自噬相关因子在糖尿病小鼠晶状体上皮细胞中的表达变化  

The expression change of autophagy-related factors in lens epithelial cells of diabetic mice

作　　者：程荣 张璐 黄钰森[3] Cheng Rong;Zhang Lu;Huang Yusen(Medical College,Qingdao University,Shandong Eye Institute,Cataract Division,Qingdao 266071,China;School of Medicine,Shandong University,Shandong Eye Institute,Jinan 250012,China;Shandong Provincial Key Laboratory of Ophthalmology,Shandong Eye Institute,Qingdao 266071,China)

机构地区：[1]青岛大学医学院山东省眼科研究所白内障科,青岛266071 [2]山东大学临床医学院山东省眼科研究所,济南250012 [3]山东省眼科研究所山东省眼科学重点实验室-省部共建国家重点实验室培育基地,青岛266071

出　　处：《中华实验眼科杂志》2018年第6期424-428,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81370996、81670839）;山东省医学科学院医药卫生科技创新工程

摘　　要：目的 通过检测自噬相关因子BECN1、LC3B和P62在糖尿病小鼠晶状体上皮细胞中的表达情况,以探讨自噬在糖尿病性白内障发生和发展中的作用.方法 取同批次的6～8周龄雄性C57BL/6小鼠80只,应用随机数字表法随机选取50只小鼠采用连续小剂量腹腔内注射链脲佐菌素（STZ）50 mg/（kg·d）诱导1型糖尿病小鼠模型作为模型组,其余30只小鼠腹腔内注射适当剂量的柠檬酸盐缓冲液作为对照组.3个月后检测模型组小鼠空腹血糖.使用透射电子显微镜观察模型组和对照组小鼠晶状体上皮细胞自噬小体的形态变化;免疫组织化学法检测2个组小鼠晶状体前囊膜组织中LC3B和P62蛋白的表达和定位;荧光定量PCR法检测各组小鼠晶状体前囊膜中BECN1、LC3B和P62 mRNA的表达量;Western blot法检测2个组小鼠晶状体前囊膜中自噬相关蛋白的相对表达量.结果 透射电子显微镜下观察发现,与对照组小鼠相比,模型组小鼠晶状体上皮细胞中的自噬体体积更大,且其内包含多个线粒体;免疫组织化学法检测结果显示,与对照组小鼠相比,模型组小鼠晶状体前囊膜中LC3B和P62蛋白表达均增强;荧光定量PCR结果显示,模型组小鼠晶状体前囊膜中BECN1 、LC3B和P62 mRNA的相对表达量分别为1.48±0.10、2.62±0.15和1.89±0.20,均高于对照组的1.10±0.02、1.10±0.05和1.01 ±0.01,差异均有统计学意义（t=6.64、14.25、6.14,均P＜0.05）;Western blot检测结果显示,模型组小鼠晶状体前囊膜中BECN1、LC3B和P62蛋白的相对表达量分别为1.50±0.10、1.24±0.09和3.19±1.04,均高于对照组的1.00±0.00、1.00±0.00和1.00±0.00,差异均有统计学意义（t=8.75、6.10、3.65,均P＜0.05）.结论 糖尿病小鼠晶状体上皮细胞中自噬现象出现异常,自噬功能失调可能在糖尿病性白内障的形成过程中起重要作用.Objective To investigate the role of autophagy in the development of diabetic cataract by detecting the expression of autophagy-related factors （BECN1,LC3B,P62） in diabetic mouse lens epithelial cells.Methods Eighty C57BL/6 male mice were selected.Fifty C57 mice were consecutively dosed with streptozotocin （STZ） 50 mg/ （kg · d） by intraperitoneal injection to induce type 1 diabetes mellitus model,and served as the model group;the remaining 30 mice were injected with appropriate dose of citrate buffer,and served as the control group.The fasting plasma glucose was tested by collecting the caudal vein blood in the model group.The morphological changes of autophagy of lens epithelial cells were observed by transmission electron microscopy.The expression and localization of LC3B and P62 protein were detected by immunohistochemistry.PCR was used to detect the expression of BECN1,LC3B and P62 mRNA in the anterior capsule.The relative expression of autophagy-related proteins in the anterior capsule was detected by Western blot.The use of animals complied with Regulations on the Management of Experimental Ainimals from Shandong Eye Institute.Results Compared with the control group,transmission electron microscopy revealed that the autophagosomes of lens epithelial cells in model group was large and contained more mitochondria.Immunohistochemical method showed that the expression of LC3B and P62 proteins in the anterior capsule tissue of experiment group was stronger than that of the control group.The relative expression level of BECN1,LC3B and P62 mRNA in the experiment group was 1.48±0.10,2.62±0.15 and 1.89±0.20,respectively,which was higher than 1.10±0.02,1.10±0.05 and 1.01±0.01 in the control group,with significant differences between the two groups （t =6.64,14.25,6.14;all at P ＜ 0.05）.The relative expression of BECN1,LC3B and P62 protein in the experiment group was 1.50±0.10,1.24±0.09 and 3.19± 1.04,respectively,which was higher than 1.00±0.00,1.00±0.00 and 1.00±0.00 in the control group

关 键 词：糖尿病性白内障 自噬 晶状体上皮细胞 BECN1 LC3B P62 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]