重组pEGFP-Mfn2真核表达载体的构建及转染人乳腺癌细胞系MCF-7

Construction of eukaryotic expression recombinant plasmid pEGFP-Mfn2 and transfected into human breast cancer cell line MCF-7

目的:构建Mfn2基因真核表达载体,将此载体转染入人乳腺癌细胞系MCF-7,并观察高表达Mfn2对MCF-7增殖的影响。方法:1)设计pEG-FP-Mfn2质粒,并将其用脂质体转染入人乳腺癌细胞系MCF-7;2)DNA测序、RT-PCR法、流式细胞术及免疫细胞化学法鉴定以上述载体是否转染成功;3)MTT法检测转染后Mfn2对MCF-7细胞增殖的影响。结果:1)重组后的pEGFP-N1质粒已成功载入小鼠Mfn2的全长编码基因,DNA序列测定的结果与预期设计基本一致。普通PCR琼脂糖凝胶电泳显示转染后MCF-7细胞中存在外源性Mfn2的表达。2)琼脂糖凝胶电泳检测显示实验组中Mfn2较对照组明显升高,转染Mfn2后MTT法检测转染pEGFP-Mfn2组的MCF-7细胞数明显低于空白对照组与转染pEGFP-N1组,P<0.05。3)细胞免疫化学显示转染Mfn2后MCF-7细胞数目减少,癌细胞出现核碎裂现象。结论:成功构建了Mfn2基因真核表达载体,并成功转染MCF-7细胞。转染Mfn2后,MCF-7细胞的增殖受到明显抑制。

OBJECTIVE：To observe the affect to cell proliferation,we we construct eukaryotic expression plasmid including Mfn2 gene and transfect it inti breast cancer cells MCF-7.METHODS：1.We constructed the pEGFP-Mfn2 plasmid and transfected it into human breast cancer cell line MCF-7 using liposomes.2.Using the method of DNA sequencing,RT-PCR,Flow Cytometry and Immunocytochemistry to identify whether the vector transfected.3.MTT was used to detect how the Mfn2 affect the cell proliferation after transfecting exogenous Mfn2 gene into the cell cycle of MCF-7 cell in vitro.RESULTS：1.The recombinant plasmid pEGFP-N1 successfully loaded full-length mouse Mfn2 gene encoding,DNA sequencing results were basically consistent with the expected design.General PCR Agarose Gel Electrophoresis of transfected MCF-7 cells were in the presence of exogenous Mfn2 expression.2.The Agarose Gel Electrophoresis（AGE） showed that the expression of Mfn2 in experimental group was significantly higher than in the control group.After transfection,the cell proliferation velocity and the OD value of pEGFP-N1 group and non-transfection group were basically at equal pace.But the pEGFP-Mfn2 group＇ 48 h was manifestly less than both of pEGFP-N1 group and non-transfection group（P0.05）.3.The Immunohistochemical was showed that cancer cells appeared nuclear fragmentation.CONCLUSIONS：The eukaryotic expression plasmid pEGFP-Mfn2 was successfully constructed,and Mfn2 was expressed in MCF-7 cells after being transfected.Exogenous Mfn2 strongly inhibited cell proliferation in MCF-7 cell line.