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目的:观察携带目的基因的大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)静脉移植在严重烫伤延迟复苏损伤体内的分布。方法:分离培养MSCs,用Ad-GFP转染MSCs,25只Wistar大鼠随机分为延迟复苏组(A组,10只)、即时复苏组(B组,10只)、假伤组(C组,5只)。A、B两组大鼠背部造成Ⅲ度30%烫伤,制备A组为延迟复苏模型,B组为即时复苏模型,同时制备C组为假伤模型,经股静脉移植转染Ad-GFP 48 h后的MSCs。24 h,7 d后取小肠、肝脏、肾脏、烫伤皮肤创缘等组织,快速冰冻切片,荧光显微镜下观察在体内的分布。结果:MSCs体外分离培养扩增5代,细胞数可达(1~2)×1011个,具有多态性和贴壁生长特性,MOI=100时,Ad-GFP转染MSCs效率可达86.4%。经股静脉移植24 h,在延迟复苏组烫伤皮肤组织创缘,小肠黏膜广泛可见绿色荧光,而在肝、肺等器官少见,即时复苏组荧光以烫伤皮肤创缘分布为主,小肠黏膜较少,假伤组中绿色荧光分布以肝脏为主。延迟复苏组小肠荧光强度明显强于即时复苏组和假伤组(P<0.05),而延迟和即时复苏组皮肤创缘荧光强度又强于假伤组(P<0.05)。结论:导入目的基因可能不会改变MSCs归巢特性,并将为后续启动基因治疗烧伤延迟复苏后的损伤研究提供参考。
OBJECTIVE: To observe the distribution of venous transplanted mesenchymal stem cells (MSCs) carrying the gene of interest in the severely scalded and delayed resuscitation injury. METHODS: MSCs were isolated and cultured, MSCs were transfected with Ad-GFP, and 25 Wistar rats were randomly divided into delayed resuscitation group (A group, 10 rats), immediate resuscitation group (B group, 10 rats) , 5). The rats in group A and B had Ⅲ degree and 30% burns on their backs. The rats in group A were delayed resuscitation, group B was immediate resuscitation, and group C was sham injury. Ad-GFP was transplanted in femoral vein for 48 h After the MSCs. After 24 h and 7 d, small intestine, liver, kidney, wound margin and other wounds were removed and frozen sections were observed. The distribution in the body was observed under a fluorescence microscope. Results: The MSCs were isolated and cultured in vitro for 5 passages, and the number of cells was up to (1 ~ 2) × 1011 with polymorphism and adherent growth characteristics. MSCs transfected with Ad-GFP reached 86.4% . Transplantation of the femoral vein for 24 h delayed the recovery of scalded skin tissue wound margin, the small intestine mucosa can be seen widely green fluorescence, and liver, lung and other organs is rare, real-time resuscitation group scalded skin wound edge distribution, small intestinal mucosa less In the group of false injury, the distribution of green fluorescence was mainly liver. Intestinal fluorescence intensity of delayed resuscitation group was significantly stronger than that of immediate resuscitation group and sham injury group (P <0.05), while delayed wound healing and immediate resuscitation group was significantly stronger than sham injury group (P <0.05). Conclusion: The introduction of the target gene may not change the homing characteristics of MSCs, and will provide a reference for the subsequent study on gene therapy of delayed injury after burn injury.