骨髓间充质干细胞来源的外泌体对糖尿病大鼠创面炎症因子的影响

Effects of exosomes derived from bone marrow mesenchymal stem cells on inflammatory factors in wounds of diabetic rats

目的探讨骨髓间充质干细胞(BMSCs)来源的外泌体移植后,对糖尿病大鼠创面愈合及炎症因子的影响。方法全骨髓贴壁法分离BMSCs,超速离心法提取BMSCs外泌体。选取清洁SD大鼠30只构建糖尿病大鼠创伤模型,成功建模后,将存活大鼠分为对照组(n=13)、外泌体组(n=14);采取尾静脉途径,予外泌体组大鼠0.2 mg/ml外泌体悬液1 ml,同时予对照组等剂量PBS溶液注射;采用Image J软件分析大鼠的创面愈合率,并通过HE染色评估创面组织中炎症程度;采用酶联免疫分析法(ELISA)检测大鼠创面组织中TNF-α、IL-1β、IL-10的浓度;采用Western blot检测创面组织中TLR-4的表达。结果流式细胞术鉴定BMSCs表面抗原,CD29、CD90高表达,CD40低表达;超速离心法获取的BMSCs外泌体直径分布于60~160 nm;与对照组比较,外泌体组大鼠创面愈合率较高,且组织中炎性因子浓度较低,TLR-4表达较低(均P<0.05)。结论BMSCs外泌体可通过调控TLR-4的表达降低糖尿病大鼠创面组织的炎症程度。

Objective To investigate the effects of bone marrow mesenchymal stem cells(BMSCs)-derived exosomes on wound healing and inflammatory factors in diabetic rats.Methods BMSCs were isolated by whole bone marrow adherence method,and BMSCs exosomes were extracted by ultracentrifugation.Thirty clean SD rats were selected to construct the diabetic rat trauma model.After successful modeling,the surviving rats were divided into a control group(n=13)and an exosome group(n=14).In the exosome group,1 ml of exosome suspension(0.2 mg/ml)was injected into the caudal vein;in the control group,the same dose of PBS solution was injected.Image J software was used to analyze the wound healing rate of the rats,and HE staining was used to analyze the degree of inflammation in the wound tissue;Enzyme-linked immunoassay(ELISA)was used to detect the concentrations of TNF-α,IL-1β,and IL-10 in wound tissues of the rats;Western blot was used to detect the expression of TLR-4 in wound tissues.Results Flow cytometry identified BMSCs surface antigens,high expression of CD29 and CD90,low expression of CD40;the diameter of BMSCs exosomes obtained by ultracentrifugation was distributed at 60-160 nm;compared with the control group,the wound healing rate of the rats in the exosome group was higher,and the concentration of inflammatory factors in the tissue was lower,and the expression of TLR-4 was lower(all P<0.05).Conclusion BMSCs exosomes can reduce the degree of inflammation in diabetic rat wound tissues by regulating the expression of TLR-4.