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

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态。(5)取hASC,分为AGE高糖联合组和蛋白高渗透压联合组,每组9个样本,同实验(3)相应组处理。培养2、4、6 d,采用花青素3-链霉亲和素双抗体夹心技术检测细胞内蛋白表达水平。对数据进行析因设计方差分析、LSD检验及Bonferroni校正。结果(1)nASC、dASC的CD44阳性表达率均>96%,CD31、CD34阳性表达率低,CD105阳性表达率为40%左右,基本符合纯度要求。(2)2组大鼠组内3种方式处理创面伤后1 d面积均相近(P>0.05)。与组内PBS处理创面的(0.682 1±0.078 9)、(0.314 3±0.113 7)、(0.064 3±0.002 1)cm^2比较,健康组大鼠nASC处理创面面积于伤后3、7、12 d显著缩小[(0.464 1±0.092 6)、(0.223 9±0.072 7)、(0.034 3±0.012 5)cm^2,P<0.05];健康组大鼠dASC处理创面面积于伤后3、12 d显著缩小[(0.514 1±0.124 1)、(0.043 7±0.032 8 )cm^2,P<0.05],伤后7 d无明显改变[(0.274 2±0.062 5)cm^2,P>0.05]。与组内dASC处理创面比较,健康组大鼠nASC处理创面面积于伤后3、7 d显著缩小(P<0.05),伤后12 d无明显改变(P>0.05)。与组内PBS处理创面的(0.853 5±0.204 8)、(0.670 5±0.164 8)、(0.131 4±0.074 4)cm2比较,糖尿病组大鼠nASC处理创面面积于伤后3、7、12 d显著缩小[(0.633 4±0.132 5)、(0.331 8±0.023 5)、(0.074 2±0.003 8)cm^2,P<0.05];糖尿病组大鼠dASC处理创面面积于伤后3 d显著缩小[(0.773 6±0.182 2)cm2,P<0.05],伤后7、12 d无明显改变[(0.510 6±0.192 2)、(0.114 4±0.003 1)cm^2,P>0.05]。与组内dASC处理创面比较,糖尿病组大鼠nASC处理创面面积伤后3、7 d无明显改变(P>0.05),伤后12 d显著缩小(P<0.05)。伤后1 d各组大鼠组内3种方式处理创面组织学形态无明显差异。伤后3 d,2组大鼠nASC、dASC处理创面都有少量微血管形成,PBS处理创面几乎未见微血管形成。伤后7 d,2组大鼠nASC、dASC处理创面内可见较多小血管及成纤维细胞(Fb),PBS处理创面小血管及Fb略少。伤后12 d,2组大鼠nASC、dASC处理创面已基本被上皮组织覆盖;健康组大鼠PBS处理创面内肉芽组织不明显,糖尿病组大鼠PBS处理创面未全部上皮化。(3)与空白对照组比较,单纯AGE组hASC细胞量于培养2、4、6 d显著减少(P<0.05),单纯高糖组hASC细胞量于培养2、4 d显著增加(P<0.05),AGE高糖联合组hASC细胞量于培养4、6 d时显著减少(P<0.05)。(4)与组内培养4 d比较,空白对照组、单纯AGE组、AGE高糖联合组hASC培养6 d时CD105阳性表达率明显下降(P<0.05)。各组hASC各时间点CD44阳性表达率均>95%,CD45阳性表达率均<2%。(5)共有7种蛋白检测值位于可信区间。2组hASC中碱性成纤维细胞生长因子、基质金属蛋白酶组织抑制剂1表达水平均随培养时间延长呈上升趋势。AGE高糖联合组hASC中人单核细胞趋化蛋白1(MCP-1)表达水平随培养时间的延长呈上升趋势,生长相关性癌基因(GRO)表达水平则在培养6 d时显著高于组内培养4 d时(P<0.05);蛋白高渗透压联合组hASC中MCP-1、GRO表达水平均随培养时间延长呈下降趋势。蛋白高渗透压联合组hASC中卵泡抑素表达水平在培养4 d时明显下降,AGE高糖联合组hASC中卵泡抑素表达水平则在培养6 d时显著低于组内培养4 d时(P<0.05)。蛋白高渗透压联合组hASC中血管内皮生长因子(VEGF)表达水平随培养时间延长逐渐下降,AGE高糖联合组hASC中VEGF表达水平在培养4 d时较组内培养2 d时显著下降(P<0.05)。蛋白高渗透压联合组hASC中尿激酶型纤溶酶原激活剂受体表达水平在培养6 d时较组内培养4 d时显著升高(P<0.05),且显著高于AGE高糖联合组培养6 d时(P<0.05)。结论nASC和dASC对大鼠单纯缺损伤创面都有促愈作用,但dASC对合并糖尿病大鼠创面的促愈作用不显著,这可能与高糖及AGE干预可抑制ASC增殖并影响其稳态及分泌功能有关。

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 medium, and cells in simple advanced glycation end products (AGEs) group, simple protein group, simple high glucose group, simple high osmotic pressure group, AGEs-high glucose combination group, and protein-high osmotic pressure combination group were cultured with mesenchymal stem cell culture medium containing a final mass concentration of 100 mg/L AGEs, 100 mg/L bovine serum albumin (BSA), 28 mmol/L D-glucose, 28 mmol/L mannitol, 100 mg/L AGEs+ 28 mmol/L D-glucose, 100 mg/L BSA+ 28 mmol/L mannitol, respectively. Cell proliferation was detected by cell counting kit 8 at post culture hour (PCH) 2 and on post culture day (PCD) 2, 4 and 6.(4) The hASCs were divided into blank control group, simple AGE group, simple high glucose group, and AGE-high glucose combination group, with 12 samples in each group, which were treated the same as corresponding groups in experiment (3). On PCD 0, 2, 4, and 6, the positive expression rates of cell surface differentiation antigens CD105, CD44, and CD45 were detected by flow cytometer to estimate their homeostasis.