ILK对幽门螺杆菌诱导胃上皮细胞GES-1凋亡的影响
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摘要
目的探讨ILK在幽门螺杆菌诱导胃上皮细胞GES-1凋亡中的作用。方法体外培养GES-1细胞,随机分为空白对照组、Cpd22组和Hp组。采用罗丹明-鬼笔环肽检测不同浓度ILK抑制剂Cpd22对胃上皮细胞形态及细胞骨架改变的影响;Cpd22作用于GES-1细胞1h后,Western blot检测细胞内ILK表达情况;Hp与Cpd22作用1h的GES-1细胞共培养(MOI=100∶1)24h后,采用流式细胞术检测细胞凋亡率,Western blot检测凋亡相关蛋白Caspase-3、PARP表达情况。结果与空白对照组相比,Cpd22浓度为2μmol/L时细胞形态变圆,间隙变大,细胞外缘伪足减少,细胞无法正常伸展。空白对照组与Cpd22组ILK相对表达量分别为0.83±0.01、0.74±0.01,比较差异有统计学意义(t=5.65,P<0.05)。空白对照组、Cpd22组、Hp组细胞凋亡率分别为(6.53±2.78)%、(21.13±5.60)%、(14.07±3.97)%,比较差异有统计学意义(t值分别为-5.52和-6.83,P<0.05);Caspase-3相对表达量分别为0.63±0.07、0.95±0.14、0.88±0.11,差异均有统计学意义(t值分别为-5.44和-5.88,P<0.05);PARP的相对表达量分别为1.17±0.07、0.93±0.16、1.09±0.12,差异有统计学意义(t值分别为3.32和3.46,P<0.05)。Cpd22与Hp共作用组细胞凋亡率为(30.37±5.17)%,Caspase-3的相对表达量为1.07±0.10,PARP的相对表达量为0.86±0.07,与Cpd22组比较差异均有统计学意义(t值分别为4.90、2.85和6.70,P<0.05)。结论抑制ILK表达可增强Hp诱导GES-1细胞凋亡的作用,其机制可能与细胞骨架重排和Caspase-3的表达有关。
Objective To examine the role of ILK in the apoptosis of GES-1 gastric epithelial cells induced by Helicobacter pylori. Methods GES-1 cells were cultured in vitro and randomly divided into a blank control group,cells treated with Cpd22,and cells co-cultured with H.pylori.The effects of different concentrations of the ILK inhibitor Cpd22 on the morphology and cytoskeleton of gastric epithelial cells were detected using rhodamine phalloidin.GES-1 cells were treated with Cpd22 for 1 h,and the expression of ILK in cells was detected using Western blotting.GES-1 cells were cocultured with H.pylori(MOI=100:1)and treated with Cpd22 for 1 h.Twenty-four h later,the rate of apoptosis was detected using flow cytometry,and expression of the apoptosis-related proteins Caspase-3 and PARP was detected using Western blotting. Results Compared to the blank control group,when the concentration of Cpd22 was 2μmol/L,the cell morphology was rounder,gaps grew,there were fewer pseudopods on the outer edge of cells,and the cells did not spread normally.The relative expression of ILK was 0.83±0.01 in the blank control group and 0.74±0.01 in cells treated with Cpd22.The level of that relative expression differed significantly in the 2 groups(t=5.65,P<0.05).The rate of apoptosis was(6.53±2.78)%in the blank control group,(21.13±5.60)%in cells treated with Cpd22,and(14.07±3.97)%in cells co-cultured with H.pylori.The rate of apoptosis differed significantly among the groups(t=-5.52,-6.83,P<0.05).The relative expression of Caspase-3 was 0.63±0.07 in the blank control group,0.95±0.14 in cells treated with Cpd22,and 0.88±0.11 in cells co-cultured with H.pylori.The level of that relative expression differed significantly among the groups(t=-5.44 and-5.88;P<0.05).The relative expression of PARP was 1.17±0.07 in the blank control group,0.93±0.16 in cells treated with Cpd22,and 1.09±0.12 in cells co-cultured with H.pylori.The level of that relative expression differed significantly among the groups(t=3.32 and 3.46;P<0.05).Therate of apoptosis was(30.37±5.17)%in the Cpd22 and H.pylori co-acting group.The relative expression of Caspase-3 was 1.07±0.10,and the relative expression of PARP was 0.86±0.07,and those levels of relative expression differed significantly from those in cells treated with Cpd22(t= 4.90,2.85,and 6.70,respectively,P<0.05). Conclusion Inhibiting the expression of ILK can enhance the apoptosis of GES-1 cells induced by H.pylori,and this may be related to cytoskeletal rearrangement and expression of Caspase-3.
