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目的:构建人CCR7真核表达载体,建立稳定转染CCR7的HeLa细胞株,初步探讨肿瘤细胞表达HLA-I类分子表达改变及相关机制。方法:从人脾脏cDNA扩增出CCR7,装入pDsRed2-N1,脂质体转染HeLa细胞。CCR7配体CCL21作用后,流式细胞术(FCM)检测肿瘤细胞表达HLA-I的表达,萤虫素酶报告基因系统检测NF-κB活化。结果:经PCR、酶切和测序证实,含CCR7真核表达载体成功构建。G418筛选后获得稳定表达CCR7蛋白的HeLa细胞。FCM证实HeLa细胞表达HLA-I类分子水平增高,萤虫素酶报告基因系统证实NF-κB活化明显。结论:成功构建人CCR7真核表达载体,CCR7促进肿瘤细胞表达HLA-I类分子,诱导肿瘤细胞NF-κB活化。
OBJECTIVE: To construct a human CCR7 eukaryotic expression vector and establish a HeLa cell line stably transfected with CCR7, to explore the alteration of HLA-I expression in tumor cells and related mechanisms. Methods: CCR7 was amplified from human spleen cDNA, loaded into pDsRed2-N1 and transfected into HeLa cells. CCR7 ligand CCL21 effect, flow cytometry (FCM) detection of tumor cells expressing HLA-I expression, firefly enzyme reporter gene system to detect NF-κB activation. Results: The CCR7 eukaryotic expression vector was successfully constructed by PCR, restriction enzyme digestion and sequencing. After G418 screening, HeLa cells stably expressing CCR7 protein were obtained. FCM confirmed that HeLa cells express increased levels of HLA-I molecules, and that the luciferase reporter gene system confirmed the significant activation of NF-κB. Conclusion: The human CCR7 eukaryotic expression vector was successfully constructed. CCR7 can promote the expression of HLA-I molecules in tumor cells and induce the activation of NF-κB in tumor cells.