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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 effect of pigment epithelium-derived factor (PEDF) on the apoptosis of retinal Müller cells stimulated by high glucose, and to provide an experimental basis for the treatment of diabetic retinopathy with PEDF. Methods The experiment was divided into three groups: normal control group, high glucose group (HG group) (medium containing 25mmol·L-1 glucose) and PEDF-treated high glucose group (HG + PEDF group) Add 25 mg · L-1 of PEDF). Retinal Müller cells were cultured and purified by trypsin digestion. Immunofluorescence was used to identify the purity of Müller cells. Morphological changes of Müller cells were observed by inverted phase contrast microscope. Annexin V-FITC and PI staining were used to detect the apoptosis rate of Müller cells. RT- PCR was used to detect the expression of bcl-2 and bax mRNA in Müller cells. Results The primary cultured Müller cells reached 95% purity at the third passage. The early apoptotic rates of Müller cells in normal control, HG and HG + PEDF groups were (1.223 ± 0.106)% and (2.657 ± 0.120)%, (1.907 ± 0.117)% and the relative gray values of bax mRNA were 0.269 ± 0.041,0.804 ± 0.052,0.528 ± 0.018, the relative gray values of bcl-2 mRNA were 0.904 ± 0.021, 0.413 ± 0.015 and 0.521 ± 0.010, respectively. Compared with the normal control group, the morphological changes of Müller cells in HG group were significantly changed, the apoptosis rate was significantly increased (P <0.001), bax mRNA expression was significantly increased, and bcl-2 mRNA expression was significantly decreased (all P <0.01). The apoptotic rate of Müller cells in HG + PEDF group was significantly lower than that in HG group (P <0.001), the expression of bax mRNA was significantly decreased and the expression of bcl-2 mRNA was significantly increased (all P <0.01), but still different from the normal control group (All P <0.05). Conclusion Exogenous administration of PEDF can significantly reduce the apoptosis of Müller cells induced by high glucose and has a significant protective effect on retinal Müller cells.