负载人脐带间充质干细胞的水凝胶对糖尿病小鼠皮肤创面愈合的疗效

Effects of hydrogel loaded with human umbilical cord mesenchymal stem cells on diabetic wound repair in mice

背景:目前,缺乏有效的治疗方法促进糖尿病患者的皮肤创面愈合。人脐带间充质干细胞已被证明对皮肤再生具有多种治疗作用,可注射水凝胶具有良好的生物相容性和可调节性,可以提高干细胞治疗的效果。目的:以负载人脐带间充质干细胞的水凝胶为切入点,观察其对小鼠糖尿病皮肤创面的疗效,并探索可能的作用机制。方法:①链脲佐菌素溶液连续腹腔注射5 d构建C57BL/6J小鼠糖尿病模型,经尾静脉采血测血糖值评估模型是否建立成功。②造模成功后,利用打孔器建立小鼠背部皮肤损伤模型,水凝胶组给予纯水凝胶敷胶治疗,复合水凝胶组给予负载有人脐带间充质干细胞的水凝胶治疗,对照组给予等量生理盐水治疗,治疗第7,14天观察创面愈合情况。结果与结论:①敷胶后的14 d内,水凝胶组、复合水凝胶组小鼠皮肤创面面积明显低于对照组(P<0.05),复合水凝胶组的皮肤创面面积明显低于水凝胶组(P<0.05);②苏木精-伊红染色结果显示:复合水凝胶组创面肉芽组织新生率明显高于水凝胶组和对照组(P<0.05);③Masson染色结果显示:与对照组和水凝胶组相比,复合水凝胶组创面组织胶原沉积率明显增加(P<0.05);④免疫组化CD31染色结果显示:复合水凝胶组创面组织中新生微血管数量明显高于水凝胶组和对照组(P<0.05);⑤免疫组化CD45染色结果显示:与对照组和水凝胶组相比,复合水凝胶组创面组织中炎症面积明显减少(P<0.05);⑥免疫荧光染色和qRT-PCR结果显示:与对照组和水凝胶组相比,复合水凝胶组创面组织中M2巨噬细胞及其标志性因子白细胞介素10表达显著升高,且M1巨噬细胞及其标志性因子白细胞介素6表达明显降低(P<0.05);⑦上述结果提示:负载人脐带间充质干细胞的氯化壳聚糖-β-甘油磷酸钠复合水凝胶通过促进糖尿病创面肉芽组织和微血管形成,减轻炎症反应,从而促进创面愈合。

BACKGROUND:There is a lack of effective treatment methods to promote the healing of the skin wounds of diabetic patients.Human umbilical cord mesenchymal stem cells have been proven to have a variety of therapeutic effects on skin regeneration.Injectable hydrogels have good biocompatibility and adjustability,which can improve the effect of stem cell therapy.OBJECTIVE:Taking the hydrogel loaded with human umbilical cord mesenchymal stem cells as a starting point,to observe its curative effect on diabetic skin wounds in mice,and explore the possible mechanism of action.METHODS:(1)A diabetic model of C57BL/6J mouse was constructed by continuous intraperitoneal injection of streptozotocin solution for 5 days,and the success of the model was evaluated by blood glucose values collected from the tail vein.(2)A mouse back skin injury model was established by using a puncher.The hydrogel group was treated with pure hydrogel.The composite hydrogel group was treated with hydrogel loaded with human umbilical cord mesenchymal stem cells.The control group was treated with the same amount of normal saline.The wound healing was observed on the 7th and 14th days of treatment.RESULTS AND CONCLUSION:(1)The skin wound area of mice in the composite hydrogel group and hydrogel group was significantly smaller than that in the control group at 14 days after gum application(P<0.05).The skin wound area of mice was significantly smaller in the composite hydrogel group than that in the hydrogel group(P<0.05).(2)Hematoxylin-eosin staining results illustrated that the new granulation tissue in the composite hydrogel group was significantly more than that in the control group and hydrogel group(P<0.05).(3)Masson’s staining results clarified that the collagen deposition in the composite hydrogel group was significantly increased compared with the control group and hydrogel group(P<0.05).(4)The results of immunohistochemical CD31 staining confirmed that the number of new microvessels in the skin trauma was significantly higher in the composite hydrogel group than that in the control group and hydrogel group(P<0.05).(5)The results of immunohistochemical CD45 staining showed that the area of inflammation in the new skin was significantly reduced in the composite hydrogel group compared with the control group and hydrogel group(P<0.05).(6)The results of immunofluorescence staining and qRT-PCR showed that compared with the control group and hydrogel group,the amount of M2 macrophages and the level of interleukin 10 in the trauma tissue were significantly higher,and the amount of M1 macrophages and the level of interleukin 6 were significantly lower in the composite hydrogel group(P<0.05).(7)The above results illustrate that the chlorinated chitosan-β-sodium glycerophosphate composite hydrogel loaded with human umbilical cord mesenchymal stem cells can promote the formation of granulation tissue and microvessels on diabetic wounds,reduce inflammatory response,and promote wound repair.