过表达B7-H6在自然杀伤细胞介导的肝细胞凋亡中的作用

发布时间：2018-05-02 20:48

本文选题：B同源物 + 真核表达载体　；参考：《中国病理生理杂志》2017年11期

【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(P0.05)。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。
[Abstract]:Aim: to investigate the role of overexpression of B7 homologue 6B 7 H 6 in natural killer NKK cells mediated hepatocyte apoptosis. Methods: the full-length B7-H6 oligonucleotide primers were designed and amplified by PCR and subcloned into the linear eukaryotic expression vector pIRES2-EGFP.The recombinant B7-H6 overexpression vector pIRES2-EGFP-B7-H6 was constructed and identified by double enzyme digestion and sequencing. The recombinant plasmid of p IRES2-EGFP-B7-H6 was transfected into the normal liver cell line L02 by liposome. The expression of EGFP was observed by fluorescence microscope, the transfection efficiency was detected by flow cytometry and the expression level of B7-H6 mRNA and protein was detected by Western blot and RT-PCR. L02 cells transfected with p IRES2-EGFP-B7-H6 recombinant plasmid were co-cultured with NK-92 cells at different target ratios. CCK-8 assay was used to detect the killing effect of NK-92 cells on L02 cells. Results: the overexpression vector of p IRES2-EGFP-B7-H6 was successfully constructed by PCR, restriction endonuclease digestion and sequencing, and strong green fluorescent expression was observed under fluorescence microscope after 48 h transfection of liposome into L02 cells. Flow cytometry showed that the transfection efficiency was 92.6g / qRT-PCR and Western blot results showed that the high expression of mRNA and protein in B7-H6 of L02 cells showed that the cytotoxicity of pIRES2-EGFPNK-92 cells to L02 cells transfected with p IRES2-EGFP-B7-H6 was significantly enhanced compared with that of pIRES2-EGFPNK-92 cells. Conclusion: the eukaryotic expression vector pIRES2-EGFP-B7-H6 was successfully constructed, and it was further confirmed that NK-92 cells had a significant killing effect on L02 cells over-expressing B7-H6.
【作者单位】：中山大学附属第三医院输血科;中山大学附属第三医院血液科;
【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)
【分类号】：R512.62

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