人脂肪干细胞与表皮生长因子促进皮肤创面愈合的研究

Human adiposederived stem cells and epidermal growth factor promoting skin wound healing

目的观察人脂肪干细胞(hASCs)与人表皮生长因子(hEGF)对皮肤缺损创面修复的影响。方法选择郑州大学第二附属医院2020年1月6例患者抽脂减肥的脂肪组织作为hASCs的来源,以酶消化法从人脂肪组织中提取hASCs,将其培养至第3代。利用倒置显微镜对细胞形态进行观察;使用流式细胞仪鉴定细胞表型及分化能力检测。随机将24只新西兰大白兔建立背部全层皮肤缺损模型,并将实验用新西兰大白兔随机分为3组,设立实验组(hASCs+hEGF组),细胞治疗对照组(hASCs组)和空白对照组[磷酸盐缓冲液(PBS)组]。实验组予以100 μl PBS重悬第3代脂肪来源干细胞(1×106个),以10 μg/L加入人表皮细胞生长因子;细胞治疗对照组予以100 μl PBS重悬第3代脂肪来源干细胞(1×106个);空白对照组取PBS,各组均注射600 μl局部移植至创面边缘及基底部。大体观察创面愈合情况,计算创面面积占初始创面面积百分比;创面组织进行标本苏木精-伊红(HE)染色,免疫组织化学CD31染色检测等方法观察和比较各组创面愈合水平。多样本均数比较采用方差分析,采用t检验对比组间计量资料。结果相差显微镜观察hASCs的细胞形态学特征:可见原代脂肪间充质干细胞(ADSCs)呈梭形,接种后2 h后开始圆形,大部分细胞变形在48 h后,细胞形态以长梭形为主,短梭形、狭长形及多角形较少,胞质丰富,胞核清楚,并分泌大量基质,基质内呈特异性深蓝色,第3代时可见细胞以长梭形为主,漩涡状生长。应用流式细胞仪检测显示CD90、CD105、CD73呈阳性表达,阳性率在95%以上,CD34、CD11b、CD19、CD45及HLADR呈阴性表达;人脂肪源性干细胞(hADSCs)在加入成脂诱导液培养后,hADSCs可向脂肪细胞分化,加入成软骨诱导培养基诱导后,hADSCs可向软骨细胞分化。表明提取培养的细胞为hADSCs。实验组创面完全愈合,被覆盖新生的上皮组织,接近正常皮肤;细胞治疗对照组创面有68%愈合,新生上皮组织较薄,易破裂出血,与实验组比较,愈合质量欠佳;空白对照组创面愈合质量差,愈合面积约41%。创面面积占初始创面面积百分比3组比较,治疗后第7、11、14、18、21天时间点,实验组愈合速度最快,与细胞治疗对照组和空白对照组差异均有统计学意义(F=21.095、115.094、199.695、204.917、600.699,P<0.05),而细胞治疗对照组的愈合速度次之,且在7、11、14、18、21 d时间点与空白对照组比较差异有统计学意义(t=13.064、13.187、14.315、16.177、14.238,P<0.05)。HE染色法行组织形态学观察实验组创面已经完全愈合,表皮修复情况良好,呈复层上皮排列,同时可见部分炎性细胞浸润;细胞治疗对照组则仍有部分创面未能愈合,肉芽组织以及创面表面仍可见较多的炎性细胞浸润;空白对照组则有相当一部分创面尚未愈合,存在较明显的炎性细胞浸润,且在尚未愈合的创口周围可见有瘢痕组织增生。CD31染色法观察新生微血管:实验组创面已经完全愈合,创面浅面可见新生血管,深部血管则呈垂直创面的方向生长,且血管密度较大;细胞治疗对照组则仍有部分创面未能愈合,肉芽组织生长良好,新生血管密度较为丰富,创面表面仍可见较多的炎性细胞浸润;空白对照组则有相当一部分创面尚未能愈合,炎性细胞浸润较明显,肉芽组织中新生血管数量较前两组有明显减少。结论 hEGF可提高脂肪干细胞的存活率,并延长细胞的存活时间,从而增强hASCs促进创面愈合的作用。

