弓形虫ROP16蛋白在人白血病细胞THP-1表达及对其细胞增殖与凋亡的影响  

Expression of Toxoplasma gondii ROP16 protein in THP-1 human leukemia cells and its effects on proliferation and apoptosis

作　　者：陈梅 贾伟 党甜甜[1] 潘亚菲 杨宁爱 苏雅静 赵志军 CHEN Mei;JIA Wei;DANG Tian-tian;PAN Ya-fei;YANG Ning-ai;SU Ya-jing;ZHAO Zhi-jun(College of Clinical Medicing,Ningxia Medical University,Yinchuan 750004,China;Ningxia Key Laboratory of Clinical and Pathogenic Microbiology,Yinchuan 750004,China;Laboratory Medical Center,General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学临床医学院,银川750004 [2]宁夏临床病原微生物重点实验室,银川750004 [3]宁夏医科大学总院医学实验中心,银川750004

出　　处：《中国人兽共患病学报》2022年第4期277-284,共8页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.81560333)。

摘　　要：目的探讨弓形虫Ⅱ型(Me49)ROP16蛋白在急性单核细胞白血病细胞株THP-1中的表达及其对THP-1细胞增殖与凋亡的影响。方法构建过表达ROP16(overExp-ROP16)和空载体(overExp-NC)慢病毒,通过转染THP-1细胞,72 h后观察荧光表达情况,采用PCR与Western blot验证ROP16在THP-1细胞中的表达。免疫荧光技术检测ROP16蛋白在THP-1细胞中的定位。CCK-8与流式细胞术检测细胞增殖及凋亡。采用qRT-PCR检测细胞凋亡相关因子Bcl-2、Bax、Caspase-3 mRNA相对表达水平。Western blot检测Bax、Bcl-2、Pro-Caspase-3和Cleaved-Caspase-3蛋白的表达变化。结果构建的过表达ROP16重组慢病毒转染到THP-1细胞后,可观察强荧光信号,ROP16蛋白在THP-1细胞中成功表达。免疫荧光结果表明ROP16蛋白定位于THP-1细胞核。CCK-8与流式细胞术证实ROP16蛋白抑制THP-1细胞的增殖并促进其凋亡(均P<0.05)。与对照组相比,过表达组促凋亡因子Bax、Caspase-3 mRNA高表达(P<0.05),抗凋亡因子Bcl-2 mRNA低表达(P<0.01),Bax与Cleaved-Caspase-3蛋白水平上调(P<0.01),Bcl-2与Pro-Caspase-3蛋白水平下调(P<0.01)。结论构建的慢病毒表达质粒overExp-ROP16可在急性单核细胞白血病细胞株THP-1中表达且通过入核调节凋亡蛋白Bcl-2、Bax、Caspase-3而抑制THP-1细胞增殖,促进其凋亡。This study aimed to explore the expression of Toxoplasma gondii type Ⅱ(Me49)ROP16 protein in the acute monocytic leukemia cell line THP-1 and to assess its effects on proliferation and apoptosis.We constructed a lentivirus for overexpression of ROP16(overExp-ROP16)and an empty vector(overExp-NC),and used them to transduce THP-1 cells.Fluorescence was observed through fluorescence microscopy after 72 h,and the expression of ROP16 in THP-1 cells was determined by PCR and western blotting.We used an immunofluorescence technique to detect the localization of ROP16 protein in THP-1 cells.CCK-8 and flow cytometry were used to detect the changes in proliferation and apoptosis.qRT-PCR was used to analyze the relative expression levels of the apoptosis-associated factors Bcl-2,Bax,and Caspase-3 mRNA.Western blotting detected the expression changes of Bax,Bcl-2,pro-Caspase-3 and cleaved Caspase-3 proteins.After the recombinant lentivirus for overexpression of ROP16 was used to transduce the THP-1 cell line,strong fluorescence signals were observed.The ROP16 protein was thus successfully expressed in THP-1 cells.Immunofluorescence showed that the ROP16 protein localized in the nuclei of THP-1 cells.CCK-8 and flow cytometry analyses indicated that the ROP16 protein inhibited the proliferation of THP-1 cells and promoted apoptosis(P<0.05).In the overexpression group compared with the control group,the expression of the pro-apoptotic factors Bax and Caspase-3 mRNA was higher(P<0.05),the expression of the anti-apoptotic factors Bcl-2 mRNA was lower(P<0.01),the expression of Bax and Cleaved-Caspase-3 proteins was higher(P<0.01),and the expression of Bcl-2 and Pro-Caspase-3 proteins was lower(P<0.01).This study suggests that the recombinant lentivirus expression plasmid overExp-ROP16 can be expressed in the acute monocytic leukemia cell line THP-1.ROP16 inhibits proliferation and promotes apoptosis by entering the nucleus and regulating the apoptosis-associated proteins Bcl-2,Bax and Caspase-3.

关 键 词：刚地弓形虫 ROP16 THP-1细胞 增殖 凋亡 

分 类 号：R382.5[医药卫生—医学寄生虫学]