PI3K抑制剂LY294002对人晶状体上皮细胞增殖的影响

发布时间：2018-07-22 11:24

【摘要】： 前言后发性白内障是现代白内障手术后最常见的并发症,可引起视力的再次低下,又称后囊膜混浊(posterior capsular opacification, PCO)。白内障术后残留的晶状体上皮细胞(lens epithelial cells, LECs)发生增殖、迁移、纤维化生并伴随细胞外基质的分泌是PCO形成的主要原因,而晶状体上皮细胞的增殖最为关键。多年来国内外学者一直致力于通过药物等方法抑制晶状体上皮细胞增殖的研究。目前,信号转导通路是细胞增殖机制研究领域的热点,PI3K/AKT(phosphati-dylinositol-3 kinase/AKT,磷脂酰肌醇-3激酶／蛋白激酶B)信号通路对细胞的生长、存活、增殖及凋亡均有重要调控作用[3],而LY294002作为该信号通路的特异性抑制剂,能够抑制细胞的增殖。S期激酶相关蛋白2(S phase kinase associated protein2,Skp2)作为该通路下游的靶调控因子,能够介导细胞周期因子降解而调控细胞的增殖。本研究应用PI3K抑制剂LY294002处理体外培养的人晶状体上皮细胞株SRA01／04,观察其对细胞增殖的影响及研究可能的相关机制,为临床后发性白内障的预防和治疗提供新的理论依据。材料与方法一、材料人晶状体上皮细胞株SRA01／04由北京中国科学院肿瘤研究所提供。二、方法 1、细胞培养,人晶状体上皮细胞株SRA01／04在含10%胎牛血清的RPMI1640培养液中,置于37℃、5%CO2、饱和湿度细胞培养箱内传代培养。 2、细胞分组,实验组加入25μmol／L的P13K抑制剂LY294002,对照组加入等体积DMSO。 3、应用噻唑蓝(MTT)比色法检测PI3K抑制剂LY294002对人晶状体上皮细胞增殖的影响,绘制生长曲线,筛选LY294002的最佳作用时间。 4、应用流式细胞术(FCM)检测PI3K抑制剂LY294002对人晶状体上皮细胞周期的影响。 5、应用Western-blot法检测PI3K抑制剂LY294002对人晶状体上皮细胞Skp2蛋白表达的影响。 6、应用实时RT-PCR法检测PI3K抑制剂LY294002对人晶状体上皮细胞Skp2mRNA表达的影响。三、统计分析实验数据采用SPSS 11.5统计学软件进行分析。各组数据以x±s表示,统计学分析应用t检验,P0.05认为差异有统计学意义。结果 1、PI3K抑制剂LY294002抑制人晶状体上皮细胞的增殖 MTT法检测得出：实验组吸光度(A)值较对照组下降,差异有统计学意义(P0.05)。HLEC生长速率明显减慢,在12、24、36 h细胞生长抑制率分别为19.6%、33.8%和24.2%。 2、PI3K抑制剂LY294002阻滞人晶状体上皮细胞周期于G1／S期 FCM检测得出：实验组G1期细胞数(66.15+2.63)%与对照组G1期细胞数(56.73+1.35)%相比增多,差异有统计学意义(P0.05)；而实验组S期细胞数(27.34+6.58)%较对照组S期细胞数(37.32+5.54)%显著减少,差异有统计学意义(P0.05)。 3、PI3K抑制剂LY294002抑制人晶状体上皮细胞Skp2蛋白表达 Western blot法检测得出：实验组细胞Skp2蛋白表达量明显减少,与对照组比较,差异有统计学意义(P0.05)。 4、PI3K抑制剂LY294002抑制人晶状体上皮细胞Skp2 mRNA表达实时RT-PCR法检测得出：实验组细胞Skp2 mRNA的表达量降低,与对照组比较,差异有统计学意义(P0.05)。结论 1、PI3K抑制剂LY294002抑制人晶状体上皮细胞株SRA01／04的增殖。 2、LY294002抑制细胞中Skp2表达,使部分细胞阻滞于细胞周期G1／S期。
[Abstract]:Preface
Posterior capsular cataract is the most common complication after modern cataract surgery, which can cause the lower visual acuity, also called posterior capsular opacification (PCO). The residual lens epithelial cells (lens epithelial cells, LECs) after cataract surgery proliferate, migrate, fibrotic and accompany the secretion of extracellular matrix. It is the main reason for the formation of PCO, and the proliferation of lens epithelial cells is the most important. For many years, domestic and foreign scholars have been working on the study of inhibiting the proliferation of lens epithelial cells through drugs and other methods.
At present, signal transduction pathway is a hot spot in the research field of cell proliferation. PI3K/AKT (phosphati-dylinositol-3 kinase/AKT, phosphatidylinositol -3 kinase / protein kinase B) signaling pathway plays an important role in regulating cell growth, survival, proliferation and apoptosis, [3], and LY294002 as a specific inhibitor of this signal pathway can be inhibited. The proliferation of.S phase kinase related protein 2 (S phase kinase associated protein2, Skp2), as a target regulator in the downstream of the pathway, can mediate cell cycle factor degradation and regulate cell proliferation. This study applied PI3K inhibitor LY294002 to treat the cultured human crystalline epithelial cell lines in vitro, SRA01 / 04, to observe the proliferation of cells. The influence and research of possible mechanisms will provide a new theoretical basis for the prevention and treatment of posterior capsule opacification.
