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目的:构建真核表达载体pcDNA3.1-Fat1,线性化稳定转染人口腔鳞癌细胞株Tca8113,检测其细胞内脂肪酸含量变化。方法:通过重叠延伸PCR方法人工合成利于真核表达的Fat-1基因,用基因重组技术构建真核表达载体pcDNA3.1-Fat-1,用脂质体转染真核细胞的方法转染人口腔鳞癌细胞株Tca8113,用气相色谱仪检测脂肪酸的变化情况。结果:测序及酶切鉴定成功合成真核偏好表达的Fat-1基因。与对照组相比,转染Fat-1基因的口腔癌细胞的n-3脂肪酸明显增多,n-6/n-3明显下降。结论:成功构建真核表达载体pcDNA3.1-Fat1,并对口腔鳞癌细胞内脂肪酸含量产生明显影响,为进一步研究Fat-1基因在口腔鳞癌中的生物学功能奠定了基础。
OBJECTIVE: To construct eukaryotic expression vector pcDNA3.1-Fat1 and to linearly stably transfect human oral squamous cell carcinoma cell line Tca8113 to detect the change of intracellular fatty acid. Methods: The recombinant eukaryotic expression vector Fat-1 was constructed by overlap extension PCR. The eukaryotic expression vector pcDNA3.1-Fat-1 was constructed by gene recombination and transfected into human Oral squamous cell carcinoma cell line Tca8113, using gas chromatography to detect changes in fatty acids. Results: The eukaryotic expression of Fat-1 gene was successfully synthesized by sequencing and restriction enzyme digestion. Compared with the control group, oral cancer cells transfected with Fat-1 gene n-3 fatty acids increased significantly, n-6 / n-3 decreased significantly. CONCLUSION: The eukaryotic expression vector pcDNA3.1-Fat1 was successfully constructed and had a significant effect on the intracellular fatty acid content in oral squamous cell carcinoma, which laid the foundation for the further study on the biological function of Fat-1 gene in oral squamous cell carcinoma.