PI3K-AKT信号通路在PDGF促进人晶状体上皮细胞增殖中的作用
发布时间：2018-08-03 21:46
【摘要】：目的:后发性白内障(posterior capsule opacification,PCO)也称后囊膜混浊,是白内障术后及晶状体外伤后最常见的并发症。研究表明残留的晶状体上皮细胞增殖在PCO的发病机制中发挥着十分重要的作用,但其增殖的具体原理尚未完全阐明,本研究探讨体外应用血小板源性生长因子(platelet derived growth factor,PDGF)及PI3K(phosphatidylinositols-kinase)信号通路抑制剂LY294002分别或同时刺激人源晶状体上皮细胞(lens epithelial cells,LECs)株SRA01/04,以探讨PI3K-AKT信号通路在PDGF促进人晶状体上皮细胞增殖中的作用。方法:人晶状体上皮细胞株SRA01/04常规传代培养,用不同浓度的PDGF(0ng/ml,0.1ng/ml,1ng/ml,10ng/ml,100ng/ml),不同浓度LY294002(0μM,5μM,10μM,20μM,40μM)以及不同浓度的PDGF(10ng/ml)+LY294002(0μM,5μM,10μM,20μM,40μM)分别作用于SRA01/04细胞24小时,MTT法检测各组细胞的增殖率或增殖抑制率;根据MTT结果,选定各药物组浓度,将实验分为四组,即空白对照组、PDGF组(PDGF浓度为10ng/ml)、LY294002组(LY294002浓度为40μM)、PDGF+LY294002组(10ng/ml的PDGF+40μM的LY294002),各组细胞培养24h,倒置显微镜下观察各组晶状体上皮细胞增殖、抑制的情况;蛋白印迹法检测各组细胞中总AKT及P-AKT的蛋白表达量差异。结果:1、人晶状体上皮细胞SRA01/04的增殖率与PDGF的浓度呈正相关(P0.001);2、人晶状体上皮细胞SRA01/04的增殖的抑制率与LY294002的浓度呈正相关(P0.001);3、PDGF促人晶状体上皮细胞SRA01/04的增殖作用与LY294002的浓度呈负相关(P0.001);4、PDGF、LY294002分别或同时干预人晶状体上皮细胞SRA01/04时细胞总AKT表达无明显差异(P=0.066);5、PDGF单独干预人晶状体上皮细胞SRA01/04时细胞p-AKT表达量增加,差异有统计学意义(P0.001);6、LY294002单独干预人晶状体上皮细胞SRA01/04时细胞p-AKT表达量减少,差异有统计学意义(P0.001);7、PDGF和LY294002同时干预人晶状体上皮细胞SRA01/04时细胞p-AKT表达量较PDGF单独干预时减少而较LY294002单独干预时增加,差异有统计学意义(P0.001)。结论:1、PDGF能促进人晶状体上皮细胞的增殖;2、LY294002能抑制人晶状体上皮细胞的增殖;3、PDGF促进人晶状体上皮细胞的增殖作用能被LY294002抑制;4、PDGF通过激活PI3K/AKT信号通路促进人晶状体上皮细胞的增殖。
[Abstract]:Objective: posterior capsular opacification (posterior capsule) is the most common complication after cataract surgery and lens trauma. Studies have shown that the residual lens epithelial cell proliferation plays a very important role in the pathogenesis of PCO, but the specific mechanism of its proliferation has not been fully clarified. In this study, we used platelet-derived growth factor (platelet derived growth factor-PDGF (PDGF) and PI3K (phosphatidylinositols-kinase) signal pathway inhibitor LY294002 to stimulate human lens epithelial cell line SRA01 / 04, respectively or simultaneously, in order to explore the effect of PI3K-AKT signaling pathway on the promotion of PDGF on human lens. The role of skin cell proliferation. Methods: human lens epithelial cell line SRA01/04 was subcultured routinely. SRA01/04 cells were treated with different concentrations of PDGF (0 ng / ml 0.1 ng / ml 10 ng / ml 10ng / ml), different concentrations of LY294002 (0 渭 M 5 渭 M 10 渭 M ~ 10 渭 M) and different concentrations of PDGF (10ng/ml) LY294002 (0 渭 M 5 渭 M 5 渭 M 10 渭 M 10 渭 M 10 渭 M 40 渭 M) for 24 hours respectively to detect the proliferation rate or inhibition rate of SRA01/04 cells. Each drug group was selected and divided into four groups: the blank control group (10ng/ml) (LY294002 40 渭 M) and PDGF LY294002 (40 渭 M). The cells were cultured for 24 hours. The proliferation and inhibition of lens epithelial cells in each group were observed under inverted microscope. The protein expression of total AKT and P-AKT was detected by Western blot. Results the proliferation rate of SRA01/04 in human lens epithelial cells was positively correlated with the concentration of PDGF (P0.001) and the inhibitory rate of SRA01/04 proliferation in human lens epithelial cells was positively correlated with the concentration of LY294002 (P0.001). There was no significant difference (P0. 066) in the total AKT expression of human lens epithelial cells (P0. 066). P0. 066 (P0. 066) P0. 