(5) The hASCs were divided into AGE-high glucose combination group and protein-high osmotic pressure combination group, with 9 samples in each group, which were treated the same as corresponding groups in experiment (3). On PCD 2, 4, and 6, the expression of intracellular protein was detected by cyanine 3-streptavidin double-antibody sandwich technique. Data were processed with analysis of variance for factorial design, least significant difference test, and Bonferroni correction. Results (1) The positive expression rates of CD44 in nASCs and dASCs were both higher than 96%, the positive expression rates of CD31 and CD34 were low, and the positive expression rates of CD105 were about 40%, which basically met the purity requirements.(2) The areas of wounds treated by three methods in rats of healthy group and diabetic group were similar on PID 1 (P>0.05). In healthy group, compared with (0.682 1±0.078 9),(0.314 3±0.113 7), and (0.064 3±0.002 1) cm^2 of the PBS-treated wounds in rats, the area of nASCs-treated wounds in rats decreased significantly on PID 3, 7, and 12 [(0.464 1±0.092 6),(0.223 9±0.072 7), and (0.034 3±0.012 5) cm^2, P<0.05], the area of dASCs-treated wounds in rats decreased significantly on PID 3 and 12 [(0.514 1±0.124 1) and (0.043 7±0.032 8) cm^2, P<0.05] but was not obviously changed on PID 7 [(0.274 2±0.062 5) cm2, P>0.05]. Compared with those of the dASCs-treated wounds of rats within the same group, the area of the nASCs-treated wounds of rats in healthy group decreased significantly on PID 3 and 7 (P<0.05) but was not obviously changed on PID 12 (P>0.05). In diabetic group, compared with (0.853 5±0.204 8),(0.670 5±0.164 8), and (0.131 4±0.074 4) cm^2 of the PBS-treated wounds in rats, the area of nASCs-treated wounds in rats decreased significantly on PID 3, 7, and 12 [(0.633 4±0.132 5),(0.331 8±0.023 5), and (0.074 2±0.003 8) cm^2, P<0.05], the area of dASCs-treated wounds in rats decreased significantly on PID 3 [(0.773 6±0.182 2) cm^2, P<0.05] but was not obviously changed on PID 7 and 12 [(0.510 6±0.192 2) and (0.114 4±0.003 1) cm^2, P>0.05]. Compared with the dASCs-treated wounds of rats within the same group, the area of the nASCs-treated wounds of rats in diabetic group was not obviously changed on PID 3 and 7 (P>0.05) but decreased significantly on PID 12 (P<0.05). There was no obvious difference in histological morphology of the wounds treated with three methods in rats of each group on PID 1. On PID 3, a small amount of microvessels were formed in the wounds treated with nASCs and dASCs of rats in both groups, but microvessel formation was almost undetected in the PBS-treated wounds. On PID 7, more small blood vessels and fibroblasts (Fbs) were observed in the wounds treated with nASCs and dASCs of rats in both groups, but the small blood vessels and Fbs were slightly less in the PBS-treated wounds. On PID 12, the wounds treated with nASCs and dASCs of rats in the two groups were covered by epithelial tissue, the granulation tissue in the PBS-treated wounds of rats in healthy group was not obvious, and the PBS-treated wounds of rats in diabetic group were not completely epithelialized.