摘要
目的:探讨过表达B7同源物6(B7-H6)在自然杀伤(NK)细胞介导的肝细胞凋亡中的作用。方法:设计针对B7-H6全长的寡核苷酸引物,经PCR扩增调取B7-H6全长,并将其亚克隆入线性化的真核表达载体p IRES2-EGFP,构建重组B7-H6过表达载体p IRES2-EGFP-B7-H6,通过双酶切、PCR及测序进行鉴定。利用脂质体将p IRES2-EGFP-B7-H6重组质粒转染正常肝细胞系L02,采用荧光显微镜观察EGFP的表达,流式细胞技术检测转染效率,qRT-PCR和Western blot检测B7-H6 mRNA和蛋白的表达水平。将转染p IRES2-EGFP-B7-H6重组质粒的L02细胞与NK-92细胞以不同的效靶比共培养,利用CCK-8实验分析检测NK-92细胞对L02细胞的杀伤效应。结果:经PCR、酶切及测序等方法证实成功构建p IRES2-EGFP-B7-H6过表达载体;经脂质体转染L02细胞48 h后,在荧光显微镜下观察到较强绿色荧光表达,流式检测显示转染效率达到92.6%;qRT-PCR和Western blot结果显示L02细胞B7-H6的mRNA和蛋白高表达;CCK-8实验证实相对于转染空载体p IRES2-EGFP,NK-92细胞对转染了p IRES2-EGFP-B7-H6的L02细胞的杀伤活性显著增强(P<0.05)。结论:成功构建过表达B7-H6的真核表达载体p IRES2-EGFP-B7-H6,并进一步证实NK-92细胞对过表达B7-H6的L02细胞具有显著的杀伤效应。
AIM：To investigate the role of B7 homologue 6（B7-H6） over-expression in natural killer（NK）cell-mediated hepatocyte apoptosis.METHODS：The full-length fragment of B7-H6 gene was amplified by PCR and subcloned into linearized eukaryotic expression vector p IRES2-EGFP to construct recombinant B7-H6 over-expression vector p IRES2-EGFP-B7-H6.The recombinant plasmid was identified by double digestion,PCR and sequencing,and was then transfected into L02 cells.The expression of EGFP was observed by fluorescence microscopy and the transfection efficiency was evaluated by flow cytometry.B7-H6 expression was confirmed by qRT-PCR and Western blot.The L02 cells transfected with p IRES2-EGFP-B7-H6 recombinant plasmid were co-cultured with NK-92 cells at different effector/target ratios,and the cytotoxicity of NK-92 cells was evaluated by CCK-8 assay.RESULTS：The strong green fluorescence in the L02 cells was observed under fluorescence microscope 48 h after transfection.The transfection efficiency reached 92.6%.The expression of B7-H6 at mRNA and protein levels was remarkably increased 48 h after transfection.The cytotoxicity of NK-92 cells against L02 cells transfected with p IRES2-EGFP-B7-H6 plasmid was significantly higher than that of the null vector transfection group（P 〈 0.05）.CONCLUSION：The recombinant eukaryotic expression vector p IRES2-EGFP-B7-H6 was constructed successfully.The cytotoxic effect of NK-92 cells against L02 cells can be enhanced by transfecting L02 cells with p IRES2-EGFP-B7-H6 plasmid.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2017年第11期2095-2098,共4页
Chinese Journal of Pathophysiology
基金
广东省科技计划资助项目(No.2014A020212575
No.2016A020215215)
广东省自然科学基金资助项目(No.2016A030313357)
