缺氧条件下自噬在视网膜血管内皮细胞血管生成中的作用

Role of autophagy in angiogenesis of retinal vascular endothelial cells under hypoxia condition

目的研究缺氧条件下自噬对体外培养的小鼠视网膜血管内皮细胞（RVECs）增生、迁移和管腔形成的影响。方法取清洁级C57BL/6J小鼠50只，采用组织块培养法分离和培养小鼠RVECs，采用CD34免疫荧光染色法对培养细胞进行鉴定。将生长良好的RVECs分为正常对照组、缺氧对照组及缺氧＋3-甲基腺嘌呤（3-MA）组。正常对照组细胞常规培养，缺氧对照组细胞在氧体积分数1%的环境下培养24 h，缺氧＋3-MA组细胞先用5 mmol/L 3-MA预处理4 h，然后在上述缺氧环境下培养24 h。采用Western blot法检测自噬标志蛋白微管相关蛋白1轻链3（LC3-Ⅱ/LC3-Ⅰ）比值及细胞中Beclin-1的表达；透射电子显微镜下观察细胞内形成的自噬小体的超微结构；分别采用Click-iT EdU试剂盒、细胞划痕法和Matrigel胶法检测细胞增生率、细胞迁移情况及细胞管腔形成情况。 结果原代培养后5～7 d培养的细胞呈铺路石样排列生长，CD34表达阳性。透射电子显微镜下缺氧对照组细胞自噬小体明显多于正常对照组和缺氧＋3-MA组。正常对照组、缺氧对照组和缺氧＋3-MA组细胞中LC3-Ⅱ/LC3-Ⅰ比值分别为0.243±0.030、0.658±0.032和0.405±0.095，Beclin-1蛋白相对表达量分别为0.260±0.040、0.650±0.071和0.461±0.089；细胞增生率分别为（45.93±6.39）%、（22.74±2.35）%和（24.12±3.59）%；细胞划痕愈合率分别为（36.02±5.84）%、（57.26±11.98）%和（18.16±9.73）%；细胞管腔形成数分别为（29.20±6.10）、（41.40±4.04）和（22.00±2.92）个；总体比较差异均有统计学意义（F＝35.86、23.53、34.28、21.12、23.27，均P〈0.01）。与正常对照组相比，缺氧对照组细胞的LC3-Ⅱ/LC3-Ⅰ比值、Beclin-1蛋白相对表达均明显增加，细胞增生率明显下降，划痕愈合率和管腔形成数明显增加，缺氧＋3-MA组细胞中LC3-Ⅱ/Ⅰ比值、Beclin-1蛋白相对表达量、划痕愈合率和细胞管腔形成数较缺氧对照组明显降低。结论缺氧激活小鼠RVECs自噬，进而促进细胞迁移和管腔形成，自噬抑制剂3-MA可抑制自噬小体活性，并抑制RVECs的迁移和管腔形成能力。

ObjectiveTo study the role of autophagy in proliferation, migration and tube formation of retinal vascular endothelial cells （RVECs） under the condition of hypoxia in vitro.MethodsMouse RVECs were isolated from 50 C57BL/6J mice primarily cultured using explant culture method, and the cells were identified by immunofluorescence of CD34.Well cultured RVECs were divided into normal control group, hypoxia control group and hypoxia＋ 3-methyladenine （3-MA） group.The cells in the normal control group were cultured in the normal environment for 24 hours, and the cells in the hypoxia control group were cultured 1% O2 environment for 24 hours, and the cells in the hypoxia＋ 3-MA group were pretreated with 5 mmol/L 3-MA for 4 hours and then exposed to 1% O2 environment for 24 hours.Microtubule-related protein 1 light chain 3 （LC3-Ⅱ/LC3-Ⅰ） and Beclin-1 protein contents in the cells were detected by Western blot analysis; the ultrastructure of autophagosome was examined under the transmission electron microscope.The proliferation, migration and tube formation of the cells were detected by Click-iTEdU kit, scratch assay and matrigel, respectively.ResultsPrimarily culture cells grew well with the cobblestone-like appearance 5-7 days after culture and showed positive response for CD34.The autophagosome number was increased in the hypoxia control group compared with the hypoxia＋ 3-MA group and normal control group.The LC3-Ⅱ/LC3-Ⅰ ratio was 0.243±0.030, 0.658±0.032 and 0.405±0.095; the relative expression of Beclin-1 protein in the cells was 0.260±0.040, 0.650±0.071 and 0.461±0.089; the proliferation rate of the cells was （45.93±6.39）%, （22.74±2.35）% and （24.12±3.59）%; the scratch healing rate of the cells was （36.02±5.84）%, （57.26±11.98）% and （18.16±9.73）%; the number of tube formation was 29.20±6.10, 41.40±4.04 and 22.00±2.92 in the normal control group, hypoxia control group and hypoxia＋ 3-MA group, respectively, with significant differences among the groups （F=35.86, 23.53, 34.28, 21.12, 23.27; all at P〈0.01）. Compared with the normal control group, the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression of Beclin-1 protein, scratch healing rate and the number of tube formation were significantly increased, and the proliferation rate was significantly reduced in the hypoxia control group （all at P〈0.05）. Compared with the hypoxia control group, the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression of Beclin-1 protein, scratch healing rate and the number of tube formation were significantly decreased in the hypoxia＋ 3-MA group. ConclusionsHypoxia environment activates autophagy of mouse RVECs, which enhances the migration and tube formation abilities of the cells.3-MA inhibits the angiogenesis abilities of mouse RVECs.