异体糖尿病大鼠来源脂肪干细胞移植治疗糖尿病大鼠创面的初步评价及其机制探讨  

Preliminary evaluation and mechanism of adipose-derived stem cell transplantation from allogenic diabetic rats in the treatment of diabetic rat wounds

作　　者：董叫云[1] 弓家弘 嵇晓芸[1] 田鸣[1] 刘英开[1] 青春[1] 陆树良[1] 宋菲[1] Dong Jiaoyun;Gong Jiahong;Ji Xiaoyun;Tian Ming;Liu Yingkai;Qing Chun;Lu Shuliang;Song Fei(Wound Repair Center,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China;Department of Orthopaedics,United Family Healthcare,Shanghai 200336,China)

机构地区：[1]上海交通大学医学院附属瑞金医院创面修复中心,200025 [2]上海和睦家医院骨科,200336

出　　处：《中华烧伤杂志》2019年第9期645-654,共10页Chinese Journal of Burns

基　　金：国家自然科学基金(81671916);重大疾病防治科学行动计划创伤修复专项(2017ZX1001-S15).

摘　　要：目的探讨异体糖尿病大鼠来源脂肪干细胞(ASC)是否对糖尿病大鼠创面有促愈作用及其机制。方法(1)取56只12~16周龄雄性Wistar大鼠,按随机数字表法(分组方法下同)分为糖尿病组和健康组,每组28只。健康组大鼠不进行任何处理;糖尿病组大鼠于腹腔一次性注射10 g/L链脲佐菌素60 mg/kg,建立糖尿病模型。按随机数字表法取健康组和糖尿病组大鼠各4只,取腹股沟处脂肪组织,培养纯化ASC以获得健康大鼠来源ASC(下称nASC)和糖尿病大鼠来源ASC(下称dASC)。取第3代nASC和dASC(样本数均为3),流式细胞仪检测细胞表面分化抗原CD105、CD31、CD34及CD44的阳性表达率,确定纯度。(2)取健康组和糖尿病组剩余大鼠各24只,每只大鼠背部制作3个直径12 mm的圆形全层皮肤缺损创面。伤后即刻,分别于每只大鼠3个创面及其创缘皮下注射磷酸盐缓冲液(PBS)、2×10^7个/mL的nASC、2×10^7个/mL的dASC各0.5 mL。伤后1、3、7、12 d,按随机数字表法每组各取6只大鼠计算创面面积;取创面组织行苏木精-伊红染色,观察创面组织学形态。(3)对人ASC(hASC)进行传代培养,采用第4~7代细胞进行后续实验。将hASC分为7组,每组12个样本。空白对照组细胞用间充质干细胞培养液培养,单纯晚期糖基化终末产物(AGE)组、单纯蛋白组、单纯高糖组、单纯高渗透压组、AGE高糖联合组、蛋白高渗透压联合组细胞分别用加入终质量浓度100 mg/L AGE、100 mg/L牛血清白蛋白(BSA)、28 mmol/L D-葡萄糖、28 mmol/L甘露醇、100 mg/L AGE+28 mmol/L D-葡萄糖、100 mg/L BSA+28 mmol/L甘露醇的间充质干细胞培养液培养。培养2 h及2、4、6 d,采用细胞计数试剂盒8法检测细胞增殖情况。(4)取hASC,分为空白对照组、单纯AGE组、单纯高糖组、AGE高糖联合组,每组12个样本,同实验(3)相应组处理。培养0、2、4、6 d,流式细胞仪检测细胞表面分化抗原CD105、CD44、CD45阳性表达率以确定其稳�Objective To investigate whether adipose-derived stem cells (ASCs) from allogeneic diabetic rats can promote wound healing in diabetic rats or not and the mechanism. Methods (1) Fifty-six male Wistar rats aged 12-16 weeks were divided into diabetic group and healthy group according to the random number table (the same grouping method below), with 28 rats in each group. Rats in healthy group were not treated with any treatment. Rats in diabetic group were injected with 10 g/L streptozotocin 60 mg/kg intraperitoneally in one time to establish the diabetic model. Four rats in diabetic group and 4 rats in healthy group were selected according to the random number table, and the adipose tissue in the inguinal region was taken to culture and purify ASCs, so as to obtain healthy rat-derived ASCs (hereinafter referred to as nASCs) and diabetic rat-derived ASCs (hereinafter referred to as dASCs). The third passage of nASCs (n=3) and dASCs (n=3) were taken, and the positive expression rates of cell surface differentiation antigens CD105, CD31, CD34, and CD44 were detected with flow cytometer for defining ASCs purity.(2) The rest 24 rats in healthy group and 24 rats in diabetic group were used to make three round full-thickness skin defect wounds with a diameter of 12 mm on the back of each rat. Immediately after injury, phosphate buffer saline (PBS), nASCs of 2×10^7/mL, and dASCs of 2×10^7/mL each in the volume of 0.5 mL were subcutaneously injected into three wounds and their margins of each rat, respectively. On post injury day (PID) 1, 3, 7, and 12, 6 rats in each group were selected according to the random number table to calculate the wound area, and the wound tissue was stained with hematoxylin-eosin to observe the histological morphology of the wound.(3) Human ASCs (hASCs) were subcultured, and the 4th to 7th passage of cells were used for the subsequent experiments. The hASCs were divided into 7 groups, with 12 samples in each group. Cells in blank control group were cultured with mesenchymal stem cell culture me

关 键 词：糖尿病 伤口愈合 脂肪干细胞 晚期糖基化终末产物 高糖 

分 类 号：R587.2[医药卫生—内分泌] R641[医药卫生—内科学] R-332[医药卫生—临床医学]