-FABP7 was constructed correctly.The count and A490 value of cells 48,72 and 96 h after transfection with pcDNA3.1-FABP7 were significantly lower,while the percentages of cells at G1 stage were signifi-cantly higher,than those with empty plasmid pcDNA3.1.Conclusion The eukaryotic expression vector for FABP7 gene was suc-cessfully constructed,which inhibited the proliferation of human breast cancer MCF-7 cells significantly.