2型糖尿病患者脂肪源性间充质干细胞对小鼠压疮创面愈合的影响

Effects of adipose-derived mesenchymal stem cells from type 2 diabetes mellitus patients on wound healing of pressure ulcers in mice

目的探讨2型糖尿病患者脂肪源性间充质干细胞(AMSC)对小鼠压疮创面愈合的影响。方法(1)2016年9月,取1例60岁女性2型糖尿病患者的皮下脂肪组织,采取胶原酶消化法提取AMSC培养,取第3代细胞用于后续实验。观察细胞形态,行成骨、成软骨、成脂诱导分化鉴定,运用流式细胞仪检测细胞表面标志物CD90、CD105、CD73及CD34的表达(样本数为3)。(2)取16只6~8周龄雌性C57BL/6野生型小鼠,采用磁铁压迫皮肤的方法在每只小鼠脊柱两侧各制成1个压疮创面,将每只小鼠的2个创面配对分为糖尿病AMSC组和阴性对照组,创面内分别注射100μL磷酸盐缓冲液(PBS)悬浮的绿色荧光蛋白标记的糖尿病AMSC(1×106个)、100μLPBS。观察注射后21d内2组创面的愈合情况并计算注射后5、13、17d的创面愈合率。注射后11、21d分别处死3只小鼠,每组切取3个创面组织,苏木精-伊红染色观察皮肤结构,Masson染色评估胶原沉积情况,免疫组织化学法计数CD31阳性表达即新生血管数。另取2组前述制备的注射后21d创面组织标本,免疫组织化学法检测S100阳性细胞率即施万细胞新生情况。另取糖尿病AMSC组前述制备的注射后11d创面组织标本,采用荧光示踪法观察AMSC定植情况。对数据行配对t检验并进行Bonferroni校正。结果(1)从2型糖尿病患者皮下脂肪组织中分离培养的第3代细胞贴壁生长,多呈长梭形,呈旋涡状生长;经诱导后具有成骨、成软骨、成脂分化功能,细胞表面CD90、CD105、CD73阳性表达率高于90.00%,CD34表达率为0.46%。细胞鉴定为AMSC。(2)小鼠糖尿病AMSC组创面愈合较快,注射后17d所有创面均完全愈合;而小鼠阴性对照组创面此时未完全闭合,表面仍有痂皮。注射后5、13、17d,小鼠糖尿病AMSC组创面愈合率分别为(35.6±6.5)%、(87.1±2.5)%、100.0%,显著高于阴性对照组的(19.8±7.2)%、(66.2±5.2)%、(86.9±5.3)%(t=6.49、14.31、9.73,P<0.05)。与阴性对照组相比,小鼠糖尿病AMSC组创面组织注射后11d炎性细胞浸润减少,注射后21d可见较厚的表皮和真皮以及再生的皮肤附属器。注射后11、21d,小鼠糖尿病AMSC组创面组织胶原百分比分别为(48.3±1.3)%、(54.1±1.7)%,明显高于阴性对照组的(41.4±1.7)%、(50.3±1.2)%(t=6.98、3.99,P<0.01)。注射后11、21d,小鼠糖尿病AMSC组创面组织新生血管数分别为(17.2±1.3)、(18.0±2.1)个,明显多于阴性对照组的(8.0±1.4)、(14.0±1.5)个(t=10.69、3.38,P<0.01)。注射后21d,小鼠糖尿病AMSC组创面组织S100阳性细胞率为(1.76±0.12)%,明显高于阴性对照组的(0.55±0.03)%(t=21.68,P<0.001)。注射后11d,小鼠糖尿病AMSC组创面组织仍有AMSC定植。结论2型糖尿病患者AMSC移植可通过促进血管再生、胶原沉积及施万细胞再生进而加快小鼠压疮创面愈合。