Objective To examine the role of ILK in the apoptosis of GES-1 gastric epithelial cells induced by Helicobacter pylori. Methods GES-1 cells were cultured in vitro and randomly divided into a blank control group,cells treated with Cpd22,and cells co-cultured with H.pylori.The effects of different concentrations of the ILK inhibitor Cpd22 on the morphology and cytoskeleton of gastric epithelial cells were detected using rhodamine phalloidin.GES-1 cells were treated with Cpd22 for 1 h,and the expression of ILK in cells was detected using Western blotting.GES-1 cells were cocultured with H.pylori(MOI=100:1)and treated with Cpd22 for 1 h.Twenty-four h later,the rate of apoptosis was detected using flow cytometry,and expression of the apoptosis-related proteins Caspase-3 and PARP was detected using Western blotting. Results Compared to the blank control group,when the concentration of Cpd22 was 2μmol/L,the cell morphology was rounder,gaps grew,there were fewer pseudopods on the outer edge of cells,and the cells did not spread normally.The relative expression of ILK was 0.83±0.01 in the blank control group and 0.74±0.01 in cells treated with Cpd22.The level of that relative expression differed significantly in the 2 groups(t=5.65,P<0.05).The rate of apoptosis was(6.53±2.78)%in the blank control group,(21.13±5.60)%in cells treated with Cpd22,and(14.07±3.97)%in cells co-cultured with H.pylori.The rate of apoptosis differed significantly among the groups(t=-5.52,-6.83,P<0.05).The relative expression of Caspase-3 was 0.63±0.07 in the blank control group,0.95±0.14 in cells treated with Cpd22,and 0.88±0.11 in cells co-cultured with H.pylori.The level of that relative expression differed significantly among the groups(t=-5.44 and-5.88;P<0.05).The relative expression of PARP was 1.17±0.07 in the blank control group,0.93±0.16 in cells treated with Cpd22,and 1.09±0.12 in cells co-cultured with H.pylori.The level of that relative expression differed significantly among the groups(t=3.32 and 3.46;P<0.05).Therate of apoptosis was(30.37±5.17)%in the Cpd22 and H.pylori co-acting group.The relative expression of Caspase-3 was 1.07±0.10,and the relative expression of PARP was 0.86±0.07,and those levels of relative expression differed significantly from those in cells treated with Cpd22(t= 4.90,2.85,and 6.70,respectively,P<0.05). Conclusion Inhibiting the expression of ILK can enhance the apoptosis of GES-1 cells induced by H.pylori,and this may be related to cytoskeletal rearrangement and expression of Caspase-3.
引文
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[6]Puente PDL,Weisberg E,Muz B,et al.Identification of ILK as a novel therapeutic target for acute and chronic myeloid leukemia[J].Leuk Res,2015,39(11):1299-308.
[7]Kudryashova T V,Goncharov D A,Pena A,et al.HIPPO-Integrin-linked kinase cross-talk controls self-sustaining proliferation and survival in pulmonary hypertension[J].Am J RespirCrit Care Med,2016,194(7):866-77.
[8]Fukuda K,Gupta S,Chen K,et al.The pseudo-active site of ILK is essential for its binding toα-parvin and localization to focal adhesions[J].Mol Cell,2009,36(5):819-30.
[9]Hussain R,Macklin WB.Integrin-linked kinase(ILK)deletion disrupts oligodendrocyte development by altering cell cycle[J].J Neuro Sci Offic,2016,37(2):397-412.
[10]Tan C,Cruethennequart S,Troussard A,et al.Regulation of tumor angiogenesis by integrin-linked kinase(ILK)[J].Cancer Cell,2004,5(1):79-90.
[11]Mamuya FA,Canope?alver JL,Li WU,et al.ILK and cytoskeletal architecture:an important determinant of AQP2Recycling and Subsequent Entry into the Exocytotic Pathway[J].Am J Physiol Renal Physiol,2016,311(6):1346-57.
[12]Persad S,Attwell S,Gray V,et al.Inhibition of integrin-linked kinase(ILK)suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells[J].Proc Natl Acad Sci U S A,2000,97(7):3207-12.
[13]吕磊,曾甫清,朱朝辉,等.siRNA特异性沉默整合素连接激酶对前列腺癌PC3细胞凋亡的影响[J].中国肿瘤,2009,18(11):908-11.
[2]刘海峰,刘为纹,房殿春.幽门螺杆菌感染对胃粘膜上皮细胞增殖和凋亡的影响[J].中华消化内镜杂志,1998,15(2):91-3.
[3]胡伏莲.中国幽门螺杆菌研究现状[J].胃肠病学,2007,12(9):516-8.
[4]Dong Z,Pan W,Wu H,et al.Activated integrin-linked kinase negatively regulates muscle cell enhancement factor 2Cin C2C12cells[J].Biomed Res Int,2015,2015(1):748470.
[5]李宁,李亚斐,吴雄飞.整合素连接激酶生物学功能研究进展[J].生命的化学,2016(3):385-9.
[6]Puente PDL,Weisberg E,Muz B,et al.Identification of ILK as a novel therapeutic target for acute and chronic myeloid leukemia[J].Leuk Res,2015,39(11):1299-308.
[7]Kudryashova T V,Goncharov D A,Pena A,et al.HIPPO-Integrin-linked kinase cross-talk controls self-sustaining proliferation and survival in pulmonary hypertension[J].Am J RespirCrit Care Med,2016,194(7):866-77.
[8]Fukuda K,Gupta S,Chen K,et al.The pseudo-active site of ILK is essential for its binding toα-parvin and localization to focal adhesions[J].Mol Cell,2009,36(5):819-30.
[9]Hussain R,Macklin WB.Integrin-linked kinase(ILK)deletion disrupts oligodendrocyte development by altering cell cycle[J].J Neuro Sci Offic,2016,37(2):397-412.
[10]Tan C,Cruethennequart S,Troussard A,et al.Regulation of tumor angiogenesis by integrin-linked kinase(ILK)[J].Cancer Cell,2004,5(1):79-90.
[11]Mamuya FA,Canope?alver JL,Li WU,et al.ILK and cytoskeletal architecture:an important determinant of AQP2Recycling and Subsequent Entry into the Exocytotic Pathway[J].Am J Physiol Renal Physiol,2016,311(6):1346-57.
[12]Persad S,Attwell S,Gray V,et al.Inhibition of integrin-linked kinase(ILK)suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells[J].Proc Natl Acad Sci U S A,2000,97(7):3207-12.
[13]吕磊,曾甫清,朱朝辉,等.siRNA特异性沉默整合素连接激酶对前列腺癌PC3细胞凋亡的影响[J].中国肿瘤,2009,18(11):908-11.