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目的:研究胎膜来源的间充质干细胞在治疗SD大鼠Ⅲ度烧伤中的作用。方法:用胰酶消化法分离足月产胎膜细胞,加入Amino-MAXⅡ培养基进行培养。流式细胞仪检测细胞表面标志物CD29、CD73、CD14、CD45。分别用浓度为3mg/ml、5mg/ml、7mg/ml、9mg/ml的明胶溶液包被脱细胞真皮(ADM),以5×106/ml的密度将细胞种植在包被和未包被的ADM表面,2小时后收集未贴附在ADM上的细胞,计算细胞种植效率。实验组:以7mg/ml的明胶包被ADM,以5×106/ml的密度种植第2～3代细胞,培养3～5天后,移植至SD大鼠Ⅲ度烧伤创面;对照组Ⅰ移植单纯的ADM,对照组Ⅱ未进行移植,术后定期观察创面大体情况,计算创面收缩率、表皮再生面积比例,第5周切取新生皮肤组织行HE染色。结果:胎膜分离的细胞以长梭形为主,表达与间充质干细胞相关的表面抗原标志物CD29、CD73,极少表达造血细胞标志物CD14、CD45。不同浓度明胶溶液包被和未包被的ADM,其细胞种植效率均无明显区别(P>0.05);相比对照组,实验组移植物成活时间更长,创面再上皮化时间较早,创面收缩率较小,再生表皮面积亦较多(P<0.05);再生表皮更厚,有明显的表皮突,真皮层毛细血管丰富。结论:成功分离、培养了胎膜来源的间充质干细胞。胎膜来源间充质干细胞复合ADM可促进SD大鼠Ⅲ烧伤创面表皮再生,加速创伤愈合,抑制创面收缩。
Objective: To study the role of fetal membrane-derived mesenchymal stem cells in the treatment of third degree burn in SD rats. Methods: Full-term fetal membranes were isolated by trypsinization and cultured in Amino-MAXⅡ medium. Cell surface markers CD29, CD73, CD14 and CD45 were detected by flow cytometry. Acellular dermis (ADM) was coated with gelatin solution at concentrations of 3 mg / ml, 5 mg / ml, 7 mg / ml and 9 mg / ml, respectively, and the cells were seeded at a density of 5 x 106 / ml on both coated and uncoated ADM surface, cells not attached to the ADM were collected 2 hours later, and the cell planting efficiency was calculated. Experimental group: ADM was coated with 7mg / ml gelatin, the second to third generation of cells were planted at the density of 5 × 106 / ml, cultured for 3-5 days and then transplanted into third degree burn wounds of SD rats; Of ADM and control group Ⅱ were not transplanted. The general condition of the wound was observed regularly, the rate of wound contraction and the area of epidermis regeneration were calculated, and the new skin tissue was excised by HE staining in the fifth week. Results: The cells isolated from fetal membranes were mainly fusiform-shaped, expressing the surface antigen markers CD29 and CD73 related to mesenchymal stem cells and rarely expressing hematopoietic cell markers CD14 and CD45. Compared with the control group, the graft survival time of the experimental group was longer, the time of wound re-epithelization was earlier, and the wound healing rate The shrinkage rate is smaller, and the area of regenerated epidermis is also more (P <0.05). The regenerated epidermis is thicker with obvious epidermal protrusion and the dermis is rich in capillaries. Conclusion: Membrane-derived mesenchymal stem cells were successfully isolated and cultured. Mesenchymal stem cells derived from fetal membranes can promote epidermal regeneration of burn wounds in SD rats Ⅲ, accelerate wound healing and inhibit wound contractions.