CRISPR/Cas9介导Smad3基因敲除对人腹膜间皮细胞增殖和凋亡的影响  

Effects of CRISPR/Cas9-mediated Smad3 gene knockout on proliferation and apoptosis of human peritoneal mesothelial cells

作　　者：梁靖梅 李福记 林鹏 廖蕴华[1] Liang Jingmei;Li FujiLin Peng;Liao Yunhua(Nephrology Department,The First Affiliated Hospital of Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学第一附属医院肾内科,南宁530021

出　　处：《广西医科大学学报》2020年第8期1398-1403,共6页Journal of Guangxi Medical University

基　　金：国家自然科学基金资助项目(No.81660133);广西自然科学基金资助项目(No.2018GXNSFAA050047)。

摘　　要：目的:以CRISPR/Cas9基因编辑技术,构建Smad3基因敲除的人腹膜间皮细胞(HPMC),研究Smad3在HPMC增殖和凋亡中的调控机制。方法:利用CRISPR Design软件设计gRNA,比对后合成,构建Smad3 gRNA-PX459质粒,用Lipofectamine 3000转染细胞,嘌呤霉素筛选后用有限稀释法获得单克隆细胞株,提取DNA进行PCR扩增后测序,同时提取总蛋白,采用蛋白免疫印迹(Western blotting)检测蛋白表达情况;CCK8法检测细胞增殖情况,流式细胞术检测细胞凋亡率,Western blotting检测凋亡相关蛋白表达。结果:成功构建Smad3稳定敲除的HPMC。Smad3基因敲除后,细胞增殖率无明显变化(P>0.05),而凋亡率从(7.92±0.26%)上升至(11.70±0.56%),促凋亡相关蛋白Bax表达量从(0.70±0.05)上调至(1.25±0.11)(P<0.05)。结论:Smad3基因在HPMC的凋亡中具有重要的调控作用。Objective:To construct Smad3 gene knockout of human peritoneal mesothelial cell(HPMC)by CRISPR/Cas9 gene editing,and to study the regulatory mechanism of Smad3 in HPMC proliferation and apoptosis.Methods:The gRNA,was designed and synthesized by CRISPR Design software,and the Smad3 gRNAPX459 plasmid was constructed.The cells were transfected with Lipofectamine 3000,and the monoclonal cell lines were obtained by limiting dilution method after puromycin screening.DNA was extracted for PCR amplification,and the total protein was extracted at the same time.The protein expression was detected by Western blotting.The cell proliferation was detected by CCK8 method,the apoptosis rate was detected by flow cytometry,and the expression of apoptosis-related protein was detected by Western blotting.Results:The HPMC of Smad3 stable knockout was successfully constructed.After Smad3 gene knockout,there was no significant change in cell proliferation rate(P>0.05),but the apoptosis rate increased from(7.92±0.26%)to(11.70±0.56%),and the expression of apoptosis-related protein Bax was up-regulated from(0.70±0.05)to(1.25±0.11)(P<0.05).Conclusion:Smad3 gene plays an important role in the regulation of HPMC apoptosis.

关 键 词：SMAD3基因 CRISPR/Cas9 人腹膜间皮细胞 

分 类 号：R692.5[医药卫生—泌尿科学]