脂肪间充质干细胞条件培养基制备温敏凝胶外用治疗皮肤烫伤  

Therapeutic effect of adipose derived mesenchymal stem cell conditioned medium thermosensitive hydrogel on skin scald

作　　者：刘培[1] 胡振生[1] 马玲[1] 王换换 李栋[2] Liu Pei;Hu Zhen-sheng;Ma Ling;Wang Huan-huan;Li Dong(1Department of Burn & Plastic Surgery Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China;Cyromedicine Lab, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China)

机构地区：[1]山东大学齐鲁医院整形美容科,山东省济南市250012 [2]山东大学齐鲁医院低温医学研究室,山东省济南市250012

出　　处：《中国组织工程研究》2017年第30期4853-4860,共8页Chinese Journal of Tissue Engineering Research

基　　金：中华医学会欧莱雅项目(H2015121409)~~

摘　　要：背景:有研究显示脂肪干细胞参与烫伤后皮肤组织的修复,但利用脂肪干细胞分泌物制备水凝胶,并治疗皮肤烫伤的研究尚未见报道。目的:分析人脂肪干细胞条件培养基水凝胶外用治疗小鼠皮肤烫伤模型的疗效。方法:采用酶解联合贴壁培养法,从脂肪组织培养获得脂肪干细胞并进行鉴定。取稳定增殖期细胞,收集其条件培养基,加入壳聚糖、甘露醇、β-甘油磷酸钠和透明质酸等制备温敏水凝胶,同时用新鲜脂肪干细胞完全培养基制备水凝胶。24只C57BL/6小鼠背部脱毛后用95℃铝块烫伤10 s快速建立3度皮肤烫伤模型,右侧以脂肪干细胞条件培养基水凝胶每天涂抹2次;左侧使用脂肪干细胞新鲜培养基水凝胶每天涂抹2次,连续涂抹7 d,观察愈合时间与愈合进程,计算愈合率,在烫伤后第4,14,28天取材进行苏木精-伊红染色观察组织病理学变化。结果与结论:①人脂肪间充质干细胞为成纤维状,增殖旺盛,平均倍增时间为55 h,可被诱导分化为成骨细胞、软骨细胞和脂肪细胞,高表达CD29、CD44、CD90和CD105,低表达CD31和CD34,证明其符合间充质干细胞的标准;②用人脂肪间充质干细胞条件培养基制备的温敏水凝胶在4-20℃呈现为清凉透明的黏稠液体,放入37℃15 min变为半固态凝胶;③95℃铝块烫伤小鼠表皮、真皮及皮下脂肪和肌肉组织的正常结构完全破坏,符合严重的3度烫伤标准,伤口面积均大致为3 cm^2;④在修复过程中发现,与新鲜培养基水凝胶对照组相比,脂肪干细胞条件培养基水凝胶组创口愈合时间短,伤口整洁,瘢痕增生少,创面组织结构恢复好;⑤脂肪干细胞条件培养基水凝胶组炎症浸润深度、炎症浸润细胞密度、肉芽组织厚度、成纤维细胞密度、新生血管密度和表皮厚度等指标显著好于新鲜培养基水凝胶组,差异有显著性意义(P<0.05);⑥以上结果表明,人脂肪间充质干细胞条件培养�BACKGROUND:Studies have shown that adipose derived mesenchymal stem cells are involved in the skin repair after scald,but the hydrogel made of the excreta by adipose derived mesenchymal stem cells is rarely reported in the treatment of skin scald.OBJECTIVE:To analyze the therapeutic effect of human adipose derived mesenchymal stem cell conditioned medium hydrogel in a mouse model of skin scald.METHODS:Adipose derived mesenchymal stem cells were obtained from adipose tissues by enzyme digestion combined with adherent culture method.Morphological and flow cytometry were used to identify phenotype and induce differentiation.Secondly,the stable proliferative phase cells were harvested to obtain the conditioned medium,and chitosan,mannitol,beta-glycerol phosphate sodium and hyaluronic acid were added to prepare the thermosensitive hydrogel.Then the95℃aluminum block was used to rapidly establish a model of degree III skin scald on the left(experimental group)and right(control group)sides of the back of24C57BL/6mice.In the experimental group,adipose derived stem cell conditioned medium hydrogel was applied twice a day on the right side of the mouse back,and in the control group,fresh medium hydrogel was applied twice a day on the left side of the mouse back.The treatment period lasted for7days.Healing time and healing process were observed to calculate the healing rate.Histopathological changes were observed by hematoxylin-eosin staining at paraffin sections at4,14,28days after skin scald.RESULTS AND CONCLUSION:(1)The human adipose derived mesenchymal stem cells had fibroblast-like morphology and proliferated vigorously,and the average doubling time was55hours.These cells could be induced to differentiate into osteoblasts,chondrocytes and adipocytes.High expression of CD29,CD44,CD90and CD105were observed on these cells with low expression of CD31and CD34,which met the standard of mesenchymal stem cells.(2)The thermosensitive hydrogel prepared by the conditioned medium was cool and transparent viscous liquid at4-20℃,

关 键 词：生物材料 缓释材料 皮肤烫伤 脂肪间充质干细胞 条件培养基 温敏水凝胶 

分 类 号：R318[医药卫生—生物医学工程]