(3) Compared with those of blank control group, the cell number of hASCs in simple AGEs group decreased significantly on PCD 2, 4, and 6 (P<0.05), which increased significantly on PCD 2 and 4 in simple high glucose group (P<0.05), and that in AGEs-high glucose combination group decreased significantly on PCD 4 and 6 (P<0.05).(4) Compared with that on PCD 4 within the same group, the positive expression rate of CD105 in hASCs decreased significantly in blank control group, simple AGEs group, and AGEs-high glucose combination group on PCD 6 (P<0.05). The positive expression rate of CD44 was higher than 95%, and that of CD45 was less than 2% in hASCs of each group at each time point.(5) Detection values of 7 proteins were located in the confidence interval. The expression levels of basic fibroblast growth factor and tissue inhibitor of metalloproteinase-1 in hASCs of AGEs-high glucose combination group and protein-high osmotic pressure combination group showed increasing trend with the prolongation of culture time. The expression level of human monocyte chemoattractant protein 1 (MCP-1) in hASCs of AGEs-high glucose combination group showed increasing trend with the prolongation of culture time, while the expression level of growth-regulated oncogene (GRO) on PCD 6 was significantly higher than that on PCD 4 within the same group (P<0.05);the expression levels of MCP-1 and GRO in hASCs of protein-high osmotic pressure combination group showed decreasing trend with the prolongation of culture time. The expression level of follistatin in hASCs of protein-high osmotic pressure combination group decreased obviously on PCD 4, while that in hASCs of AGEs-high glucose combination group was significantly lower on PCD 6 than that on PCD 4 (P<0.05). The expression level of vascular endothelial growth factor (VEGF) in hASCs of protein-high osmotic pressure combination group decreased gradually with the prolongation of culture time, while that in hASCs of AGEs-high glucose combination group on PCD 4 decreased significantly as compared with that on PCD 2 (P<0.05). The expression level of urokinase-type plasminogen activator receptor in hASCs of protein-high osmotic pressure combination group on PCD 6 was significantly higher than that on PCD 4 within the same group (P<0.05) and that of AGEs-high glucose combination group on PCD 6 (P<0.05). Conclusions Both nASCs and dASCs can promote wound healing in rats with simple defect injury, but dASCs have no significant effect on wound healing in rats with diabetes mellitus, which may be related to the inhibition of ASCs proliferation and the influence of high glucose and AGEs intervention on their homeostasis and secretory function.