高糖环境诱导视网膜细胞衰老的实验研究

Experimental study on retinal cell senescence induced by high glucose

背景 细胞衰老在血管功能失调性疾病中的作用越来越受到人们的重视,人们推测细胞衰老可能参与糖尿病相关血管并发症的发生及发展,而目前检测细胞衰老的主要标志是β-半乳糖苷酶表达的上调.目的 证实高糖环境对牛视网膜血管内皮细胞（BRVECs）及糖尿病小鼠视网膜衰老状态的影响.方法 对BRVECs进行体外培养和传代,取第7代细胞用于实验.将培养的细胞分为对照组和高糖培养组,分别用含5.5 mmol/L或25.0mmol/L葡萄糖的M199内皮细胞培养液培养细胞7d,用5-溴-4-氯-3-吲哚-β-D半乳糖苷（X-Gal）染色法观察各组离体细胞中β-半乳糖苷酶的阳性表达情况,计算方法为β-半乳糖苷酶阳性细胞/总细胞＆#215;100％.将SPF级8～10周龄C57BL/6J雄性小鼠12只,采用随机数字表法分为对照组和模型组,用链脲佐菌素腹腔内注射法制备糖尿病小鼠模型,3个月后取其双眼视网膜平铺于48孔板,采用X-Gal染色法进行染色,光学显微镜下比较两个组小鼠视网膜细胞中β-半乳糖苷酶阳性表达细胞数.结果 BRVECs复苏并培养24 h后每孔内细胞70％～80％铺满,细胞排列不规则;培养后48 h细胞融合成片,紧密排列.高糖培养组和对照组β-半乳糖苷酶染色阳性细胞比例分别为（51.4±5.4）％和（36.6±3.8）％,差异有统计学意义（t=-3.204,P=0.033）;糖尿病鼠视网膜中β-半乳糖苷酶染色阳性细胞数为（94.0±15.1）个/视野,明显多于对照组的（60.0±5.7）个/视野,差异有统计学意义（t=-2.974,P=0.041）.结论 高糖环境下离体BRVECs和在体小鼠视网膜细胞中β-半乳糖苷酶表达明显上调,高糖引起的细胞早衰可能参与糖尿病视网膜病变的发生.

Background Researchers are paying increasing attention to the effect of cellular senescence in vascular dysfunctional diseases,and it is hypothesized that cellular senescence may also be involved in the development of diabetes related vascular complications.The outstanding feature of cellular senescence is the upregulation of beta-galactosidase.Objective This study was to investigate the effects of high glucose on cell senescent in vitro and in vivo on bovine retinal endothelial cells （BRVECs） and mouse retina.Methods BRVECs were cultured and passaged,and the seventh generation of cells were employed in this study.The cells were divided into the control group and the high glucose culture group and cultivated using M199 medium containing 5.5 mmol/L or 25.0 mmol/L glucose,respectively.5-bromine-chlorine-4-3-indole-beta D galactose glucoside （X-Gal） staining was used to examine the expression of beta-galactosidase in the cells.Diabetic models were established in the SPF male C57BL/6J mice aged 8-10 weeks by intraperitoneal injection of streptozotocin （STZ）,and the age-and gendermatched normal mice served as controls.The mouse retinas were collected and starched in the 48-well plates 3 months later.X-Gal staining was employed to calculate the positive cells.Results BRVECs grew well 24 hours after culture but showed irregular arrangement.Forty-eight hours later,the cells reached confluence with a tight connect.The ratios of positive BRVECs and total cells were （51.4±5.4） % and （36.6-±3.8） % in the high glucose culture group and the control group,with a significant difference between the two groups （t =-3.204,P =0.033）.The number of positive cells for X-Gal in mouse retinas was （94.0± 15.1） /field in the diabetic group,which was higher than that in the control group （[60.0 ± 5.7]/field） （t =-2.974,P =0.041）.Conclusions High glucose environment accelerates senescence of retinal cells,and high glucose induces premature cell senescence,which likely plays an important role in the pathogenesis of diabetic retinopathy.