摘要背景糖尿病性白内障是糖尿病的主要眼部并发症之一,包括皮质性、核性、囊膜下性及混合型白内障,其表型可能与晶状体上皮细胞(LECs)的不同病理改变有关,而糖尿病囊膜下性白内障是常见的表型之一.研究糖尿病囊膜下性白内障LECs的生物学行为对相关药物的研发和相关疾病的治疗有重要临床意义. 目的 探讨Src-家族酪氨酸蛋白激酶(SFKs)在高糖诱导的人LECs凋亡及上皮-间质转分化(EMT)中的作用. 方法 将人LECs系HLE-B3细胞分为正常对照组、高糖组和PP1组,分别在含5.5 mmol/L葡萄糖的DMEM、含35.5 mmol/L葡萄糖的DMEM及含35.5 mmol/L葡萄糖+10 μmol/L SFK特异性抑制剂PP1的DMEM中培养24 h.分别于细胞培养后3、6、12和24 h采用流式细胞术检测各组细胞的凋亡率;用倒置显微镜观察各组细胞的形态学变化;采用免疫荧光染色检测细胞中EMT标志物E-钙黏蛋白(E-cadherin)及α-平滑肌肌动蛋白(α-SMA)的相对表达量;并用Western blot法检测p-Src418(活化的c-Src激酶)及凋亡相关蛋白bcl-xl、survivin、caspase-3,EMT相关蛋白E-cadherin、α-SMA的表达变化. 结果 高糖组人LECs中p-Src418的表达增高,并在培养6h时达峰值,正常对照组、高糖组和PP1组人LECs中p-Src418的相对表达量(相对灰度值)分别为0.042±0.011、0.125±0.036和0.035 ±0.018,正常对照组和PP1组人LECs中p-Src418的表达量明显低于高糖组,差异均有统计学意义(均P＜0.01).细胞培养后6、12和24 h,高糖组人LECs凋亡率稍高于正常对照组,但差异均无统计学意义(均P＞0.05),PP1组人LECs凋亡率分别为(6.433±2.084)％、(10.333±2.610)％和(8.033±2.967)％,明显高于高糖组的(3.233±1.320)％、(3.533±1.159)％、(5.733±0.230)％及正常对照组的(3.133±1.170)％、(2.833±0.751)％、(3.333±1.201)％,差异均有统计学意义(均P＜0.05),而高糖组与正常对照组间比较差异均无统计学意义(均P＞0.05).细胞培养后6h和12h,PP1组细胞中凋亡抑制蛋白bcl-xl、survivin的相对表达量(相对灰度值)明显低于高糖组和正常对照组,而凋亡促进蛋白caspase-3的相对表达量明显高于高糖组和正常对照组,差异均有统计学意义(均P＜0.05).高糖组培养后24 h,LECs呈梭形,发生纤维细胞样改变,PP1组纤维样细胞减少.Western blot法检测和免疫荧光染色均显示,培养后6h高糖组细胞中EMT标志物E-cadherin蛋白的相对表达量低于PP1组和正常对照组,而α-SMA的相对表达量高于PP1组和正常对照组,差异均有统计学意义(均P＜0.05). 结论 高糖环境可激活LECs中的c-Src激酶,进而诱导LECs发生EMT,同时LECs的凋亡减少;用PP1抑制c-Src激酶的异常激活有助于维持高糖环境中LECs的上皮特性.

abstractsBackground Diabetic cataract is one of the major ocular complications in diabetes mellitus,including cortical cataract,nuclear cataract,subcapsular cataract and mixed cataract,and different cataractogenesis may be associated with lens epithelial cells (LECs).Subcapsular cataract is one of diabetic cataracts.Studying the biological behavior of LECs in subcapsular cataract is crucial for prevention and treatment.Objective This study was to investigate the effects of Src-family tyrosine kinases (SFKs) on apoptosis and epithelial-mesenchymal transition (EMT) of human LECs cultured by high glucose.Methods Human LECs (HLE-B3) were cultured for 24 hours with DMEM containing 5.5 mmol/L glucose (normal control group),DMEM containing 35.5 mmol/L glucose (high glucose group) and DMEM containing 35.5 mmol/L glucose + 10 μmol/L PP1,a specific inhibitor of SFKs (PP1 group).In 3,6,12 and 24 hours after culture,the apoptosis of human LECs was detected by flow cytometry assay;morphological change of human LECs was observed under the inverted microscope,and the expressions of the markers of EMT,E-cadherin and a-smooth muscle actin (α-SMA),in the cells were detected by immnofluorescence staining.In addition,the alternations of p-Src418 (active c-Src),bcl-xl,survivin,caspase-3,E-cadherin and α-SMA proteins were assayed by Western blot analysis.Results An elevated expression level of p-Src418 was found in LECs in the high glucose group and peaked 6 hours after cultured.The expressions of p-Src418(grey levels) were 0.125 ±0.036 in the high glucose group,and which was significantly higher than 0.042±0.011 in the normal control group and 0.035 ± 0.018 in the PP1 group,respectively (both at P＜0.01).No remarkable differences were seen in the apoptotic rates between the high glucose group and normal control group in 6,12 and 24 hours after culture (all at P＞0.05).The apoptotic rates of human LECs were(6.433±2.084)％,(10.333±2.610)％ and (8.033±2.967)％ in the PP1 group,which were higher than (3.233 ± 1.320) ％,(3.533 ± 1.159) ％,(5.733 ±0.230) ％ in the high glucose group and (3.133±1.170)％,(2.833±0.751)％,(3.333±1.201)％ in the normal control group (all at P＜0.05),however,there were significant differences in the apoptosis between the high glucose group and the normal control group (all at P＞0.05).In 6 hours and 12 hours after cell culture,the expression levels of bcl-xl and survivin (grey values) in human LECs were significantly declined,but the expression of caspase-3 was increased in the PP1 group compared with the the high glucose group and the normal control group (all at P＜0.05).The LECs showed slender in shape 24 hours after culture in the high glucose group,but the cell shape was close to the normal in the PP1 group.Western blot and immunofluorescence assay revealed that the expression of E-cadherin in human LECs was significantly reduced and that of α-SMA was significantly increased 6 hours after culture in the high glucose group compared with the PP1 group and the normal control group (all at P＜0.05).Conclusions High glucose activates c-Src kinase of LECs in high glucose environment and therefore induces EMT and inhibits apoptosis.However,PP1 impedes the biological process of EMT and apoptosis of LECs to maintain the epithelial characteristics even under the stress of high glucose.