Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (10): 1484-1491.doi: 10.12307/2023.268
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Effect of adipose-derived stem cells stimulated by direct current electric field on refractory wound healing in diabetic rats
Zhang Rui, Liu Lan, Xie Defu, Lin Yingxi, Ren Hongjing, Yan Hong
- Department of Plastic and Burn Surgery, Affiliated Hospital of Southwest Medical University, National Key Clinical Construction Specialty, Wound Repair and Regeneration Laboratory, Luzhou 646000, Sichuan Province, China
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Received:2022-04-07Accepted:2022-05-13Online:2023-04-08Published:2022-09-07 -
Contact:Yan Hong, MD, Chief physician, Department of Plastic and Burn Surgery, Affiliated Hospital of Southwest Medical University, National Key Clinical Construction Specialty, Wound Repair and Regeneration Laboratory, Luzhou 646000, Sichuan Province, China -
About author:Zhang Rui, Master candidate, Department of Plastic and Burn Surgery, Affiliated Hospital of Southwest Medical University, National Key Clinical Construction Specialty, Wound Repair and Regeneration Laboratory, Luzhou 646000, Sichuan Province, China Liu Lan, Master, Physician, Department of Plastic and Burn Surgery, Affiliated Hospital of Southwest Medical University, National Key Clinical Construction Specialty, Wound Repair and Regeneration Laboratory, Luzhou 646000, Sichuan Province, China Zhang Rui and Liu Lan contributed equally to this article. -
Supported by:the Strategy and Cooperation Project of Southwest Medical University-Luzhou People’s Government, No. 2019LZXNYDZ08 (to YH); School-Level Scientific Research Project of Southwest Medical University, No. 2021ZKQN074 (to LL)
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Zhang Rui, Liu Lan, Xie Defu, Lin Yingxi, Ren Hongjing, Yan Hong. Effect of adipose-derived stem cells stimulated by direct current electric field on refractory wound healing in diabetic rats[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(10): 1484-1491.
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2.1 大鼠脂肪来源干细胞形态观察及鉴定结果 大鼠脂肪来源干细胞提取后接种在培养皿中，24 h后倒置显微镜观察显示，细胞形态大多呈梭形，少数呈三角形或多边形，见图1A。第3代大鼠脂肪来源干细胞成脂诱导过程中，细胞内逐渐出现形态大小不一的脂滴，诱导2周后油红O染色呈阳性；成骨诱导4周的过程中，大鼠脂肪来源干细胞的形态逐渐由梭形变为多边形，可观察到细胞间有钙结节形成，茜素红染色呈阳性，见图1B，C。流式细胞术结果表明大鼠脂肪来源干细胞表面抗原CD29、CD90阳性表达率分别为84.51%，99.95%，CD34、CD45的阳性表达率分别为0.52%，0.06%，见图1D。 2.2 糖尿病大鼠模型评估结果 注射链脲佐菌素后第3，5，7天大鼠随机血糖值均大于正常值16.7 mmol/L，大鼠第7天随机血糖(26.66±4.91) mmol/L较造模前(5.68±0.18) mmol/L明显增加(P < 0.001)，说明糖尿病大鼠模型是合格的，见图1E。"
2.3 直流电场作用后的大鼠脂肪来源干细胞对糖尿病大鼠难愈性创面愈合的影响 2.3.1 大鼠脂肪来源干细胞促进糖尿病大鼠创面愈合 测量大鼠治疗前(0 d)和治疗后3，7，14 d创面愈合率，结果表明：PBS组各时间点创面愈合率均明显低于未刺激脂肪来源干细胞组和直流电场刺激脂肪来源干细胞组(P < 0.05)，与未刺激脂肪来源干细胞组相比，直流电场刺激脂肪来源干细胞组的创面愈合速度更快(P < 0.05)。第14天，直流电场刺激脂肪来源干细胞组创面已经基本愈合，愈合率达到95%，未刺激脂肪来源干细胞组愈合率为87%，PBS组为81%，见图2A，B，表明直流电场作用后的脂肪来源干细胞可促进糖尿病大鼠创面愈合，其促进糖尿病大鼠创面愈合的效率显著高于未刺激组。 2.3.2 大鼠脂肪来源干细胞促进创面上皮化 大鼠皮肤全层缺损后表现为表皮、真皮层结构明显缺失。术后第3天，各组大鼠创面均未见明显上皮化，可见大量炎症细胞；治疗第7天，直流电场刺激脂肪来源干细胞组表皮及真皮增生更加明显；治疗第14天，各组基本形成表皮及真皮结构，其中直流电场刺激脂肪来源干细胞组的表皮较其他组更完整，而PBS组表皮则不连续，未刺激脂肪来源干细胞组则介于二者之间，各组炎性细胞均明显减少，见图2C。"
2.3.3 大鼠脂肪来源干细胞促进创面血管生成 通过高倍镜下苏木精-伊红染色视野计数和免疫荧光观察CD31的表达来评估创面新生血管的形成，结果显示：术后第3，7，14天，与未刺激脂肪来源干细胞组和PBS组相比，直流电场刺激脂肪来源干细胞组血管密度和CD31表达均明显增加(P < 0.05)， 未刺激脂肪来源干细胞组在第7，14天时与PBS组相比稍多(P < 0.05)，见图3(箭头表示血管生成)。"
2.3.4 大鼠脂肪来源干细胞促进血管内皮生长因子的生成 血管生成与血管内皮生长因子等细胞因子关系密切，为此通过免疫组化方法对不同组间创面组织血管内皮生长因子的表达进行图像软件分析。数据显示：随着创面愈合的进展各组血管内皮生长因子的表达逐渐增加，治疗第3天，直流电场刺激脂肪来源干细胞组中可见较少血管内皮生长因子表达，但多于未刺激脂肪来源干细胞组和PBS组(P < 0.05)；第7天时各组大鼠创面血管内皮生长因子表达明显增加，与未刺激脂肪来源干细胞组和PBS组相比，直流电场刺激脂肪来源干细胞组血管内皮生长因子表达增加明显(P < 0.05)，未刺激脂肪来源干细胞组和PBS组间也存在明显差异(P < 0.05)；第14天时创面组织血管内皮生长因子表达丰富，其中直流电场刺激脂肪来源干细胞组表达最多的血管内皮生长因子(P < 0.05)，其余两组无明显差异，见图4。"
2.3.5 大鼠脂肪来源干细胞促进创面血流灌注 激光散斑对比成像仪测量不同时间点大鼠创面及周围正常皮肤血流灌注，结果显示，治疗后第3，7，14天，直流电场刺激脂肪来源干细胞组创面与周围正常皮肤血流灌注比值均高于PBS组(P < 0.05)。与未刺激脂肪来源干细胞组相比，第3，7天时直流电场刺激脂肪来源干细胞组创面血流灌注明显增高(P < 0.05)，第14天时两组则无明显差异。未刺激脂肪来源干细胞组在第7天也表现出较PBS组更高的血流灌注(P < 0.05)，而在第3，14天时则无统计学意义。此外，3组大鼠的血流灌注在第3天时稍低，第7天达到峰值，随后出现不同程度下降，表现出相同的趋势，见图5。"
2.4 生物相容性 术后没有发生与植入细胞有关的不良反应。"
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