Background Vitronectin is a glycoprotein that has a variety of functions.Its expression was markedly higher in the retina of oxygen induced mice,which was confirmed in our animal model,and also increased in human umbilical vein endothelial cells (人 UVECs) that were cultured in high glucose.However,there was no evidence that showed vitronectin was involved in retinal neovascularization.Objective This study was to observe the influence of vitronectin on cytoskeleton remodeling,cell migration and blood vessel formation in 人 UVECs conditioned by high glucose.Methods 人 UVECs were cultured in high glucose and the expression of vitronectin was knocked down using RNA interference technology.The experiments were divided into the high glucose group (人 UVECs were conditioned with DMEM medium that contained 50 mmol/L glucose),negative interference group (人 UVECs were transfected with control siRNA in advance,and then were conditioned with DMEM medium that contained 50 mmol/L glucose) and positive interference group (HUVEC were transfected with vitronectin siRNA in advance,and then were conditioned with DMEM medium that contained 50 mmol/L glucose).The protein expression of vitronectin was measured by Western blot,and the microfilament cytoskeleton of 人 UVECs was examined by immunofluorescence cytochemical staining followed by fluorescence microscopy.Cell migration ability in a scratch wound assay and blood vessel formation ability in a matrigel assay of 人 UVECs were evaluated.The general differences were analysed by One-Way ANOVA ;further contrasts of the two groups were analysed by the LSD-t test.Results The differences in vitronectin expression of the three groups were not obvious at 0 hour (F=1.064,P＞0.05).After 24 hours,vitronectin expression was highest in the high glucose group,lower in the negative interference group,and the lowest in the positive interference group,and the differences were significant (F =15.519,P＜0.05).After 48 hours,vitronectin expression of the three groups displayed the same pattern,and the differences were also significant (F=37.521,P＜0.05).Immunofluorescence showed that the cytoskeleton structure was most obvious in the high glucose group,moderate in the negative interference group,and was the least obvious in the positive interference group,after both 24 hours and 48 hours.In the scratch wound assay,the cell migration ability of the high glucose group was the highest,lower in the negative interference group,and the lowest in the positive interference group after 24 hours,and the differences were significant (F=90.685,P＜0.05).After 48 hours,the cell migration abilities of the three groups displayed the same pattern,and the differences were also significant (F=67.880,P＜0.05).In the matrigel assay,after 6 hours,the number of blood vessels formed in the high glucose group was more than that in the negative interference group,and the least amount was found in the positive interference group.The differences among of them were significant (F =86.653,P＜0.05).The number of blood vessel formed in the positive interference group was also the lowest after 12 hours,and the differences were also significant (F=18.992,P＜0.05).Conclusions Vitronectin can bring about cytoskeleton remodeling,increase in cell migration,and enhancement of blood vessel formation in 人 UVECs conditioned in high glucose.It may be one of the important influence factors of diabetic retinopathy.%背景 玻连蛋白是一种多功能糖蛋白.本研究组前期的动物实验表明,玻连蛋白在缺氧小鼠视网膜中表达明显增高,体外细胞实验表明高糖可以诱导人脐静脉内皮细胞人(UVECs)中玻连蛋白表达的增加.但目前尚未证实玻连蛋白在视网膜新生血管病变中的作用机制. 目的 观察玻连蛋白对高糖培养下人UVECs的细胞骨架结构、细胞迁移、细胞成管能力的影响. 方法 建立高糖培养的人UVECs体外模型,利用干扰RNA技术干扰玻连蛋白的表达.实验分为高糖组(含50 mmol/L葡萄糖的DMEM培养液培养人UVECs)、阴性干扰组(对照用siRNA预干扰人UVECs后,再用含50 mmol/L葡萄糖的DMEM培养液培养)和阳性干扰组(玻连蛋白siRNA预干扰人UVECs后,再用含50 mmol/L葡萄糖的DMEM培养液培养),在各组中分别利用Western blot法检测玻连蛋白的表达量,免疫荧光细胞化学染色结合荧光显微镜观察人UVECs微丝骨架,划痕法观察人UVECs的迁移情况,Matrigel法检测人UVECs的成管能力.各组的总体差异比较采用单因素方差分析,组间的多重比较采用LSD-t检验. 结果 Western blot结果显示,0h时3个组间玻连蛋白表达量的差异无统计学意义(F=1.064,P＞0.05)；培养24 h时高糖组、阴性干扰组、阳性干扰组中玻连蛋白的表达量依次降低,差异有统计学意义(F=15.519,P＜0.05)；培养48 h时高糖组、阴性干扰组、阳性干扰组中玻连蛋白表达量也依次降低,3个组间的差异亦有统计学意义(F=37.521,P＜0.05).免疫荧光结果显示,24 h时高糖组中的细胞微丝骨架结构最明显,阴性干扰组次之,阳性干扰组最不明显；培养48 h细胞微丝骨架分布亦然.划痕实验表明,培养24 h时高糖组、阴性干扰组、阳性干扰组中迁移入划痕区的细胞数量依次减少,3个组间的差异有统计学意义(F=90.685,P＜0.05)；培养48 h时高糖组、阴性干扰组、阳性干扰组中迁移入划痕区的细胞数量也呈递减趋势,3个组间的差异有统计学意义(F=67.880,P＜0.05).Matrigel法证实培养6h高糖组、阴性干扰组、阳性干扰组中细胞形成的血管腔数目逐渐减少,3个组间的差异有统计学意义(F=86.653,P＜0.05)；培养12h时高糖组、阴性干扰组、阳性干扰组中细胞形成的血管腔数目也依次减少,3个组间的差异有统计学意义(F=18.992,P＜0.05). 结论 玻连蛋白可以引起高糖培养的人UVECs发生细胞骨架重塑、细胞迁移能力增加、细胞成管能力增强,可能是糖尿病视网膜病变(DR)新生血管形成的一个重要影响因素.
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