Materials and methods
First, material
Human lens epithelial cell line SRA01 / 04 was provided by the Institute of cancer research, Chinese Academy of Sciences, Beijing.
Two, method
1, cell culture, human lens epithelial cell line SRA01 / 04 was cultured in RPMI1640 culture medium containing 10% fetal bovine serum, and placed at 37, 5%CO2, saturated humidity cell culture box.
2, the cells were grouped, the experimental group was added with 25 mol / L P13K inhibitor LY294002, and the control group was added equal volume DMSO..
3, the effect of PI3K inhibitor LY294002 on the proliferation of human lens epithelial cells was detected by MTT colorimetric assay, and the growth curve was plotted and the optimal time of LY294002 was screened.
4, flow cytometry (FCM) was used to detect the effect of PI3K inhibitor LY294002 on the cell cycle of human lens epithelial cells.
5, Western-blot method was used to detect the effect of PI3K inhibitor LY294002 on the expression of Skp2 protein in human lens epithelial cells.
6, real-time RT-PCR assay was used to detect the effect of PI3K inhibitor LY294002 on Skp2mRNA expression in human lens epithelial cells.
Three, statistical analysis
The experimental data were analyzed by SPSS 11.5 statistical software. The data of each group were expressed as x + s, and t analysis was used for statistical analysis. P0.05 showed that the difference was statistically significant.
Result
1, PI3K inhibitor LY294002 inhibits proliferation of human lens epithelial cells.
The MTT method showed that the absorbance (A) value of the experimental group was lower than that of the control group. The difference was statistically significant (P0.05), the growth rate of.HLEC was significantly slowed, and the growth inhibition rate of 12,24,36 h cells was 19.6%, 33.8% and 24.2%., respectively.
2, PI3K inhibitor LY294002 blocks human lens epithelial cell cycle in the G1 / S phase.
FCM detection showed that the number of cells in G1 phase (66.15+2.63) in the experimental group was more than that of the control group (56.73+1.35)%, and the difference was statistically significant (P0.05), while the number of S phase (27.34+6.58)% in the experimental group was significantly lower than that in the control group (37.32+5.54)%, and the difference was statistically significant (P0.05).
3, PI3K inhibitor LY294002 inhibits the expression of Skp2 protein in human lens epithelial cells.
Western blot assay showed that the expression of Skp2 protein in the experimental group was significantly decreased compared with that in the control group, and the difference was statistically significant (P0.05).
4, PI3K inhibitor LY294002 inhibits the expression of Skp2 mRNA in human lens epithelial cells.
Real time RT-PCR assay showed that the expression of Skp2 mRNA in experimental group decreased, and the difference was statistically significant compared with the control group (P0.05).
conclusion
1, PI3K inhibitor LY294002 inhibits the proliferation of human lens epithelial cell line SRA01 / 04.
2, LY294002 inhibited the expression of Skp2 in cells and blocked some cells in the G1 / S phase.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R776.1

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