摘要
目的探讨色素上皮衍生因子(pigment epithelium-derived factor,PEDF)对高糖刺激下视网膜Müller细胞凋亡的影响,为PEDF治疗糖尿病视网膜病变提供实验依据。方法实验分3组,分别是正常对照组、高糖模型(HG)组(培养基内含有25mmol·L-1的葡萄糖)和PEDF处理高糖模型(HG+PEDF)组(在高糖培养基础上加入25 mg·L-1的PEDF)。反复胰蛋白酶消化法培养纯化SD大鼠视网膜Müller细胞,免疫荧光双标鉴定纯度;应用倒置相差显微镜观察Müller细胞形态,AnnexinV-FITC、PI染色流式细胞技术检测Müller细胞的凋亡率,RT-PCR测定Müller细胞bcl-2、bax mRNA的表达变化。结果原代培养的Müller细胞传至3代时纯度为95%以上。正常对照组、HG组、HG+PEDF组Müller细胞早期凋亡率分别为(1.223±0.106)%、(2.657±0.120)%、(1.907±0.117)%,bax mRNA相对灰度值分别为0.269±0.041、0.804±0.052、0.528±0.018,bcl-2 mRNA相对灰度值分别为0.904±0.021、0.413±0.015、0.521±0.010。HG组与正常对照组相比,Müller细胞形态发生明显改变,细胞凋亡率显著增加(P<0.001),bax mRNA表达显著升高,bcl-2 mRNA表达显著降低(均为P<0.01);HG+PEDF组Müller细胞凋亡率较HG组显著降低(P<0.001),bax mRNA表达显著降低,bcl-2 mRNA表达显著升高(均为P<0.01),但仍有别于正常对照组(均为P<0.05)。结论外源给予PEDF可以显著减少高糖刺激引起的Müller细胞的凋亡,对视网膜Müller细胞具有显著的保护作用。
Objective To investigate the effects of pigment epithelium-derived factor（PEDF） on high glucose-stimulated rat retinal Mtiller cells apoptosis, and provide experimental basis for the treatment of diabetic retinopathy with PEDF. Methods The cells were divided into normal control group ,high glucose model（HG） group（ containing 25 mmol· L^- 1 glucose ill medium） and high glucose plus PEDF （ HG ＋ PEDF） group （ containing 25 mmol · L^- 1 glucose and 25 mg· L^- 1 PEDF in medium）. Repeated pancreatic enzyme-degisting method was used to culture and purify the retinal Mueller cells of SD rats. The purity was indentified by double-labeled immunofluorescence. The morphology was observed by confocalmicroscopy. The changes of Mtiller cells morphology and apoptosis were assayed with inverted phase contrast microscope, AnnexinV- FITC ＆ PI staining and flow cytometric, the expression of bcl-2 and bax mRNA were detected by RT-PCR. Results The purity of passage 3 retinal Mueller cells was above 95%. The apoptotic rate of MOiler ceils in normal control group ,HG group,HG ＋ PEDF group were（ 1. 223 ±0. 106 ） % , （ 2.657 ±0. 120 ） % , （ 1. 907 ±0. 117 ） % , respectively, the relative gray values of bax mRNA were 0. 269 ±0.041,0. 804 ±0. 052,0. 528 ±0.018, respectively,the relative gray values of bcl-2 mRNA were 0.904 ±0.021,0.413 ±0.015, 0. 521 ±0.010, respectively. Compared with normal control group, the morphology of retinal Mueller cells in HG group were changed, and the apoptotic rate was obviously increased（P 〈 0. 001 ）;The expression of bax mRNA in Mtiller cells was obviously increased,while bcl-2 mRNA was obviously decreased（ all P 〈 0.01 ） ;Compared with HG group,the apoptotic rate in HG ＋ PEDF group was obviously decreased（P 〈0. 001 ） ,the expression of bel-2 mRNA was significantly decreased, while bax mRNA was significantly increased （ all P 〈 0. 01 ）, but still had statistical difference compared with normal control group （ all P 〈 0.05 ）. Conclusion Giving exogenous PEDF can significantly reduce the high glucose-stimulated retinal Mtiller cells apoptosis, so PEDF has a significant protective effect on retinal Miiller cells.
出处
《眼科新进展》
CAS
北大核心
2014年第6期526-529,共4页
Recent Advances in Ophthalmology