To investigate the effects of adipose-derived mesenchymal stem cells (AMSCs) from type 2 diabetes mellitus patients on wound healing of pressure ulcers in mice. Methods (1) In September 2016, the subcutaneous adipose tissue of a 60-year-old woman with type 2 diabetes mellitus was harvested, and then AMSCs were extracted by collagenase digestion and cultured. The third passage of cells were used for subsequent experiments. The morphology of cells was observed, and their osteogenic, chondrogenic, and adipogenic differentiation abilities were identified. The expressions of cell surface markers CD90, CD105, CD73, and CD34 were detected by flow cytometer (n=3). (2) Sixteen female C57BL/6 wild-type mice aged 6-8 weeks were selected, and one pressure ulcer wound was created on each side of the spine of each mouse by pressing the skin with two magnets. The two wounds of each mouse were paired and divided into diabetic AMSCs group and negative control group, injected with 100 μL phosphate buffer solution (PBS) containing green fluorescent protein-labeled AMSCs (1×106 cells) and 100 μL PBS, respectively. The wound healing status of the two groups within post injection day (PID) 21 was observed, and their wound healing rates on PID 5, 13, and 17 were calculated. Three mice were sacrificed on PID 11 and 21, respectively, and tissue of three wounds was harvested from each group. The skin structure was observed by hematoxylin-eosin staining, the collagen deposition was evaluated by Masson staining, and the positive expression of CD31, i. e., the number of new blood vessels was counted by immunohistochemistry. Wound tissue samples of two groups prepared on PID 21 as above-mentioned were harvested, and the positive cell rate of S100, representing the regeneration of Schwann cells, was detected by immunohistochemistry. Wound tissue samples of diabetic AMSCs group prepared on PID 11 as above-mentioned were harvested, and the colonization of AMSCs was observed by fluorescence tracer method. Data were processed with paired t test and Bonferroni correction. Results (1) The third passage of cells isolated and cultured from the subcutaneous adipose tissue of a type 2 diabetes mellitus patient grew adherently to the wall in a long spindle and vortex-like manner. After induction, the cells showed osteogenic, chondrogenic, and lipogenic differentiation abilities. The positive expression rates of CD90, CD105, and CD73 on the cell surface were higher than 90.00%, and the expression rate of CD34 was 0.46%. The cells were identified as AMSCs. (2) The mice wounds of diabetic AMSCs group healed quickly, and all the wounds healed completely on PID 17, while the mice wounds in negative control group were not completely closed at this time, and there was still scab on the surface. On PID 5, 13, and 17, the healing rates of mice wounds of diabetic AMSCs group were (35.6±6.5)%, (87.1±2.5)%, and 100.0%, respectively, significantly higher than (19.8±7.2)%, (66.2±5.2)%, and (86.9±5.3)% of negative control group (t=6.49, 14.31, 9.73, P<0.05). Compared with that of negative control group, the inflammatory cell infiltration was reduced in mice wounds tissue of diabetic AMSCs group on PID 11, and thicker epidermis and dermis as well as regenerated skin appendages were observed on PID 21. On PID 11 and 21, the collagen percentages of mice wounds tissue in diabetic AMSCs group was (48.3±1.3)% and (54.1±1.7)%, respectively, significantly higher than (41.4±1.7)% and (50.3±1.2)% of negative control group (t=6.98, 3.99, P<0.01). On PID 11 and 21, the numbers of new blood vessels in mice wounds tissue of diabetic AMSCs group were 17.2±1.3 and 18.0±2.1, respectively, significantly more than 8.0±1.4 and 14.0±1.5 of negative control group (t=10.69, 3.38, P<0.01). On PID 21, the S100 positive cell percentage in mice wounds tissue of diabetic AMSCs group was (1.76±0.12)%, significantly higher than (0.55±0.03)% of negative control group (t=21.68, P<0.001). On PID 11, the colonization of AMSCs in mice wounds tissue of diabetic AMSCs group was observed. Conclusions Transplantation of AMSCs from type 2 diabetic mellitus patients can accelerate wound healing of pressure ulcers in mice by promoting angiogenesis, collagen deposition, and Schwann cell regeneration.