05 PDGF alone interfered with the increase of p-AKT expression in SRA01/ 04:00 cells of human lens epithelial cells. The difference was statistically significant (P0.001). LY294002 alone interfered with the decrease of p-AKT expression in SRA01/ 04:00 cells of human lens epithelial cells. The difference was statistically significant (P0. 001). The p-AKT expression of SRA01/ 04:00 cells in human lens epithelial cells treated with P0. 001 PDGF and LY294002 at the same time was lower than that with PDGF alone and increased compared with LY294002 alone. The difference was statistically significant (P0. 001). Conclusion LY294002 can inhibit the proliferation of human lens epithelial cells by activating PI3K/AKT signaling pathway.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R776.1
本文编号：2163087
[Abstract]:Objective: posterior capsular opacification (posterior capsule) is the most common complication after cataract surgery and lens trauma. Studies have shown that the residual lens epithelial cell proliferation plays a very important role in the pathogenesis of PCO, but the specific mechanism of its proliferation has not been fully clarified. In this study, we used platelet-derived growth factor (platelet derived growth factor-PDGF (PDGF) and PI3K (phosphatidylinositols-kinase) signal pathway inhibitor LY294002 to stimulate human lens epithelial cell line SRA01 / 04, respectively or simultaneously, in order to explore the effect of PI3K-AKT signaling pathway on the promotion of PDGF on human lens. The role of skin cell proliferation. Methods: human lens epithelial cell line SRA01/04 was subcultured routinely. SRA01/04 cells were treated with different concentrations of PDGF (0 ng / ml 0.1 ng / ml 10 ng / ml 10ng / ml), different concentrations of LY294002 (0 渭 M 5 渭 M 10 渭 M ~ 10 渭 M) and different concentrations of PDGF (10ng/ml) LY294002 (0 渭 M 5 渭 M 5 渭 M 10 渭 M 10 渭 M 10 渭 M 40 渭 M) for 24 hours respectively to detect the proliferation rate or inhibition rate of SRA01/04 cells. Each drug group was selected and divided into four groups: the blank control group (10ng/ml) (LY294002 40 渭 M) and PDGF LY294002 (40 渭 M). The cells were cultured for 24 hours. The proliferation and inhibition of lens epithelial cells in each group were observed under inverted microscope. The protein expression of total AKT and P-AKT was detected by Western blot. Results the proliferation rate of SRA01/04 in human lens epithelial cells was positively correlated with the concentration of PDGF (P0.001) and the inhibitory rate of SRA01/04 proliferation in human lens epithelial cells was positively correlated with the concentration of LY294002 (P0.001). There was no significant difference (P0. 066) in the total AKT expression of human lens epithelial cells (P0. 066). P0. 066 (P0. 066) P0. 05 PDGF alone interfered with the increase of p-AKT expression in SRA01/ 04:00 cells of human lens epithelial cells. The difference was statistically significant (P0.001). LY294002 alone interfered with the decrease of p-AKT expression in SRA01/ 04:00 cells of human lens epithelial cells. The difference was statistically significant (P0. 001). The p-AKT expression of SRA01/ 04:00 cells in human lens epithelial cells treated with P0. 001 PDGF and LY294002 at the same time was lower than that with PDGF alone and increased compared with LY294002 alone. The difference was statistically significant (P0. 001). Conclusion LY294002 can inhibit the proliferation of human lens epithelial cells by activating PI3K/AKT signaling pathway.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R776.1
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