[1]李蓉,姚杨,杜军辉,等.自噬在高糖条件下视网膜色素上皮细胞表达血管内皮生长因子促进RF/6A细胞血管生成中的作用[J].眼科新进展,2019,39(8):714-718.[doi:10.13389/j.cnki.rao.2019.0163]
　LI Rong,YAO Yang,DU Jun-Hui,et al.Autophagy promotes angiogenesis of RF/6A cells following upregulating the expression of vascular endothelial growth factor in retinal pigment epithelial cells under high glucose conditions[J].Recent Advances in Ophthalmology,2019,39(8):714-718.[doi:10.13389/j.cnki.rao.2019.0163]
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自噬在高糖条件下视网膜色素上皮细胞表达血管内皮生长因子促进RF/6A细胞血管生成中的作用/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
39卷
期数:
2019年8期
页码:
714-718
栏目:
实验研究
出版日期:
2019-08-05

文章信息/Info

Title:
Autophagy promotes angiogenesis of RF/6A cells following upregulating the expression of vascular endothelial growth factor in retinal pigment epithelial cells under high glucose conditions
作者:
李蓉; 姚杨; 杜军辉; 姚国敏; 王小娣; 张进
710077　陕西省西安市，西安医学院第一附属医院眼科（李蓉，姚国敏，王小娣），中心实验室（姚杨）；710054　陕西省西安市，西安交通大学附属西安市第九医院眼科（杜军辉）；719000　陕西省榆林市，榆林市第一医院眼科（张进）
Author(s):
LI Rong; YAO Yang; DU Jun-Hui; YAO Guo-Min; WANG Xiao-Di; ZHANG Jin
Department of Ophthalmology，the First Affiliated Hospital of Xi’an Medical University (LI Rong，YAO Guo-Min，WANG Xiao-Di)；Department of Central Laboratory，the First Affiliated Hospital of Xi’an Medical University(YAO Yang)；Department of Ophthalmology，Xi’an Ninth Hospital，Affiliated to Medical College of Xi’an Jiaotong University (DU Jun-Hui)；Department of Ophthalmology，the First Hospital of Yulin (ZHANG Jin)
关键词:
自噬; 血管生成; 高糖; 血管内皮生长因子; 视网膜新生血管; 糖尿病视网膜病变; 细胞培养
Keywords:
autophagy; angiogenesis; high glucose; vascular endothelial growth factor; retinal neovascularization; diabetic retinopathy; cell culture
分类号:
R774.1
DOI:
10.13389/j.cnki.rao.2019.0163
文献标志码:
A
摘要:
目的　研究高糖条件下诱导的人视网膜色素上皮细胞株（ARPE-19）自噬对其表达血管内皮生长因子（vascular endothelial growth factor，VEGF），及恒河猴脉络膜视网膜血管内皮细胞（RF/6A）迁移和管腔形成的影响。方法　根据不同干预将ARPE-19细胞随机分为对照组、高糖组及3-甲基腺嘌呤（3-MA）+高糖组。对照组细胞在无糖DMEM培养基中常规培养24 h，高糖组细胞在DMEM培养基中加入30 mmol·L-1葡萄糖溶液处理24 h，3-MA+高糖组细胞先用10 mmol·L-1 3-MA处理24 h，然后更换培养基再加入30 mmol·L-1葡萄糖溶液处理24 h。Western blot法检测细胞自噬标志性蛋白微管相关蛋白1 轻链 3（microtubule-related protein 1 light chain 3，LC3）II型和I型的比值(LC3-II/LC3-I)、Beclin-1及细胞中自噬相关基因3（autophagy-related gene 3，Atg3）的蛋白表达。ELISA法检测ARPE-19细胞上清液中VEGF的蛋白表达。比较三组ARPE-19细胞LC3-II/LC3-I、Beclin-1、Atg3及VEGF的蛋白表达量。然后将以上三组ARPE-19细胞上清液分别加入RF/6A细胞培养基中，将RF/6A细胞也按以上方法分组，分别采用Transwell法及Matrigel胶法检测并比较三组RF/6A细胞迁移数及细胞管腔形成数。结果　对照组、高糖组和3-MA+高糖组ARPE-19细胞中LC3-II/LC3-I比值分别为0.405±0.095、0.932±0.024和0.635±0.048；Beclin-1蛋白相对表达量分别为0.205±0.035、0.590±0.120和0.425±0.082；Atg3蛋白相对表达量分别为0.277±0.035、0.539±0.071和0.389±0.019。ARPE-19细胞上清液中VEGF的蛋白表达量三组分别为（44.03±9.08）ng·L-1、（205.70±17.90）ng·L-1和（112.52±21.06）ng·L-1；RF/6A细胞迁移数三组分别为（125.60±6.35）个、（153.60±19.20）个和（67.40±7.95）个；细胞管腔形成数三组分别为（12.22±0.84）个、（18.44±1.68）个和（5.44±0.51）个；整体比较差异均有统计学意义（均为P<0.01）。与对照组相比，高糖组ARPE-19细胞的LC3-II/LC3-I比值、Beclin-1及Atg3蛋白相对表达量均明显增加（均为P<0.01），VEGF表达水平均明显升高（P<0.01），RF/6A细胞迁移数和管腔形成数均明显增加（均为P<0.01），3-MA+高糖组ARPE-19细胞中LC3-II/I比值、Beclin-1和Atg3蛋白相对表达量、VEGF表达量、RF/6A细胞迁移数和细胞管腔形成数均较高糖组明显降低（均为P<0.01）。结论　高糖激活ARPE-19细胞自噬，进而上调VEGF的表达并促进RF/6A细胞迁移和管腔形成，自噬抑制剂3-MA可抑制ARPE-19细胞自噬，并下调VEGF的表达从而抑制RF/6A细胞的血管形成能力。
Abstract:
