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

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

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。

Objective To construct a eukaryotic expression vector for the gene encoding human brain fatty acid binding protein 7 （FABP7） and observe its effect on the proliferation of human breast cancer MCF-7 ceils. Methods Amplify FABP7 gene from stellate cell tumor tissue by RT-PCR, digest with BamH Ⅰ and 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 expression of FABP7 mRNA by semi-quantitative RT-PCR. Count the ceils 48, 72 and 96 h after transfeetion respectively, and determine 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 peDNA3. 1-FABP7 were significantly lower, while the percentages of cells at G1 stage were significantly higher, than those with empty plasmid pcDNA3. 1. Conclusion The eukaryotic expression vector for FABP7 gene was successfully constructed, which inhibited the proliferation of human breast cancer MCF-7 cells significantly.