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目的构建抑癌基因人第10号染色体缺失的磷酸酶及张力蛋白同源的基因(PTEN)及其突变体(F341V)的真核表达载体,检测其在乳腺癌细胞系MCF-7中的表达,观察PTEN及其突变体(F341V)基因对MCF-7细胞增殖及凋亡的影响。方法设计PTEN基因引物,PCR扩增PTEN及其突变体(F341V)编码基因,构建p CMV-Myc重组载体,转染MCF-7细胞系,Western blot检测其表达,酶标仪及流式细胞术检测细胞的增殖及凋亡。结果 PTEN及其突变体(F341V)真核表达载体构建成功,且在MCF-7细胞中成功表达。转染PTEN基因的MCF-7细胞生长速度比对照组MCF-7细胞减慢,转染PTEN(F341V)基因的MCF-7细胞恢复了快速的生长速度;相反的,转染PTEN基因的MCF-7细胞凋亡率增加,转染PTEN(F341V)基因的MCF-7细胞凋亡率相比PTEN基因明显降低。结论 PTEN及其突变体真核表达载体成功转染入MCF-7细胞系中,且显著影响其增殖及凋亡。
Objective To construct the eukaryotic expression vector of PTEN and its mutant (F341V) with the deletion of phosphatase and tensility protein on chromosome 10 and to detect its expression in breast cancer cell line MCF-7 To observe the effect of PTEN and its mutant (F341V) gene on the proliferation and apoptosis of MCF-7 cells. Methods The PTEN gene primers were designed and the gene encoding PTEN and its mutant (F341V) was amplified by PCR. The recombinant plasmid pCMV-Myc was constructed and transfected into MCF-7 cells. Western blot was used to detect the expression of PTEN gene and flow cytometry Detection of cell proliferation and apoptosis. Results The eukaryotic expression vector of PTEN and its mutant (F341V) was successfully constructed and successfully expressed in MCF-7 cells. The growth of MCF-7 cells transfected with PTEN gene was slower than MCF-7 cells in control group, and the rapid growth of MCF-7 cells transfected with PTEN (F341V) gene was restored. In contrast, MCF- 7 cells, the apoptosis rate of MCF-7 cells transfected with PTEN (F341V) gene was significantly lower than that of PTEN gene. Conclusion The eukaryotic expression vector of PTEN and its mutant was successfully transfected into MCF-7 cell line and significantly affected its proliferation and apoptosis.