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当前位置: 首页 > 文章 > 慢病毒介导沉默PD-L1基因在乳腺癌细胞中的表达 生物技术通报 2017 (9) 259-266
Position: Home > Articles > Low Expression of PD-L1 Gene in Human Breast Carcinoma Cell Mediated by Lentiviral Vector Biotechnology Bulletin 2017 (9) 259-266

慢病毒介导沉默PD-L1基因在乳腺癌细胞中的表达

作  者：
欧阳婧;孙园园;唐泽民;唐政山;李放军;杨寅柯
单  位：
湖南省人民医院社会医学部;湖南大学生物学院
关键词：
乳腺癌;细胞程序性死亡因子配体1基因;慢病毒载体;聚乙烯亚胺;共转染
摘  要：
构建含细胞程序性死亡因子配体1(PD-L1)基因的慢病毒载体三质粒体系,并检测其在乳腺癌MDA-MB-231细胞中的表达情况。p MAGic 7.1慢病毒表达载体与质粒△8.91和p VSVG用聚乙烯亚胺(PEI)共转293T包装病毒,感染并筛选获得MDA-MB-231稳转细胞系,检测PD-L1表达情况。(1)MDA-MB-231在3种乳腺癌细胞中PD-L1表达量最高;(2)三质粒转染293T细胞48 h后荧光强度增强,达到90%以上;用病毒感染MDA-MB-231细胞,48 h获得20%左右荧光强度;(3)经筛选的MDA-MB-231稳转细胞系在m RNA水平和蛋白水平低表达PD-L1,其中由p MAGic-sh1包装的病毒干扰能力最强;(4)分别加入病毒后,干扰组细胞增殖能力下降,p MAGic-sh1干扰组细胞在加入病毒7 d后生长速度约为阴性对照的64.77%;(5)混合淋巴实验显示,sh-1干扰组的肿瘤抑制率为35.8%,是正常组的3.06倍,且干扰组的T淋巴细胞活性显著增强。成功通过PEI转染试剂建立了三质粒慢病毒包装体系,该体系获得的慢病毒可有效干扰乳腺癌MDA-MB-231细胞中PD-L1的表达量,所得的干扰细胞株细胞增殖能力下降,可增强T细胞增殖活性及肿瘤细胞杀伤活性。
译  名：
Low Expression of PD-L1 Gene in Human Breast Carcinoma Cell Mediated by Lentiviral Vector
作  者：
OUYANG Jing;SUN Yuan-yuan;TANG Ze-min;TANG Zheng-shan;LI Fang-jun;YANG Yin-ke;College of Biology,Hunan University;Department of Social Medicine,Hunan Province People's Hospital;
关键词：
breast cancer;;programmed death factor ligant 1 gene;;lentiviral vector;;PEI;;co-transfected
摘  要：
This study aims at constructing three-plasmid system of the lentiviral vector carrying the programmed death ligant 1(PD-L1)gene and investigating the expression of PD-L1 in human breast carcinoma cell MDA-MB-231. A p MAGic 7.1 recombinant lentiviral expressionvector,plasmid △ 8.91 and p VSVG were co-transfected into 293 T cells for packaging virus,using polyethylenimine(PEI)as transfectedsolution. The lentiviral products were added to infect and extract stably-expressed MDA-MB-231 cells,and finally the PD-L1 expression ofm RNA and protein levels were detected. The results showed as followings :(1)The PD-L1 expression in MDA-Mb-231 was the highest,compared with other two breast cancer cells.(2)At 48 h after co-transfecting three plasmids,the fluorescence intensity reached over 90%.Moreover,the fluorescence intensity reached almost 20% at 48 h after using virus infecting MDA-MB-231 cells.(3)There were low PDL1 expressions of m RNA and protein levels in the stably-expressed MDA-MB-231 cell lines,and the p MAGic-sh1 had the best effect on itamong the three plasmids.(4)After adding viruses,the cell proliferation declined in interfering groups,and the cell proliferation rate inthe p MAGic-sh1 interfering groups was 64.77% at 7 days of adding virus compared with the negative group.(5)Mixed lymphatic experimentsshowed that the tumor inhibition rate of sh-1 group was 35.8%,which was 3.06 times of that in normal group,and the T lymphocyteproliferation rate significantly enhanced. In conclusion,the three-plasmid system of lentiviral vector containing PD-L1 gene using PEI reagentis successfully established. The study demonstrates that the PD-L1 expression of m RNA and protein is lower through the virus infecting system,and interfering groups have lower cell proliferation rate and they enhance the T lymphocyte proliferation rates and its ability to kill tumor cells.

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