Objective To observe the effects of human adipose-derived stem cells(hASCs)and human epidermal growth factor(hEGF)on the repair of defects.Methods Adipose tissue from 6 patients with liposuction and weight loss in the Second Affiliated Hospital of Zhengzhou University in January 2020 was selected as the source of human adipose stem cells,and hASCs were extracted from human adipose tissue by enzymatic digestion and cultured to the third generation.The cell morphology was observed by an inverted microscope,and phenotypes were identified by flow cytometry,and differentiation ability was detected.Twenty-four New Zealand white rabbits were used to establish a full-thickness skin defect model of the back and were randomly divided into three groups:experimental group(hASCs+hEGF),cell therapy control group(hASCs),and blank control group(PBS).The experimental group was transplanted to the edge and the base of the wound by injection of 600μl that the passage 3 adipose-derived stem cells(1×106)were suspended through 100μl PBS was added with human epidermal growth factor for 10μg/L.The cell therapy control group was transplanted to the edge and the base of the wound by injection of 600μl that the passage 3 adipose-derived stem cells(1×106)were suspended through 100μl PBS.The blank control group was transplanted to the edge and the base of the wound by injection of 600μl PBS.They were used to observe and compare the wound healing level of each group that the wound healing was observed and the percentage of wound area in the initial wound area was calculated,and hematoxylin and eosin(HE)staining and CD31 immunohistochemical staining were used to detect the wound tissue.The analysis of variance was used to compare the means of multiple samples,and the T-test was used to compare the measurement data between groups.Results The morphological characteristics of hASCs were observed by a phase-contrast microscope:the primary ADSCs were spindle-shaped and began to be round after 2 hours of inoculation.Most of the cells were deformed after 48 hours and the cell morphology was mainly long spindle-shaped,with less short spindle-shaped,narrow long shaped and polygonal,rich cytoplasm,clear nucleus,and secreted a large amount of matrix,which shows as dark blue.In the third phase,the cells were mainly spindle-shaped and whirlpool-shaped.Flow cytometry showed that CD90,CD105,CD73 were positive,the positive rate was more than 95%,CD34,CD11b,CD19,CD45,and HLA DR were negative;hADSCs could differentiate into adipocytes when cultured in adipogenic medium,and chondrocytes when cultured in chondrogenic medium.The results showed that the cells were hADSCs.In the experimental group,the wound was completely healed,covered with new epithelial tissue,close to normal skin.In the control group,68%of the wounds were healed,and the new epithelial tissue was thin and easy to rupture and bleed.The wound healing quality of the blank control group was poor,and the healing area was about 41%.The percentage of wound area in the initial wound area of the three groups:the experimental group had the fastest healing speed on the 7th,11th,14th,18th,and 21d time points after treatment,which was significantly different from the cell therapy control group and the blank control group(F=21.095,115.094.199.695,204.917,600.699,P<0.05).The healing speed of the cell therapy control group was the second,and there were significant differences between the cell therapy control group and the blank control group at 7 d,11 d,14 d,18 d and 21 d(t=13.064,13.187,14.315,16.177,14.238,P<0.05).HE staining was used to observe the histomorphology.In the experimental group,the wound was completely healed,the epidermis was well repaired,arranged in the stratified epithelium,and some inflammatory cells infiltrated.In the cell therapy control group,there were still some wounds that could not be healed,and there were still many inflammatory cells infiltration in granulation tissue and wound surface.In the blank control group,a considerable part of the wounds was not healed,with obvious inflammatory cell infiltration,and scar tissue hyperplasia around the unhealed wounds.CD31 staining was used to observe the neovascularization:in the experimental group,the wound was completely healed,neovascularization could be seen on the superficial surface of the wound,and the deep vessels grew vertically to the wound,and the vascular density was high.In the cell therapy control group,there were still some wounds that could not be healed,granulation tissue grew well,neovascularization density was rich,and there was still more inflammatory cells infiltration on the surface of the wound.In the blank control group,a considerable part of the wounds could not be healed,the inflammatory cell infiltration was obvious,and the number of new blood vessels in granulation tissue was significantly reduced compared with the former two groups.Conclusion Epidermal growth factors can improve the survival rate of adipose-derived stem cells and prolong the survival time of adipose-derived stem cells,thus enhancing the effect of human adipose-derived stem cells on wound healing.