幽门螺杆菌及其毒力因子空泡细胞毒素A对人胃腺癌细胞DNA同源重组修复和细胞周期的影响  

Effects of Helicobacter pylori and its virulence factor vacuolating cytotoxin A on DNA homologous recombination repair and cell cycle in human gastric adenocarcinoma cells

作　　者：莫慧 黄煌[1,2,3] 梅璐[1,2,3] 米阳 刘斌[2,3] 白利梅 于泳[1,2,3] MO Hui;HUANG Huang;MEI Lu;MI Yang;LIU Bin;BAI Limei;YU Yong(Department of Gastroenterology,the Fifth Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Marshall BJ Medical Research Center of Zhengzhou University,Zhengzhou 450052,China;Key Laboratory of Helicobacter pylori&Microecology and Gastrointestinal Cancer of Henan Province,Zhengzhou 450052,China)

机构地区：[1]郑州大学第五附属医院消化内科,河南郑州450052 [2]郑州大学马歇尔医学研究中心,河南郑州450052 [3]河南省幽门螺杆菌及微生态与消化道肿瘤重点实验室,河南郑州450052

出　　处：《肿瘤基础与临床》2023年第1期7-13,共7页journal of basic and clinical oncology

基　　金：河南省科技英才海外研修工程项目(2018072)。

摘　　要：目的研究幽门螺杆菌(Hp)标准株Hp P12和其毒力因子空泡细胞毒素A(VacA)对人胃腺癌细胞DNA损伤、同源重组修复和细胞周期的影响。方法取对数生长期的人胃腺癌AGS细胞,应用Hp标准株(Hp P12)和其VacA基因敲除株(Hp P12ΔVacA)以感染复数(multiplicity of infection,MOI)100分别感染AGS细胞,使用Western Blot方法检测DNA损伤标记蛋白γH2AX、HR修复关键蛋白(Rad51、CtIP、pCtIP)、细胞周期检测点激酶2(pCHK2)的表达水平,进一步用流式细胞术检测细胞周期,验证Hp P12及其毒力因子VacA对HR修复和细胞周期的影响。结果DNA损伤标记蛋白γH2AX的表达,在Hp P12感染组较未感染组上调(6 h:t=4.870,P=0.001;12 h:t=4.118,P=0.003),在Hp P12ΔVacA感染组较Hp P12感染组下调(6 h:t=4.669,P=0.002;12 h:t=3.117,P=0.013)。Rad51、CtIP、pCtIP和pCHK2的表达在Hp P12感染12 h组较未感染组下调(Rad51:t=22.740,P<0.001;CtIP:t=31.970,P<0.001;pCtIP:t=3.777,P=0.020;pCHK2:t=10.740,P<0.001),而Rad51、pCHK2的表达在Hp P12ΔVacA感染12 h组均较Hp P12感染12 h组上调(Rad51:t=5.637,P=0.005;pCHK2:t=3.726,P=0.006)CtIP的表达在Hp P12ΔVacA感染6 h组较Hp P12感染6 h组上调(t=4.580,P=0.002)。进一步采用流式细胞术检测细胞周期发现Hp P12感染组G 1期细胞数较未感染组下调(t=11.530,P<0.001),S期细胞数上调(t=8.583,P=0.001)。而Hp P12ΔVacA感染组G 1期细胞数较Hp P12感染组上调(t=5.781,P=0.004),S期细胞数下调(t=5.481,P=0.005)。结论Hp P12标准株感染人胃腺癌细胞后引起DNA损伤,抑制了细胞HR修复,并引起细胞周期阻滞,其中VacA毒力因子起到了重要作用。Objective To study the effects of the standard strain of Helicobacter pylori(Hp)Hp P12 and its virulence factor vacuolating cytotoxin A(VacA)on DNA damage,homologous recombination(HR)repair and cell cycle in human gastric adenocarcinoma cells(AGS).Methods Hp P12 and its VacA gene knockout strain(Hp P12ΔVacA)were used to infect AGS cells which in the logarithmic growth phase with multiplicity of infection(MOI)100.Then western blot was used to detect the expression level of DNA damage marker proteinγH2AX,HR repair key protein(Rad51,CtIP,pCtIP),cell cycle detection point kinase 2(pCHK2).Flow cytometry was performed to detect cell cycle.The above methods were used to verify the effect of Hp P12 and its virulence factor VacA on HR repair and cell cycle.Results The expression of the DNA damage marker proteinγH2AX was increased in the Hp P12-infected cells compared with the non-infected cells(6 h:t=4.870,P=0.001;12 h:t=4.118,P=0.003)and reduced in the Hp P12ΔVacA-infected cells compared with the Hp P12-infected cells(6 h:t=4.669,P=0.002;12 h:t=3.117,P=0.013).The expression of Rad51,CtIP,pCtIP and pCHK2 were reduced(Rad51:t=22.740,P<0.001;CtIP:t=31.970,P<0.001;pCtIP:t=3.777,P=0.020;pCHK2:t=10.740,P<0.001)in the Hp P12-infected 12 h cells compared with the non-infected cells.The expression of Rad51 and pCHK2 were increased in Hp P12ΔVacA-infected 12 h cells compared with the Hp P12-infected cells(Rad51:t=5.637,P=0.005;pCHK2:t=3.726,P=0.006).The expression of CtIP were increased in the Hp P12ΔVacA-infected 6 h cells compared with the Hp P12-infected 6 h cells(t=4.580,P=0.002).Then flow cytometry was used to detect the cell cycle,and it was found that the number of G 1-phase cells in the Hp P12-infected cells was reduced than that in the non-infected cells(t=11.530,P<0.001),and the number of cells in the S-phase was increased(t=8.583,P=0.001).The number of cells in G 1-phase of Hp P12ΔVacA infected cells was increased than that of Hp P12-infected cells(t=5.781,P=0.004),and the number of cells in S-phase was reduced(t

关 键 词：幽门螺杆菌 同源重组修复 胃腺癌 空泡细胞毒素A 

分 类 号：R735.2[医药卫生—肿瘤]