骨髓瘤U266细胞中核干细胞因子NS与程序性细胞死亡配体PD-L1关联性的研究  

Correlation of Nucleostemin with Programmed Death-ligand-1 in Multiple Myeloma U266 Cells

作　　者：李佳 高晓娟 王亚奇 刘帅 岳保红 LI Jia;GAO Xiao-Juan;WANG Ya-Qi;LIU Shuai;YUE Bao-Hong(Department of Laboratorial Medicine,the First Affiliated Hospital of Zhengzhou University,Faculty of Laboratorial Medicine,Zhengzhou University,Key Laboratory of Laboratorial Medicine,Henan Province,Faculty of Laboratorial Medicine of Zhengzhou University,Open Laboratory,Henan Province Key Subject of Clinical Medicine,Zhengzhou 450052,Henan province,China)

机构地区：[1]郑州大学第一附属医院检验科郑州大学医学检验系河南省高等学校临床医学重点学科开放实验室河南省检验医学重点实验室

出　　处：《中国实验血液学杂志》2019年第6期1894-1900,共7页Journal of Experimental Hematology

基　　金：河南省自然科学基金项目(16230041299)

摘　　要：目的:探讨骨髓瘤U266细胞内Nucleostemin(NS)基因和程序性死亡配体-1(programmed deathligand-1,PD-L1)的关联性以及NS表达下调对骨髓瘤细胞的凋亡影响,评价NS、PD-L1关联性与MM细胞生物学行为改变的关系以及二者联合作为反映MM肿瘤细胞状态标志物的可行性。方法:通过NS-RNAi-GV248重组慢病毒下调U266细胞的NS基因表达;采用real-time PCR检测转染后NS、PD-L1及相关PI3K/AKT/mTOR通路基因变化;应用Western blot、流式细胞术检测NS及PD-L1蛋白表达;采用Annexin V-APC/7-AAD双染法检测敲除NS基因前后的U266细胞的凋亡变化。结果:在MOI=10条件下,NS-RNAi-GV248重组慢病毒转染U266细胞后,荧光显微镜下观察显示,转染效率达到75%以上;real-time PCR显示,与阴性对照组(1.002±0.026)相比,转染组(0.415±0.089)的NS、PD-L1及PI3K/AKT/mTOR基因的m RNA相对表达量明显减低(P<0.05);流式细胞术和Western blot结果表明,慢病毒转染后PD-L1蛋白表达量明显下调,U266细胞凋亡增加(P<0.05)。结论:骨髓瘤细胞U266中存在NS及PD-L1基因的异常高表达。下调NS基因表达后出现PD-L1基因及相关PI3K/AKT/mTOR通路基因表达的下调,说明二者引起的细胞生物学改变存在联动效应。Objective:To investigate the correlation of nucleostemin(NS)gene with programmed death ligand-1(PDL1)in myeloma cells and the effect of NS expression down-regulation on the apoptosis of multiple myeloma cells,and to evaluate the associations among NS,PD-L1 and biological behavior of MM cells,and the feasibility of both NS and PD-1 as markers reflecting the status of MM cells.Methods:The NS gene expression in U266 cells was down-regulated by NS-RNAi-GV248 recombinant lentivirus,the real-time PCR was used to detect the mRNA expression of NS,PD-L1 and PI3 K/AKT/mTOR.The Western blot and flow cytometry were used to detect the expression of NS and PD-L1.The Annexin V-APC/7-AAD staining method was used to detect the apoptosis of U266 cells before and after knocking out the NS gene.Results:Under the condition of MOI=10,the transfection efficiency was more than 75%by means of the fluorescent microscopy;real-time PCR showed that compared with the negative control group(1.002±0.026),the mRNA expression of NS,RD-L1 and PI3 K/AKT/mTOR gene in the transfection group(0.415±0.089)was significantly reduced(P<0.05).The results of flow cytometry and Western blot showed that the protein expression of PD-L1 was significantly down-regulated after transfection.After down-regulation of NS gene expression,the apoptosis of U266 cells increased(P<0.05).Conclusion:The abnormal high expression of NS and PD-L1 genes exists in U266 cells,moreover,the downregulation of PD-L1 and the related PISK/AKT/mTOR pathway gene expression appears after down-regulation of NS gene expression,which suggest that the cell biological changes resulted from above-mentioned results,show a synergestic effect on U266 cells.

关 键 词：多发性骨髓瘤 NS PD-L1 基因敲除 慢病毒载体 

分 类 号：R733.3[医药卫生—肿瘤]