高糖对牛眼小梁细胞形态、增殖及凋亡的影响

Effect of high glucose on morphology, proliferation and apoptosis in cultured bovine trabecular meshwork cells

目的:建立牛眼小梁细胞体外培养体系,观察高浓度葡萄糖对小梁细胞的影响,研究高糖与开角型青光眼(primary open angle glaucoma,POAG)以及糖尿病与POAG之间的关系,探讨糖尿病开角型青光眼的发病机制,从而为指导开角型青光眼的临床用药提供重要依据。方法:以新鲜牛眼为材料,分离并培养小梁细胞,用光学镜、电镜观察正常小梁细胞的形态。将传至第3代的小梁细胞制作成细胞悬液、计数并接种于培养板(或培养瓶)中,待细胞贴壁后,将细胞分为两组:对照组(葡萄糖浓度为5.5mmol/L)和高糖组(葡萄糖浓度为35mmol/L),分别培养7d后收集细胞,用透射电子显微镜、MTT法、流式细胞仪(FCM)检测法,研究高糖对小梁细胞形态、增殖和凋亡的影响。结果:高糖组与对照组相比较,小梁细胞胞浆内细胞器减少,内质网、高尔基复合体、线粒体等细胞器肿胀,溶酶体及脂肪滴增多,核浆比减小,可见凋亡小体;高浓度葡萄糖对小梁细胞的增殖有明显的抑制作用,能够促进小梁细胞的凋亡,均有统计学意义(P<0.05)。结论:高糖可能通过使小梁细胞的增殖能力下降,凋亡率增加,使近管小梁网的网状结构改变,网孔变小,改变房水流出途径的阻力导致眼内压升高,可能是糖尿病患者PO-AG发病率增高的原因之一。

AIM： Establish bovine trabecular meshwork cells （TMC） culturing systerm in vitro, to observe the effect of high glucose （HG） on cultured bovine trabecular meshwork cells, and study the relationship between high glucose and primary open angle glaucoma （POAG）, between diabetes and POAG, probe open angle the pathogenesis of glaucoma with POAG, then provide important base- ment for guiding clinical medication of POAG.METHODS： Trabecular meshwork dissected from eyes of fresh killed cattle was separated, and then TMC were cultured. TMC＇s morphology was observed by light microscope and electron microscope. After primarily cultured and subcultured, make cell suspension, count and ino- culate on culture plate（ or culture flask）. When TMC were pasting wall, divided them into two groups randomly, control group （the density of glucose is 5.5mmol/L） and HG group （the density of glucose is 35mmol/L）. These cells were grown in normal medium （5.5mmol/L）, HG （35mmol/L） medium for 7 days to be collected, then were observed by transmission electron microscope, detected by MTT method and flow cytometry （FCM）, to study the effect of HG on morphology, proliferation and apoptosis in cultured bovine TMC.RESULTS： High glucose group compared with the control group, trabecular meshwork cells to reduce cell cytoplasm, endoplasmic reticulum, Golgi complex and mitochondria and so on were swelled, lysosome and fat drop increased, nuclear cytoplasm ratio diminished, apo- ptotic body also be founded. HG had evident inhibitory effect on TMC＇s proliferations （57. 5% of control, P 〈 0.01）, even contributed to apoptosis of TMC, all had statistical significance （P〈0.05）.CONCLUSION. HG may cause reproductive activity decreasing and apoptosis rate increasing of TMC through changing the structure of formatio reticularis in near tube of trabecular meshwork and shrinking mesh, changing the way rooms flow resistance lead to ocular hypertension. It may be one of the reasons of the high incidence of POAG in diabetes.