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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。
Objective To construct eukaryotic expression vector pEGFP-C3-n-3 and detect the expression and function of CvFad3 gene of Chlorella n-3 fatty acid dehydrogenase in human breast cancer MCF-7 cells. Methods The CvFad3 gene was amplified by RT-PCR. The cDNA of CvFad3 gene was inserted into eukaryotic expression vector pEGFP-C3. The recombinant plasmid pEGFP-C3-n-3 was constructed and transfected into MCF-7 cells , Empty vector pEGFP-C3 as the control group. The expression of CvFad3 gene was detected by RT-PCR. The effect of CvFad3 gene on the proliferation of MCF-7 cells was analyzed by MTT assay. The effect of CvFad3 gene on the content of n-3 polyunsaturated fatty acids in MCF-7 cells was detected by gas chromatography. Results The recombinant plasmid pEGFP-C3-n-3 was transfected into breast cancer MCF-7 cells for 48 h, and the mRNA expression of CvFad3 gene was detected in MCF-7 cells transfected with pEGFP-C3 but not detected after 48 h CvFad3 gene mRNA bands. Compared with the control group, the MTT absorbance of transfected pEGFP-C3-n-3 cells was significantly decreased (t = 6.039, P <0.01), and the content of n-3 polyunsaturated fatty acids in MCF-7 cells was significantly increased. Conclusion The recombinant vector pEGFP-C3-n-3 was successfully constructed. CvFad3 gene was effectively heterogenously expressed in MCF-7 cells and inhibited the proliferation of MCF-7 cells and increased the content of n-3 polyunsaturated fatty acids in MCF-7 cells.