人脐带间充质干细胞对高糖环境中恒河猴视网膜血管内皮细胞的免疫调节作用  

The immunological regulation effects of human umbilical cord mesenchymal stem cells on RF/6A cultured in high glucose

作　　者：陈莉[1] 陈松[2] 张惟[2] 姜鉴洪 王健[2] 武斌[2] 何广辉[2] 杨婧[2] 马映雪[2] 

机构地区：[1]邵阳市第一人民医院眼科 [2]天津市眼科医院天津市眼科学与视觉科学重点实验室天津市眼科研究所天津医科大学眼科临床学院,300020

出　　处：《中华眼底病杂志》2016年第6期611-614,共4页Chinese Journal of Ocular Fundus Diseases

基　　金：天津市科技计划项目（13ZCZDSY01500）

摘　　要：目的 观察人脐带间充质干细胞（hUCMSC）对高糖培养的恒河猴视网膜血管内皮细胞（RF/6A细胞）的免疫调节作用.方法 利用复合培养体系将hUCMSC加入Transwell系统上层小室,下室加入含有25 mmol/L的葡萄糖及RF/6A细胞;将hUCMSC按照1：1比例与RF/6A细胞共培养.实验分为对照组、高糖组及hUCMSC与高糖共培养组进行,3组RF/6A细胞分别采用Dulbecco改良Eagle培养基（DMEM）/F12完全培养液、DMEM/25 mmol/L D-葡萄糖完全培养液、hUCMSC与25 mmol/LD-葡萄糖完全培养液进行培养.采用噻唑蓝比色法检测共培养后RF/6A细胞的增生活力;蛋白免疫印迹法（Western blot）检测RF/6A细胞中叉头蛋白（Foxp3）的相对表达量;酶联免疫吸附法（ELISA）检测细胞培养上清液中白细胞介素（IL）-17的浓度.结果 培养后第1天,对照组、高糖组及hUCMSC与高糖共培养组RF/6A细胞存活率比较,差异无统计学意义（F=0.030,P＞0.05）.培养后第3、7天,高糖组RF/6A细胞存活率较对照组明显降低,差异有统计学意义（t=36.072、27.890,P＜0.05）;hUCMSC与高糖共培养组RF/6A细胞存活率较高糖组明显提高,差异有统计学意义（t=13.280、19.650,P＜0.05）.Western blot检测结果显示,培养后第7天,高糖组RF/6A细胞中Foxp3蛋白相对表达量较对照组明显下降,差异有统计学意义（t=7.826,P＜0.05）;hUCMSC与高糖共培养组RF/6A细胞中Foxp3蛋白相对表达量较高糖组明显提高,差异有统计学意义（t=19.936,P＜0.05）.ELISA检测结果显示,培养后第7天,高糖组、hUCMSC与高糖共培养组细胞培养上清液中IL-17浓度较对照组明显升高,差异有统计学意义（F=1 267.503,P＜0.05）.hUCMSC与高糖共培养组细胞培养上清液中IL-17浓度较高糖组明显下降,差异有统计学意义（t=17.386,P＜0.05）.结论 hUCMSC能提高被高糖抑制的RF/6A细胞增生活力,可通过调控Foxp3、IL-17表达来平衡修复RF/6A细胞.Objective To observe the immunological regulation effects of human umbilical cord mesenchymal stem cells （hUCMSC） on glucose-damaged rhesus retinal vascular endothelial cells （RF/6A）.Methods hUCMSC and RF/6A were co-culture according to 1：1 ratio in the co-culture system （Transwell plates）,hUCMSC cells were added to upper chamber,while the lower chamber containing 25mmol/L glucose and RF/6A.There were three groups including RF/6A blank control group,high glucose treated RF/6A group,and high glucose treated RF/6A with hUCMSC co-culture group.MTT was used to measure the RF/6A cell viability.Western blot was used to to detect protein level of Foxp3.Enzyme-linked immunosorbent assay （ELISA） was used to detect the concentration of interleukin （IL）-17.Results MTT assay revealed that at the first day,the survival rate of the three groups had no significant difference （F=0.030,P〉0.05）.On day 3 and day 7,the cell viability of the high glucose group was significantly lower than that of the control group（t=36.072,27.890;P〈0.05）,the cell viability of the high glucose treated RF/6A with hUCMSC co-culture group was higher than that of high glucose group （t=36.072,19.650;P〈0.05）.Western blot analysis showed that Foxp3 in high glucose RF/6A group was significantly lower than that in the control group at day 7 after culture （t=7.826,P〈0.05） and high glucose RF/6A with hUCMSC group （t=19.936,P〈0.05）.ELISA showed that IL-17 in the high glucose group,high glucose with hUCMSC co-culture group was significantly higher than that of the control group （F=1 267.503,P〈0.05）,while IL-17 in the hUCMSC co-culture group was significantly lower than that in high glucose group （t=17.386,P〈0.05）.Conclusion hUCMSC can regulate the expression of Foxp3 and IL-17 to increase the proliferative ability of RF/6A,which was suppressed by high glucose.

关 键 词：间质干细胞 内皮细胞/免疫学 免疫调节 动物实验 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]