表皮生长因子对POAG小梁网细胞增殖和凋亡的影响及其与高眼压形成的关系

发布时间：2018-10-13 11:07

【摘要】：目的探讨体外培养原发性开角型青光眼(POAG)小梁网细胞的培养方法及其生物学特性,并在此基础上研究原发性开角型青光眼小梁网细胞是否表达表皮生长因子(EGF)及其受体(EGFR),EGF对POAG小梁网细胞增殖和凋亡的影响及EGF对小梁网细胞表达EGFR的影响。方法采用组织块培养法进行POAG小梁网细胞的体外培养,用倒置显微镜、透射电镜、免疫细胞化学法染色等方法观察体外培养的POAG小梁网细胞生物学特性并进行细胞鉴定。采用ELISA法检测体外培养的POAG小梁网细胞是否表达EGF及EGFR;采用CCK-8法和流式细胞术检测体外培养的第5代小梁网细胞经EGF终浓度分别为0ng/ml(对照组),5ng/ml,10ng/ml,20ng/ml,50ng/ml和100ng/ml的DMEM/F12处理48小时后小梁网细胞的增殖率和凋亡率;ELISA法检测小梁网细胞经EGF终浓度为0ng/ml,50ng/ml的DMEM/F12处理48小时后小梁网细胞EGFR的表达。结果 1.组织块培养10天左右可见细胞从其边缘向外生长,透射电镜、免疫细胞化学染色证明其为POAG小梁网细胞。 2.ELISA检测结果显示小梁网细胞表达EGF和EGFR,其ELISA检测的吸光度值(OD值)分别为0.13、0.33,计算出POAG小梁网细胞培养上清液中EGF及EGFR的表达量分别为1.25ng/ml、0.89ng/ml。 3.运用CCK-8法对经EGF终浓度为0g/ml,5ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/ml的DMEM/F12处理的实验组细胞进行检测,其吸光度值(OD)分别为(0.75±0.05),(0.80±0.07),(0.84±0.05),(0.89±0.02),(0.77±0.04),与对照组(0.66±0.05)相比,差异有统计学意义(P0.05)。 4.运用流式细胞仪对经EGF终浓度为0 ng/ml(对照组),5ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/ml的DMEM/F12处理48h的细胞进行检测,小梁网细胞的凋亡率分别为(16.14±0.44)%,(14.22±0.18)%,(10.90±0.49)%,(5.98±0.14)%,(5.58±0.21)%,(9.93±0.17)%,各浓度组与阴性对照组比较,凋亡率下降具有统计学意义(P0.05)。 5.经EGF终浓度为50ng/m的DMEM/F12处理的细胞EGFR的表达量的OD值为(0.20±0.01),与阴性对照组的OD值(0.17±0.01)相比,差异具有统计学意义(P0.05)。结论 l.掌握POAG小梁网细胞的体外细胞培养技术及其生长特点、超微结构,能够成功地进行POAG小梁网细胞的体外培养及鉴定。 2.体外培养的POAG小梁网细胞上清液中表达EGF及EGFR。 3.EGF能促进小梁网细胞的增殖,减缓其凋亡,并在一定的范围内呈现剂量依赖性。 4.EGF能够促进小梁网细胞表达EGFR。
[Abstract]:Objective to investigate the culture methods and biological characteristics of (POAG) trabecular meshwork cells in primary open-angle glaucoma in vitro. The effects of EGF (EGF) and its receptor (EGFR), EGF on the proliferation and apoptosis of POAG trabecular reticulum cells and the effect of EGF on the expression of EGFR in trabecular meshwork cells of primary open-angle glaucoma were studied. Methods POAG trabecular meshwork cells were cultured in vitro by tissue mass culture. The biological characteristics of POAG trabecular meshwork cells cultured in vitro were observed and identified by inverted microscope, transmission electron microscope and immunocytochemical staining. ELISA assay was used to detect the expression of EGF in cultured POAG trabecular meshwork cells and EGFR; was used to detect the final EGF concentration of the 5th generation trabecular meshwork cells cultured in vitro by CCK-8 and flow cytometry. The final concentrations of EGF were 0ng/ml (control group), 5 ng / ml 10 ng / ml and 50 ng / ml 100ng/ml DMEM/F12 respectively. ELISA assay was used to detect the expression of EGFR in trabecular meshwork cells treated with DMEM/F12 of 0 ng / ml 50 ng / ml for 48 hours. Result 1. About 10 days after the tissue mass was cultured, the cells grew from the edge to the outside, and transmission electron microscopy (TEM) was used. Immunocytochemical staining showed that the cells were POAG trabecular meshwork cells. 2.ELISA showed that trabecular meshwork cells expressed EGF and EGFR, their ELISA absorbance values (OD) were 0.13 ~ 0.33, respectively. The expression of EGF and EGFR were 1.25 ng 路ml ~ (-1) 路ml ~ (-1) and 0.89 ng 路ml ~ (-1) 路ml ~ (-1), respectively. CCK-8 method was used to detect the cells of the experimental group treated with 10 ng / ml DMEM/F12 of 0 g / ml 10 ng / ml DMEM/F12. The absorbance (OD) of the experimental group was (0.75 卤0.05), (0.80 卤0.07), (0.84 卤0.05), (0.89 卤0.02), (0.77 卤0.04), which was significantly higher than that of the control group (0.66 卤0.05) (P 0.05), and the mean value of (OD) was 0.75 卤0.05), (0.80 卤0.07), (0.84 卤0.05), (0.89 卤0.02), (0.77 卤0.04, compared with the control group (0.66 卤0.05). The apoptosis rate of trabecular meshwork cells was (16.14 卤0.44)%, (14.22 卤0.18)%, (10.90 卤0.49)%, (5.98 卤0.14)%, (5.58 卤0.21)%, (9.93 卤0.17)%, respectively. The apoptosis rates of cells treated with DMEM/F12 of 50 ng / ml DMEM/F12 for 48 h were (16.14 卤0.44)%, (14.22 卤0.18)%, (10.90 卤0.49)%, (5.98 卤0.14)%, (5.58 卤0.21)%, (9.93 卤0.17)%, respectively. The decrease of apoptosis rate was statistically significant (P0.05). The OD value of EGFR expression in DMEM/F12 treated with 50ng/m was (0.20 卤0. 01), which was significantly higher than that in negative control group (0. 17 卤0. 01) (P0.05). Conclusion _ _ _. The in vitro culture technique and growth characteristics and ultrastructure of POAG trabecular meshwork cells can be successfully carried out in vitro culture and identification of POAG trabecular meshwork cells. 2. The expression of EGF and EGFR. 3.EGF in the supernatant of POAG trabecular meshwork cells in vitro can promote the proliferation of trabecular meshwork cells and slow down their apoptosis. 4.EGF can promote the expression of EGFR. in trabecular meshwork cells.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R775

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