朱小丽，孙小凤，赖铭莹



·实验论著·

虫草素对高糖培养的恒河猴脉络膜-视网膜血管内皮细胞血管新生的影响

朱小丽*，孙小凤*，赖铭莹

•METHODS: Cultured RF/6A cells were divided into normal control group, high glucose group and high glucose (HG) + different concentration cordycepin groups (HG+10μg/mL group, HG+50μg/mL group, HG+100μg/mL group). The cell proliferation was assessed using cholecystokinin octapeptide dye after treated for 48h. The cell migration was investigated by a Transwell assay. The tube formation was measured on Matrigel. Furthermore, the impact of cordycepin on high glucose-induced activation of VEGF and VEGF receptor 2 (VEGFR-2) was tested by Western blot analysis.

•RESULTS: Compared with normal control group, cell viability markedly increased in high glucose group (P<0.05). Cordycepin inhibited RF/6A cell proliferation in a dose-dependent fashion: 10.2±0.9%, 23.4±1.5% and 31.1±1.2% inhibition as the concentrations of cordycepin were 10, 50 and 100μg/mL, respectively. The difference had statistically significant (P<0.05) compared with high glucose group. The number of cell migration were 55.6±2.70, 87.4±2.40, 65.4±2.7, 57.8±2.38, 62.4±2.77 in normal control group, high glucose group and HG+10μg/mL group, HG+50μg/mL group, HG+100μg/mL group respectively. Migration of RF/6A conspicuously increased in high glucose group(P<0.05) compared with normal control group; while showing a gradually reducing trend with the increase of cordycepin dose and a statistically significant difference compared with high glucose group(P<0.05). The number of tube formation were 18.7±2.08, 25.7±1.52, 19.9±1.57, 16.3± 2.51, 5.67±1.72 in the abovementioned group. Similarly showing a gradually reducing trend with the increase of cordycepin dose and a statistically significant difference with high glucose group (P<0.05). In addition, the number of tube formation of RF/6A in high glucose group significant increased compared with normal control group (P<0.05). The expression of VEGF and VEGFR-2 dramaticlly increased in high glucose groupvsnormal control group, oppositely gradually decreased with the increase of cordycepin concentrations, and had a statistically significant differencevshigh glucose group (P<0.05).

•CONCLUSION: Cordycepin can suppress the proliferation, migration and tubu formation of RF/6A in high glucose condition, might via inhibiting expression of VEGF and VEGFR-2.

目的：观察虫草素对高糖培养的恒河猴脉络膜-视网膜血管内皮细胞(RF/6A)血管新生作用的影响，探讨其可能的作用机制。

方法：培养RF/6A细胞，分为正常对照组(NC组)、高糖对照组(HG组)、高糖加不同浓度的虫草素组(HG+10μg/mL组、HG+50μg/mL组、HG+100μg/mL组)。采用CCK8法检测各组细胞的增殖活性；采用Transwell实验检测各组细胞的迁移；采用Matrigel检测各组管腔形成的情况；采用Western-blot检测各组细胞中VEGF和VEGFR2蛋白表达的情况。

结果：与NC组相比，HG组RF/6A的增殖活性增加(P<0.05)。不同浓度的虫草素对RF/6A的增殖均有抑制作用，随着虫草素浓度增加，细胞活性下降。HG+10μg/mL组、HG+50μg/mL组、HG+100μg/mL组的增殖活性抑制率分别为(10.2±0.9)%、(23.4±1.5)%、(31.1±1.2)%，与HG组比较差异均有统计学意义(P<0.05)。NC组、HG组、HG+10μg/mL组、HG+50μg/mL组、HG+100μg/mL组细胞迁移个数分别为55.6±2.70、87.4±2.40、65.4±2.7、57.8±2.38、62.4±2.77个。与NC组相比，HG组迁移细胞数量增加(P<0.05)；不同浓度虫草素组细胞迁移数量随着虫草素浓度的增加而减少，与HG组相比，差异具有统计学意义(P<0.05)。NC组、HG组、HG+10μg/mL组、HG+50μg/mL组、HG+100μg/mL组的细胞管腔形成个数分别为18.7±2.08、25.7±1.52、19.9±1.57、16.3±2.51、5.7±1.72个。与NC组相比，HG组细胞管腔形成个数增加(P<0.05)；不同浓度虫草素组细胞管腔形成个数随着虫草素浓度的增加而减少，与HG组相比，差异具有统计学意义(P<0.05)。与NC组相比，HG组VEGF和VEGFR2蛋白表达的量增加(P<0.05)；不同浓度虫草素组中细胞内VEGF和VEGFR2蛋白表达的量均低于高糖对照组(P<0.05)。

结论：虫草素可能通过抑制高糖下VEGF和VEGFR2蛋白的表达，抑制RF/6A细胞增殖、迁移和管腔形成，进而抑制血管新生。

虫草素；高糖培养；恒河猴脉络膜-视网膜血管内皮细胞；血管内皮生长因子；血管新生

引用：朱小丽，孙小凤，赖铭莹.虫草素对高糖培养的恒河猴脉络膜-视网膜血管内皮细胞血管新生的影响.国际眼科杂志2016;16(7):1237-1241

0引言

糖尿病视网膜病变(diabetic retinopathy，DR)是糖尿病最常见和严重的微血管并发症之一。DR发病机制复杂，其中以血管内皮生长因子(vascular endothelial growth factors，VEGF)为主的促血管生长因子在眼部的过度表达是导致DR发生、发展的重要因素[1]。在DR病理过程中，高糖可导致视网膜血管内皮细胞过度分泌VEGF，引起血管内皮细胞损伤，血管渗透性增加，毛细血管闭塞，促进视网膜组织的缺血缺氧，最终引起新生血管的形成[2-3]。过度分泌的VEGF与其受体(主要是VEGFR2受体)结合磷酸化后，激活下游信号通路，诱导内皮细胞的增殖、迁移和管腔形成，导致新生血管形成增加[4]。……