低浓度过氧化氢预处理骨髓间充质干细胞对创面愈合的促进作用

Effect of bone mesenchymal stem cells pre-incubated with low-concentration hydrogen peroxide on promoting wound healing

目的：观察低浓度过氧化氢（H2O2）预处理骨髓间充质干细胞（BMSCs）移植到大鼠组织缺损创面后BMSCs在创面的存活情况及其对创面的促愈合作用。方法：75只SD大鼠，于背部中线靠颈侧制备直径1cm的圆形全层皮肤缺损。按随机数字表法分为3组（每组25只）：生理盐水组、单纯BMSCs组和H2O2预处理BMSCs组。在伤后即刻，分别经尾静脉注射等体积（0．5ml）的生理盐水、CM-Dil标记的BMSOs悬液或50umol／LH2O2预处理12h并经CM—Dil标记的BMSCs。伤后0～15d创面拍照，用ImageProplus5．0图像分析软件分析图像，计算刨面愈合率；伤后1、2、5d荧光显微镜下观察两BMSCs组创面BMSCs的数量；CD31免疫组化染色观察创面微血管密度的变化。结果：①创面干细胞数量：移植H2O2预处理的BMSCs后，创面干细胞数量明显多于单纯BMSCS移植组（P〈0．05和P〈0．01）。与伤后第1天比较，单纯BMSCs组伤后第2天和第5天创面干细胞数量显著减少（P〈0．05）；而预处理组创面干细胞数量虽然也呈进行性减少，但3个时相点创面的干细胞数量差异没有显著性（P〉0．05）。②创面愈合率：创伤后5～15d，单纯BMSCs组和H2O2预处理BMSOs组创面愈合率均明显高于生理盐水组（P〈0．05或P〈0．01）；创伤后5～10d，H2O2预处理组创面愈合率明显高于单纯BMSOs组（P〈0．05）。③创面微血管数量：CD31免疫组化结果显示，伤后3d，两BMSOs治疗组之间血管数量差异无显著性；伤后5、7、10d，H2O2预处理组创面微血管数量显著多于单纯BMSCs组（P〈0．05）。结论：BMSCs经低浓度H2O2预处理再移植到创面，可以延长移植干细胞在创面的存活时间，并具有更好的促进创面愈合和血管生成的修复潜能。

Objective： To observe the survival of transplanted bone marrow mesenchymal stem cells （BMSCs） pre- incubated with low-concentration hydrogen peroxide （H202） in skin wounds of rats with acute skin defect, and to evaluate its efficiency for promoting wound healing. Methods： Seventy-five SD rats with acute full-thickness skin defect （round, 1 cm in diameter） at the dorsal midline were randomly divided into three groups： normal saline （NS） group, BMSCs alone group and H2O2 pre-incubated BMSCs group, with 25 animals in each group. After the excisional wounds were performed, rats in the three groups were immediately injected with normal saline, BMSCs （1 × 107）, and BMSCs （1 ×10^7） pre-incubated by 50μmol/L H2O2 for 12 hours, respectively. In order to trace the transplanted BMSCs, BMSCs were labeled with CM-Dil before transplantation. The wounds were photographed on day 0 to 15, the image was analyzed by Image Proplus 5.0 software, and the healing rate of the wound was calculated. The abundance of transplanted cells at the wound sites was observed under fluorescence microscope on days 1, 2, and 5. Immunohistochemical staining of CD31 was used to evaluate the changes in microvessel density at the wound sites.Results： Compared to the BMSCs alone group, the abundance of transplanted cells at wound sites was significantly increased when BMSCs were pre-incubated by H2O2 before transplantation （P〈0.05 or P〈0.01）. The numbers of BMSCs at the wound sites were significantly decreased on days 2 and 5 after injection of BMSCs compared to day 1 （P〈0.05） in BMSCs alone group, while the decrease was not statistically significant in the H2O2-preincubated BMSCs group （P〉0.05）. The healing rate of wound in BMSCs alone group and H2O2-preincubated BMSCs group were significant higher than that in the NS group on day 5 to day 15 post-injury （P〈0.05 or P〈0.01）, and it was higher in the H2O2-preincubated BMSCs group than in the BMSCs alone group on day 5 to day 10 post-injury （P〈0.05）. The density of microvessels at wound sites showed no significant difference between the two BMSCs groups on day 3 post-injury. However, the microvessel density was significantly increased in H2O2-preincubated BMSCs group compared with the BMSCs simple group on days 5, 7 and 10 post-injury （P〈0.05）. Conelusions： A low-concentration H2O2 pre-incubation could prolong the survival time of implanted BMSCs, and promote angiogenesis and wound healing, thus providing a novel strategy to improve the efficacy of mesenchymal stem cells-based therapy for cutaneous wound.