骨髓间充质干细胞-透明质酸复合物对放射性溃疡创面愈合的影响

Effect of mesenchymal stem cells-hyaluronic acid composition on the full-thickness cutaneous wounds healing of radiation induced ulcer

目的探讨骨髓间充质干细胞(MSCs)-透明质酸(HA)复合物对大鼠放射性溃疡创面的影响及其可能的作用机制。方法分离培养SD大鼠MSCs,以HA为载体构建复合物,观察复合物中MSCs的分化特性。选取30只SD大鼠以Sr-90皮肤敷贴器制作皮肤放射性溃疡模型,臀部对称制作2个直径1cm全层皮肤缺损创面。将大鼠随机分为3组,每组10只。MSCs-HA组:创面涂布200μlMSCs-HA;HA组:创面涂布200μlHA;对照组:创面涂布200μlDMEM培养基。比较各组制创术后第1、2、3、4周的创面愈合率,Ⅷ因子免疫组化微血管计数,创面组织羟脯氨酸含量。苏木精-伊红染色进行组织学观察。结果所有大鼠MSCs与HA共培养后生长良好,复合物中的MSCs可保持其特性:向脂肪细胞分化。制创术后第1周,MSCs-HA组、HA组的创面愈合率分别为(42.20±1.34)%、(34.20±2.06)%,均高于对照组的(23.45±1.66)%(P<0.05),制创术后第2、3周MSCs-HA组高于HA组及对照组(P<0.05),制创术后第4周MSCs-HA组、HA组创面完全愈合,与对照组比较,差异有统计学意义(P<0.05)。制创术后第2周,MSCs-HA组、HA组的微血管计数分别为(24.27±2.84)%、(20.54±2.55)%,均高于对照组的(16.11±2.30)%(P<0.05),制创术后第3、4周MSCs-HA组微血管计数高于HA组及对照组(P<0.05)。制创术后第2周,MSCs-HA组、HA组的羟脯氨酸含量分别为(24.52±0.60)%、(19.41±0.58)%,均高于对照组的(14.19±0.65)%(P<0.05),制创术后第3、4周MSCs-HA组羟脯氨酸含量高于HA组及对照组(P<0.05)。组织学观察显示,制创术后第4周MSCs-HA组表皮层厚度(6～7层)大于HA组(4～5层)及对照组(2～3层)。结论HA与MSCs可促进创面微血管及胶原的增生,MSCs-HA复合物可促进大鼠放射性皮肤溃疡创面愈合。

Objective To study the effect and the mechanism of transplantation of mesenchymal stem cells （MSCs）-hyaluronic acid （HA） composition in repairing radiation induced ulcer. Methods Mesenchymal stem cells were isolated and cultured from SD rats and mixed with HA. The differentiation characteristics of MSCs in the culture were observed. Sr-90 skin applicator was used to construct animal model as radioactive dermal ulcer in 30 SD rats. Two symmetrical full-thickness cutaneous wounds （1.0 cm in diameter） were made on the back hip of each SD rat and randomly divided into MSCs-HA group（n=10, with treatment of 200 μl MSCs- HA）, HA group （n=10, with treatment of 200 μl HA）, and control group （n=10, with treatment of 200μl DMEM culture medium）. Wound healing rate, microvessel counts, hydroxyproline level and histological changes were compared among each group. Results MSCs grew well when cultured with HA.The characteristic of MSCs to differentiation into lipocyte remained in the culture. The wound healing rates in MSCs-HA group [（42.20±1.34）%] and HA group [（34.20±2.06）%] were higher at 1 post-treat week（PTW） than in control group [（23.45± 1.66）%] （P 〈0.05 ）, and that in MSCs-HA group were higher compared with HA group and control group at 2,3 PTW（P 〈0.05 ）. The wound healing rates in MSCs-HA and HA groups were （100±0.00）% at 4 PTW, which were higher than that of control group （P 〈0.05）. The microvessel counts in MSCs- HA group E（24.27±2.84）%] and HA group [（20.54±2.55）%] were higher at2 PTWthanin control group [（16.11 ±2.30）%] （P 〈0.05 ）, and that in MSCs-HA group were higher compared with HA group and control group at 3, 4, PTW（P 〈0.05）. The HYP content in MSCs-HA group [ （24.52±0.60）%] and HA group [ （19.41± 0.58）%] were higher at 2 PTW than in control group [（14.19±0.65）%] （P 〈0.05 ）, and that in MSCs-HA group were higher compared with HA group and control group at 3,4 PTW（P 〈0.05）. Histological observation showed that the epidermis in MSCs-HA group （6-7 layers） was thicker than that in HA group （4-5 layers） and control group （2-3 layers） at 4 PTW. Conclusions HA and MSCs can promote the proliferation of microvascular and collagen in wound; MSCs-HA composition can promote wound healing in radiation induced ulcer rats.