缺氧条件下乙酰肝素酶抑制剂PI-88对人视网膜血管内皮细胞自噬的影响  

The Effect of Heparinase Inhibitor PI-88 on Human Retinal Endothelial Cells Autophagy under Hypoxia

作　　者：苑玉鑫 李树华 玄猛 苏焜仪 吕林[1] 胡洁[1] YUAN Yu-xin;LI Shu-hua;XUAN Meng;SU Kun-yi;LV Lin;HU Jie(State Key Laboratory of Ophthalmology,Zhongshan Ophthalmic Center,Sun Yat-sen University,Guangzhou 510060,China;Guangdong Province Traditional Chinese Medical Hospital,The Second Affiliated Hospital of Traditional Chinese Medicine University Of Guangzhou,Guangzhou 510120,China)

机构地区：[1]中山大学中山眼科中心//眼科学国家重点实验室,广东广州510060 [2]广州中医药大学第二附属医院//广东省中医院,广东广州510120

出　　处：《中山大学学报（医学科学版）》2020年第3期397-402,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(81271009);广东省科技计划项目公益研究与能力建设专项(2017A030303016);广东省中医药局科研项目(20201064)。

摘　　要：【目的】探讨乙酰肝素酶(Hpa)竞争性抑制剂PI-88在缺氧条件下对人视网膜血管内皮细胞(HREC)自噬水平的影响。【方法】以HREC为研究对象,设置3组:正常组,缺氧组(CoCl2处理的缺氧组)和实验组(CoCl2+PI-88组)。观察免疫荧光染色检测自噬体特异性标记物LC3蛋白的表达;Western blot检测自噬相关蛋白LC3-Ⅰ与LC3-Ⅱ的表达;通过GFP-LC3腺病毒转染HREC,直接观察活细胞中由GFP-LC3标记的自噬水平。【结果】缺氧状态下,HREC内自噬标记蛋白LC3表达水平上调,LC3-Ⅱ/LC3-Ⅰ比值升高(灰度值,与正常组比较,4.69±0.54比2.14±0.33,P=0.001<0.05),表明LC3-Ⅰ向LC3-Ⅱ转化明显增加。Hpa抑制剂PI-88可以抑制HREC的LC3表达,使LC3-Ⅱ/LC3-Ⅰ比值下降(灰度值,与缺氧组比较,3.51±0.20比4.69±0.54,P=0.027<0.05),表明LC3-Ⅰ向LC3-Ⅱ的转化水平下降,且活细胞内自噬体的形成减少。【结论】在缺氧状态下,抑制乙酰肝素酶可下调HREC中的自噬水平。【Objective】To explore the potential role of heparinase inhibitor PI-88 in autophagy of human retinal endo?thelial cells under hypoxia.【Methods】The human retinal endothelial cells were cultured in vitro and divided into normal culture group,hypoxia group(CoCl2),and hypoxia plus heparinase inhibitor(CoCl2+PI-88)group.Immunofluorescence was used to observe the expression of LC3.Western blot was adopted to assess the LC3-Ⅰand LC3-Ⅱexpression.Green fluorescent protein(GFP)-LC3 adenoviruses were transfected into living human retinal endothelial cells to dynamically visualize the autophagy level.【Results】LC3 exposed to hypoxia were upregulated,the ratio of LC3-II/LC3-I in HREC were upregulated(grey value,hypoxia group vs.normal culture group,4.69±0.5 vs.2.14±0.33,P=0.001<0.05).The ratio of LC3-II/LC3-I in HREC were inhibited in response to heparinase inhibitor PI-88(grey value,hypoxia plus hepari?nase inhibitor group vs hypoxia group,3.51±0.20 vs.4.69±0.54,P=0.027<0.05).Autophagosome marked by LC3-GFP in HREC exposed to heparinase inhibitor(PI-88)were decreasing compared to cells only exposed to hypoxia.【Conclusion】Inhibiting heparinase could downregulate the level of autophagosome in human retinal endothelial cells under hypoxia.

关 键 词：乙酰肝素酶 PI-88 人视网膜血管内皮细胞 自噬 缺氧 

分 类 号：R77[医药卫生—眼科]