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目的观察人脐带MSCs(human umbilical cord MSCs,hUCMSCs)在海藻酸盐凝胶支架中的生长特性,探讨hUCMSCs-海藻酸盐敷料修复创面的可行性。方法取人脐带标本体外分离培养hUCMSCs,取第4代细胞与海藻酸钙凝胶支架复合培养(实验组)。倒置相差显微镜下观察细胞在凝胶支架中的生长特征,培养第0、3、6、9天检测细胞上清液VEGF含量并行细胞计数,并检测细胞迁移能力;以正常培养hUCMSCs作为对照组。取32只8周龄Balb/c雄性小鼠,制备全层皮肤缺损模型,随机分为4组(n=8);创面分别以hUCMSCs-海藻酸钙凝胶复合物(MSC-凝胶组)、细胞上清液-海藻酸钙凝胶复合物(CS-凝胶组)、10%FBS-海藻酸钙凝胶复合物(FBS-凝胶组)及0.01 mol/L PBS-海藻酸钙凝胶复合物(PBS-凝胶组)修复。观察创面愈合情况,计算第5、10、15天创面愈合率;于第15天切取创面组织行组织学及免疫组织化学染色,评估新生皮肤情况。结果 hUCMSCs在凝胶支架中可生长增殖达1周以上,形似葡萄样;细胞迁移实验示7 d内未见细胞从凝胶支架中迁移出。培养第3天实验组细胞数及VEGF表达量均明显少于对照组(P<0.05),之后逐渐增加,至第9天时均明显多于对照组(P<0.05)。动物实验显示,与FBS-凝胶组及PBS-凝胶组相比,MSC-凝胶组及CS-凝胶组第5、10、15天创面愈合率均显著增高(P<0.05),新生皮肤鳞状上皮、成纤维细胞、皮脂腺及毛细血管均增多,第15天时VEGF表达量也显著增加(P<0.05);而MSC-凝胶组及CS-凝胶组观察情况相似。结论 hUCMSCs能在海藻酸钙凝胶中持续表达创面愈合所必需的VEGF,hUCMSCs-海藻酸钙凝胶复合物能促进小鼠创面愈合,有望成为理想的创面敷料。
OBJECTIVE: To observe the growth characteristics of human umbilical cord MSCs (hUCMSCs) in alginate gel scaffolds and to explore the feasibility of using hUCMSCs-alginate dressing to repair wounds. Methods Human umbilical cord hUCMSCs were isolated from human umbilical cord in vitro and the fourth generation cells were cultured with calcium alginate gel (experimental group). The growth characteristics of the cells in the gel scaffolds were observed under inverted phase contrast microscope. The VEGF levels in the supernatant of the cells were detected on day 0, day 3, day 6, day 6, and the cell counts were counted. The hUCMSCs were cultured as normal control group. Thirty-two male Balb / c mice of 8 weeks old were used to prepare full-thickness skin defect models and were randomly divided into 4 groups (n = 8). The wounds were treated with hUCMSCs-calcium alginate gel complex , Cell supernatant-calcium alginate gel complex (CS-gel group), 10% FBS-calcium alginate gel complex (FBS-gel group) and 0.01 mol / L PBS-calcium alginate gel Glue complex (PBS-gel group) was repaired. The wound healing was observed. The wound healing rate was calculated on the 5th, 10th and 15th days. On the 15th day, the wounds were excised for histological and immunohistochemical staining to evaluate the new skin condition. Results hUCMSCs could grow and proliferate for more than 1 week in the gel scaffold, which was similar to grape-like samples. The cell migration assay showed that no cells migrated from the gel scaffold within 7 days. On the third day, the number of cells and the expression of VEGF in the experimental group were significantly less than those in the control group (P <0.05), and then gradually increased on the third day (P <0.05). Animal experiments showed that wound healing rates of MSC-gel group and CS-gel group at 5, 10 and 15 days were significantly higher than that of FBS-gel group and PBS-gel group (P <0.05) Skin squamous epithelium, fibroblasts, sebaceous glands and capillaries increased, and the expression of VEGF increased significantly on the 15th day (P <0.05), while the MSC-gel group and the CS-gel group were similar. Conclusion hUCMSCs can continuously express VEGF, which is necessary for wound healing in calcium alginate gel. HUCMSCs-calcium alginate gel complex can promote wound healing in mice and is expected to be an ideal wound dressing.