人乳癌细胞小球藻CvFad3基因的表达和作用

目的构建真核表达载体pEGFP-C3-n-3,检测小球藻n-3脂肪酸脱氢酶基因CvFad3在人乳癌MCF-7细胞中的表达和作用。方法通过RT-PCR法扩增得到CvFad3基因,将CvFad3基因cDNA插入到真核表达载体pEGFP-C3中,构建重组表达载体pEGFP-C3-n-3,并将其转染入MCF-7细胞中,设空载体pEGFP-C3为对照组。采用RT-PCR检测CvFad3基因的表达,MTT法分析CvFad3基因对MCF-7细胞增殖的影响,气相色谱检测CvFad3基因对MCF-7细胞n-3多不饱和脂肪酸含量的影响。结果构建的重组载体pEGFP-C3-n-3转染入乳癌MCF-7细胞48 h后可检测到CvFad3基因mRNA的条带,而转染pEGFP-C3的MCF-7细胞48 h后检测不到CvFad3基因mRNA的条带。与对照组比较,转染pEGFP-C3-n-3的细胞MTT吸光度明显降低（t=6.039,P〈0.01）;人乳癌细胞MCF-7细胞膜n-3多不饱和脂肪酸的含量显著升高。结论重组载体pEGFP-C3-n-3构建成功,CvFad3基因能在MCF-7细胞内有效异源表达,且抑制了MCF-7细胞的增殖,增加了细胞膜n-3多不饱和脂肪酸的含量。

EXPRESSION AND EFFECT OF CHLORELLA VULGARIS CvFad3 GENE IN HUMAN BREAST CANCER CELLS

Objective To construct a eukaryotic expression vector pEGFP-C3-n-3,and study the expression and effect of n-3 fatty acid desaturase gene CvFad3 MCF-7 cells of human breast cancer. Methods n-3 fatty acid desaturase gene CvFad3 of Chlorella vulgaris was cloned by RT-PCR from Chlorella vulgaris.The cDNA of the gene was inserted into eukaryotic expression vector pEGFP-C3 to form a recombinant,it was then transfected into breast cancer cell MCF-7 by transfection reagent.The expression of n-3 gene was detected by RT-PCR.MTT was used to examine the proliferation of cells,and lipid analysis was used to detect the changes of n-3 PUFAs in MCF-7 cell membrane. Results The proposed recombinant plasmid was obtained through DNA recombinant technique;CvFad3 gene expressed effectively in human breast cancer cells;CvFad3 gene mRNA band appeared after 48 hours of transfection of pEGFP-C3-n-3,and CvFad3 gene mRNA band didn＇t appear after transfection of pEGFP-C3.Comparing with the control cells（pEGFP-C3-control）,MTT showed the proliferation of MCF-7 cells was markedly inhibited（21%,P〈0.01）;Moreover,lipid analysis showed that CvFad3 gene significantly increased the content of n-3 PUFAs in MCF-7 cell membrane. Conclusion pEGFP-C3-n-3 is successfully constructed;n-3 fatty acid desaturase gene CvFad3 of Chlorella vulgaris can express in human breast cancer cells MCF-7 heterologously and inhibit proliferation of MCF-7 cells.