摘要
目的:探讨在具有T790M突变的非小细胞肺癌(NSCLC)细胞中应用CRISPR/Cas9基因编辑技术敲除程序性细胞死亡配体1(PD-L1)后对吉非替尼耐药敏感性的影响,阐明PD-L1对PC-9/T790M细胞耐药敏感性的作用机制。方法:常规体外培养PC-9细胞和PC-9/T790M细胞,经CRISPR/Cas9基因编辑技术敲除PC-9/T790M细胞中PD-L1后,分为PC-9、PC-9/T790M和Cas9 PC-9/sgRNA 3组细胞。采用Western blotting法检测各组细胞中PD-L1蛋白表达水平,CCK-8法检测5、10和20 mmol·L-1吉非替尼干预24、48和72 h后各组细胞存活率。体内实验中检测吉非替尼干预后各组移植瘤体积,HE染色观察移植瘤组织病理形态表现。结果:Western blotting法检测,经CRISPR/Cas9编辑后敲除PD-L1的sgRNAl#组与PC-9、PC-9/T790M、sgRNA2#和sgRNA3#组比较,PD-L1蛋白表达水平最低。CCK-8法检测,在应用药物干预前Cas9 PC-9/sgRNA组细胞增殖活性与PC-9组、PC-9/T790M组比较,差异无统计学意义(P>0.05),10 mmol·L-1吉非替尼干预72 h后PC-9组和Cas9 PC-9/sgRNA1#组细胞存活率与PC-9/T790M组比较明显降低(P<0.01)。体内实验,与PC-9/T790M组比较,吉非替尼干预后PC-9组和Cas9 PC-9/sgRNA1#组裸鼠移植瘤体积明显减少(P<0.05);PC-9组和Cas9 PC-9/sgRNA1#组移植瘤细胞呈现中、重度坏死,而PC-9/T790M组移植瘤组织病理形态无明显变化。结论:在耐药PC-9/T790M细胞中应用CRISPR/Cas9编辑技术敲除PD-L1能够明显提高吉非替尼的药物敏感性。
Objective:To investigate the effect of CRISPR/Cas9 gene-editing knockout of programmed cell death ligand 1(PD-L1)on the geffitinib resistance sensitivity in the non-small cell lung cancer(NSCLC)cells with T790M mutation,and to clarify the mechanism of the effect of PD-L1 on the drug resistance sensitivity of PC-9/T790M cells.Methods:The PC-9 cells and PC-9/T790M cells were cultured in vitro.After CRISPR/Cas9 gene editing technology was used to knock out PD-L1 in the PC-9/T790M cells,the cells were divided into three groups:PC-9 group,PC-9/T790M group and Cas9 PC-9/sgRNA group.The expression levels of PD-L1 protein in the cells in various groups were detected by Western blotting method,and CCK-8 method was used to determine the survival rates of the cells in various groups after 24,48 and 72 h of intervention with 5,10 and 20 mmol·L-1 gefitinib.In vivo experiments,the tumor volumes in vorious groups were detected after gefitinib intervention,and the pathomorphology of transplanted tumor tissue in various groups was observed.Results:The Western blotting results showed that the expression level of PD-L1 protein in sgRNA1#group edited by CRISPR/Cas9 was the lowest compared with PC-9,PC-9/T790M,sgRNA2#and sgRNA3#groups.There were no statistically significant differences in the cell proliferation activities in Cas9 PC-9/sgRNA group compared with other two groups before drug intervention detected by CCK-8 method(P>0.05).After10 mmol·L-1 gefitinib intervention for 72 h,the survival rates of cells in PC-9 and Cas9 PC-9/sgRNA1#groups were significantly lower than that in PC-9/T790M group(P<0.01).In vivo experiment,compared with PC-9/T790M group,the tumor volumes in PC-9 group and Cas9 PC-9/sgRNA1#group were significantly reduced after gefeitinib intervention(P<0.05);moderate to severe cell necrosis was found in PC-9 group and Cas9 PC-9/sgRNA1 group,while no significant changes in transplanted tumor tissue were found in PC-9/T790M group.Conclusion:Using CRISPR/Cas9 editing technique to knock out PD-L1 in the drug-resistant PC-9/T790M cells can significantly improve the sensitivity of gefitinib.
作者
任爱华
刘婉
王大伟
REN Aihua;LIU Wan;WANG Dawei(Department of Anatomy,College of Medical Sciences,Beihua University,Jilin 132013,China;Department of Pathology,College of Medical Sciences,Beihua University,Jilin 132013,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2021年第2期292-298,共7页
Journal of Jilin University：Medicine Edition
基金
吉林省科技厅自然科学基金项目(20190201052JC)
作者简介
任爱华(1976-),男,河北省唐山市人,讲师,医学硕士,主要从事解剖病理学方面的研究。;通信作者:王大伟,教授,硕士研究生导师(E-mail:dw_wang@163.com)。
