整合素β1通过ILK/FAK/F-actin通路影响幽门螺杆菌诱导的GES-1细胞自噬的研究  

Study on the effect of integrinβ1 on autophagy induced by Helicobacter pylori via the ILK/FAK/F-actin pathway

作　　者：杜运秋 张锐清 曲磊[1] 程雪 吴玉龙[1] 赵慧琳[1] 张莹[1] 李波清[1] DU Yun-qiu;ZHANG Rui-qing;QU Lei;CHENG Xue;WU Yu-long;ZHAO Hui-lin;ZHANG Ying;LI Bo-qing(Department of Pathogen Biology,Binzhou Medical University,Yantai,Shandong,China 264003)

机构地区：[1]滨州医学院病原生物学教研室,山东烟台264003

出　　处：《中国病原生物学杂志》2020年第9期1028-1032,1037,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81771709);山东省高等学校青创科技支持计划项目(No.2019KJK012)。

摘　　要：目的观察整合素β1对ILK/FAK/F-actin通路的调节作用,分析其对幽门螺杆菌(Hp)诱导GES-1细胞自噬的影响。方法利用siRNA沉默GES-1细胞整合素β1的表达后与幽门螺杆菌共培养6 h,Western blot检测ILK的表达。以ILK特异性抑制剂Cpd22抑制细胞ILK的表达后与幽门螺杆菌共培养6 h,Western blot检测LC3B、p62、FAK、Paxillin的表达;制备细胞爬片,以iFluor-594标记的鬼笔环肽对F-actin进行染色,观察细胞形态。结果沉默整合素β1后,空白组、Hp组、沉默组、阴性对照组、沉默+Hp组、阴性对照+Hp组ILK相对表达量分别为1.012±0.073、0.637±0.216、0.727±0.173、1.173±0.177、0.712±0.160、0.983±0.119,与空白组相比,Hp组ILK的相对表达量降低(t=3.288,P<0.05),沉默组较阴性对照组,沉默+Hp组较阴性对照+Hp组ILK相对表达量均显著降低(t值分别为-4.420和-3.238,均P<0.01)。抑制ILK表达后,空白组、Cpd22组、Hp组、Cpd22+Hp组LC3B的相对表达量分别为0.437±0.064、0.808±0.116、0.812±0.130、1.247±0.201,p62相对表达量分别为0.904±0.141、1.121±0.105、1.392±0.110、0.960±0.103,与空白组相比,Cpd22和Hp组细胞内LC3B及p62相对表达量均显著增加(t值分别为-4.838、-4.479、-3.269、3.313,均P<0.01),Cpd22+Hp组较Hp组细胞LC3B的相对表达量增高(t=-3.147,P<0.05),p62相对表达量降低(t=2.907,P<0.05)。Cpd22组FAK、Paxillin相对表达量较空白组均增加(t值分别为-2.303、-3.460,均P<0.05),Cpd22+Hp组FAK、Paxillin相对表达量较Hp组均降低(t值分别为2.446、-2.648,P<0.05),Cpd22抑制ILK后感染Hp,细胞F-actin染色呈现明显杂乱碎丝状。结论整合素β1通过上调ILK的表达抑制幽门螺杆菌诱导的GES-1细胞自噬,其机制可能与FAK/Paxillin/F-actin通路有关。Objective To observe the regulatory effect of integrinβ1 on the ILK/FAK/F-actin pathway and analyze its effect on Helicobacter pylori-induced autophagy in GES-1 cells.Methods SiRNA was used to knock down the expression of Integrinβ1 in GES-1 cells that were co-cultured with H.pylori for 6 h.Western blotting was used to detect the changes in ILK protein expression.Cpd22,an inhibitor of ILK,was used to inhibit the expression of ILK in cells that were co-cultured with H.pylori for 6 h.Western blotting was used to detect changes in expression of the proteins LC3 B,p62,FAK,and Paxillin.Cell climbing slices were prepared and F-actin was stained with iFluor-594-labeled ghost pen cyclic peptide.Results After silencing Integrinβ1,the relative expression of ILK was 1.012±0.073 in the blank group,0.637±0.216 in the H.pylori group,0.727±0.173 in the silent group,1.173±0.177 in the negative group,0.712±0.160 in the silent+H.pylori group,and 0.983±0.119 in the negative+H.pylori group.The relative expression of ILK in the H.pylori group decreased compared to that in the blank group(t=3.288,P<0.05).The relative expression of ILK decreased significantly in the silent group compared to the negative group,the negative+H.pylori group,and the silent+H.pylori group(t values were-5.043,-3.553,-4.420,-3.238,P<0.01,P<0.05).After inhibiting the expression of ILK,the relative level of LC3 B-II expression was 0.437±0.064 in the blank group,0.808±0.116 in the Cpd22 group,0.812±0.130 in the H.pylori group,and 1.247±0.201 in the Cpd22+H.pylori group.The relative level of p62 expression was 0.904±0.141 in the blank group,1.121±0.105 in the Cpd22 group,1.392±0.110 in the H.pylori group,and 0.960±0.103 in the Cpd22+H.pylori group.The relative levels of expression of the LC3 B and p62 proteins in the Cpd22 group and H.pylori group were significantly higher than those in the blank group(t values:-4.338,-4.479,-3.269,and 3.313;P<0.01).The relative level of LC3 B expression in the Cpd22+H.pylori group was higher than that in the H.py

关 键 词：幽门螺杆菌 整合素Β1 GES-1细胞 整合素连接激酶 细胞自噬 

分 类 号：R378[医药卫生—病原生物学]