脐血间充质干细胞经转染绿色荧光蛋白后治疗SCID小鼠烫伤的创面效应示踪

摘要

目的 利用该实验室先期已建成的人脐血源间充质干细胞株 (FUCB-MSCs) , 标记上增强型绿色荧光蛋白 (EGFP) , 以对烫伤SCID小鼠行外源性FUCB-MSCs移植治疗的创面区进行移植效应示踪.方法 FUCB-MSCs经腺病毒转染标记GFP;建立深Ⅱ度烫伤SCID小鼠模型, 将小鼠随机分为低剂量移植组、高剂量移植组和空白对照组, 每组6只, 每只2个创面, 共12个创面, 分别将转染后的GFP-MSCs按照0.2mL 1×106个、0.2mL 2×106个以及等体积培养基通过尾静脉注射的方式进行创面移植.移植9d后取各组创面组织行冰冻切片HE染色, 比较各组创面面积和创面细胞数, 荧光显微镜下观察绿色荧光蛋白表达和创面修复损伤情况.结果 FUCB-MSCs经逆转录病毒感染后, 48h荧光显微镜下可见到EGFP表达, 阳性率在80％以上, 且6周后仍能观察到GFP稳定表达, 移植后创面部位可观察到人源性GFP阳性表达, 移植3周后荧光未见衰减;移植标记了GFP的FUCB-MSCs后SCID小鼠未出现排斥反应, 低剂量组、高剂量组与对照组比较, 在创面面积和细胞数上差异均有统计学意义 (P<0.05) , 可提示尾静脉移植注射的GFP-FUCB-MSCs参与了创面愈合.结论利用GFP对人脐血源间充质干细胞移植治疗过程中的标记示踪, 表明烫伤后外源性FUCB-MSCs可通过SCID小鼠血液循环系统, 迁移聚集到烫伤创面持续参与修复.%Objective Using the previously established mesenchymal stem cells strain derived from human fetal umbilical cord blood (FUCB-MSCs) to culture then label enhanced green fluorescent protein (EGFP) , and to observe skin repair effects of FUCB-MSCs by GFP tracing after exogenous FUCB-MSCs transplantation on to scald wound models of SCID mice.Methods FUCB-MSCs were labeled GFP by transfection with the recombinant retrovirus containing EGFP gen;The established SCID scald mice model were randomLy divided into 3groups, low dose group, high dose group and control group, 6rats each group, 2wounds each mouse, 12wounds in total, then were tail intravenous injected into 0.2mL 1×106, 0.2mL 2×106 GFP-FUCB-MSCs cells, and same volume of medium respectively.On 9days after transplantation, the sections from scald wound area were observed the expression of GFP under the fluorescence microscope and the others were analyzed by the bright-field microscopy after HE staining, and the area of wound surface and the number of wound cells were compared simultaneously.Results After 48h, expression of EGFP in FUCB-MSCs can be seen under the fluorescence microscope, positive rate of GFP was>80％, and after 6weeks GFP expression is still stable, besides, the positive expression of human GFP can be observed after transplantation and there were no fluorescence decay in transplantation after 3weeks.Compared with the control group, there was a significant difference in wound area and wound cell number in the low and high-dose group (P<0.05) .ConclusionGFP can be used as a tracking marker to label FUCB-MSCs during transplantation treatment.It indicates that exogenous FUCB-MSCs can migrate to the scalded wounds via blood circulation system and continuously participate in the repair through SCID mouse.