Objective　To study the effects of autophagy of human retinal pigment epithelial cell line (ARPE-19) induced by high glucose conditions on the expression of vascular endothelial growth factor (VEGF) as well as on the migration and tube formation of rhesus choroid-retinal endothelial cells (RF/6A).Methods　ARPE-19 cells were divided into the control group,high-glucose group and 3-methyladenine (3-MA)+high-glucose group.The cells in the control group were cultured in DMEM medium without glucose for 24 hours.The cells in the high-glucose group were cultured in DMEM medium adding 30 mmol·L-1 glucose for 24 hours.The cells in the 3-MA+high-glucose group were pretreated with 10 mmol·L-1 3-MA for 24 hours and then exposed to replaced medium adding 30 mmol·L-1 glucose for 24 hours.The ratio of II and I form of microtubule-related protein 1 light chain 3 (LC3) (LC3-II/LC3-I),Beclin-1 and autophagy-related gene 3 (Atg3) protein expressions in the cells were detected by Western blot,and the expression of VEGF in the cell supernatants was examined by ELISA assay.The expressions of LC3-II/LC3-I,Beclin-1,Atg3 and VEGF among the three groups were compared.Then,the supernatant of ARPE-19 cells in the above three groups was respectively added into RF/6A cell culture medium and RF/6A cells were also grouped by the above method.The migration and tube formation of RF/6A cells were detected by transwell and matrigel assay,respectively.The number of migrated cells and tube formation of the RF/6A cells were compared.Results　In the control group,high-glucose group and 3-MA+high-glucose group,the LC3-II/LC3-I ratio in ARPE-19 cells was 0.405±0.095,0.932±0.024 and 0.635±0.048,respectively;the relative expression of Beclin-1 protein in the cells was 0.205±0.035,0.590±0.120 and 0.425±0.082,respectively;and the relative expression of Atg3 protein in the cells was 0.277±0.035,0.539±0.071 and 0.389±0.019,respectively.The protein expression of VEGF in the supernatant of ARPE-19 cells was (44.03±9.08)ng·L-1,(205.70±17.90)ng·L-1 and (112.52±21.06)ng·L-1 for the three groups,respectively.The number of migrated RF/6A cells was 125.60±6.35,153.60±19.20 and 67.40±7.95;the number of tube formation was 12.22±0.84,18.44±1.68 and 5.44±0.51 for the three groups,respectively.There were significant differences among the groups (all P<0.01).Compared with the control group,the ratio of LC3-II/LC3-I and the expression levels of Beclin-1,Atg3 in ARPE-19 cells were significantly increased (all P<0.01) and the expression level of VEGF increased (P<0.01) in the high-glucose group.The number of migrated cells and tube formation of RF/6A cells were significantly increased in the high-glucose group (all P<0.01) compared with the control group.Compared with the high-glucose group,the ratio of LC3-II/LC3-I and the expression of Beclin-1,Atg3 and VEGF protein in ARPE-19 cells,the number of migrated cells and tube formation of RF/6A cells were significantly decreased in the 3-MA+high-glucose group (all P<0.01).Conclusion　High glucose conditions activate autophagy of ARPE-19 cells,which enhances the expression of VEGF and the migration and tube formation of RF/6A cells.Autophagy inhibitor 3-MA inhibits the angiogenesis abilities of RF/6A cells through inhibiting autophagy of ARPE-19 cells and the expression of VEGF.

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备注/Memo

备注/Memo:
国家自然科学基金青年基金项目（编号：81500726）；陕西省卫生健康科研基金项目（编号：2018D074）；西安市科技局医学研究项目［编号：201805097YX5SF31(4)］
更新日期/Last Update: 2019-07-31