弓形虫Ⅰ型和Ⅱ型ROP16蛋白对人肺腺癌A549细胞增殖、周期和凋亡的影响  

Role of ToxoplasmatypeⅠandⅡROP16on regulating the proliferation,cycle and apoptosis of human lung adenocarcinoma A549cells

作　　者：李琴惠 曾瑾[1,2] 汪澎涛 杨宁爱 马磊[1,2] 李佳铭 马慧慧 赵志军 LI Qin-hui;ZENG Jin;WANG Peng-tao;YANG Ning-ai;MA Lei;LI Jia-ming;MA Hui-hui;ZHAO Zhi-jun(Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western,Ningxia University,Yinchuan,750021,China;College of Life Science,Ningxia University;Ningxia Key Laboratory of Clinical and Pathogenic Microbiology;General Hospital of Ningxia Medical University)

机构地区：[1]西部特色生物资源保护与利用教育部重点实验室,宁夏银川750021 [2]宁夏大学生命科学学院 [3]宁夏医科大学总医院宁夏临床病原微生物重点实验室 [4]宁夏医科大学总院医学实验中心

出　　处：《中国病原生物学杂志》2022年第2期180-185,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81560333),2018年宁夏高等学校科学研究项目(No.NGY2018-103)。

摘　　要：目的探讨弓形虫Ⅰ型和Ⅱ型ROP16蛋白对人肺腺癌A549细胞增殖、周期和凋亡的影响及分子机制。方法将构建好的Ⅰ型和Ⅱ型ROP16重组慢病毒表达载体(A549-RH ROP16、A549-Me49ROP16)感染A549细胞,经嘌呤筛选稳定后获得单克隆过表达细胞株。实时荧光定量PCR和Western blot法鉴定过表达效果,免疫荧光法检测其定位。CCK-8法测定A549细胞的增殖活性。流式细胞术检测细胞凋亡和周期变化水平。Western blot法检测细胞凋亡相关蛋白Bcl-2、Bax、Cleaved Caspases-3、Caspase9、p53和细胞周期相关蛋白p21、CDK6、CyclinD1的蛋白水平。结果Western blot和q-PCR检测结果显示,重组慢病毒表达载体转染A549细胞72h后其ROP16mRNA和蛋白都有明显表达(P<0.05)。cck8检测结果显示,Ⅰ型ROP16蛋白抑制A549细胞增殖(P<0.05)。流式细胞术检测显示,与对照组比较,Ⅰ型ROP16过表达组中细胞凋亡率下降20.69%,G1期细胞百分由原来的63.2%升高到83.2%。Western blot结果显示促细胞周期G1/S转化因子CDK6、CyclinD1蛋白表达明显降低,而细胞周期蛋白依赖激酶抑制因子p21表达明显升高(P<0.05);促凋亡因子Bax、Cleaved Caspases-3、Caspase9、p53蛋白的表达明显升高,抗凋亡因子Bcl-2表达受到抑制(P<0.05)。而Ⅱ型ROP16过表达组与对照组无显著性差异(P>0.05)。结论Ⅰ型ROP16蛋白过表达可诱导人肺腺癌A549细胞周期停滞和细胞凋亡。Objective To study the effects on the proliferation,cycle and apoptosis of human lung adenocarcinoma A549cell line by overexpression the genes of Toxoplasma gondii typeⅠandⅡROP16.Methods A549cell lines were transfected with the recombinant lentiviral vectors(A549-RH ROP16,A549-Me49ROP16)and afeter stable screening with purine,monoclonal overexpression cell lines were obtained.The efficiency of overexpression of ROP16was detected by real-time fluorescent quantitative PCR and Western blot,moreover,its localization was detected by immunofluorescence staining.CCK-8assay was performed to detected the cell proliferation and flow cytometry was used to test cell apoptosis and cell cycle.The protein levels of apoptosis-related proteins including Bcl-2,Bax,Cleaved Caspases-3,Caspase9,p53and cell cycle-related proteins including p21,CDK6,CyclinD1were analyzed by using western blotting.Results The results of Western blot and q-PCR showed that the ROP16mRNA and protein were significantly expressed higher in A549cells 72hafter the recombinant lentiviral expression vector was transfected(P<0.05).The results of cck8test showed that type I ROP16protein could inhibit the proliferation of A549cells significantly(P<0.05).As for flow cytometry results,compared with the control group,the apoptosis rate in the type I ROP16overexpression group decreased by 20.69%,and the percentage of cells in G1phase increased from 63.2%to 83.2%.Western blot results showed that the protein expressions of pro-cell cycle G1/S transforming factors CDK6and CyclinD1were significantly decreased,while the expression of cyclin-dependent kinase inhibitor p21was significantly increased(P<0.05);pro-apoptotic factors Bax,Cleaved Caspases-3,Caspase9and p53protein were significantly increased,and the expression of anti-apoptotic factor Bcl-2was inhibited(P<0.05).However,there was no significant difference between the type II ROP16overexpression group and the control group(P>0.05).Conclusion Type I ROP16overexpression gruoup can induce phosphorylation of STAT3in

关 键 词：肺腺癌 A549细胞 ROP16蛋白 细胞增殖 细胞周期 细胞凋亡 

分 类 号：R382.5[医药卫生—医学寄生虫学]