人脱细胞羊膜复合脂肪源性干细胞修复大鼠全层皮肤缺损的实验研究  

AN EXPERIMENTAL STUDY ON REPAIRING FULL-THICKNESS SKIN WOUND BY HUMAN ACELLULAR AMNIOTIC MEMBRANE LOADED WITH ADIPOSE-DERIVED STEM CELLS IN RATS

作　　者：鞠晓军[1] 潘锋[1] 柏树令[1] 田晓红[2] 佟浩[1] 王军[1] 

机构地区：[1]中国医科大学人体解剖学教研室 [2],沈阳110001 [2]中国医科大学组织工程学教研室,沈阳110001

出　　处：《中国修复重建外科杂志》2010年第2期143-149,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：辽宁省自然科学基金资助项目(20051033)~~

摘　　要：目的人脱细胞羊膜(human acellular amniotic membrane,HAAM)含胶原、糖蛋白、蛋白多糖、整合素和板层体等多种成分,这些成分能为细胞增殖、分化提供丰富营养。研究HAAM与脂肪源性干细胞(adipose-derived stem cells,ADSCs)的生物相容性,探讨HAAM复合ADSCs构建真皮替代物修复全层皮肤缺损的可能性。方法取健康4月龄SD大鼠30只,雌雄不限,体重250～300g,于腹股沟处获取脂肪行ADSCs体外分离、培养和传代;取第3代细胞行HE染色及CD44、CD49d、CD34免疫细胞化学染色鉴定。应用物理和酶消化法处理自愿捐赠的人羊膜获得HAAM,行HE染色观察细胞是否脱除干净,扫描电镜观察HAAM结构特征。将第3代ADSCs以2×105个/cm2密度接种于HAAM上皮面,体外共培养1～5d后分别行扫描电镜和MTT检测,不加材料组设为对照组。将大鼠随机分为3组,每组10只,建立背部直径为2cm的全层皮肤缺损模型。A组创面植入复合ADSCs的HAAM,B组植入单纯HAAM,C组常规包扎。术后行大体观察,于术后1、2、4周计算创面愈合率,并取创面皮肤行HE染色和细胞角蛋白19(cytokeratin 19,CK19)免疫组织化学染色观察。结果HE染色示第3代ADSCs呈梭形,胞核为椭圆形,位于细胞中央;免疫细胞化学染色示第3代ADSCs表达CD44和CD49d,不表达CD34。HAAM行HE染色未见细胞残留;扫描电镜下见HAAM两面具有不同的三维结构,上皮面为较致密的网状纤维;基质面为较稀疏的胶原纤维,未见纤维发生融合、断裂和降解。ADSCs-HAAM复合培养3d后扫描电镜下可见细胞生长状态良好,胞体肥大呈梭形隆起,胞膜外绒毛致密略有弯曲,胞体下方形成丰富的伪足牢固黏附于HAAM上;培养1～5d,MTT检测示实验组和对照组吸光度值比较差异无统计学意义(P>0.05)。移植术后1、2、4周,A组创面愈合率均高于B、C组,B组高于C组,差异均有统计学意义(P<0.05)。HE染色示A组创面愈合速度快于B、C组;A组CK19表达亦明Objective Human acellular amniotic membrane（HAAM）contains collagens,glucoproteins,proteinpolysaccharide,integrin,and lamellar,which can supply rich nutrition to cell proliferation and differentiation.To explore the possibility of HAAM with adipose-derived stem cells（ADSCs）as a good engineered skin substitute for repairing skin defect.Methods Primary ADSCs were obtained from inguinal fat of 30 healthy 4-month-old SD rats,male or female,weighing 250-300 g,and cultured in vitro and purified.The 3rd passage ADSCs were used to detect CD44,CD49d and CD34 by immunocytochemistry staining.After physical and trypsin preparation,the HAAM was observed by HE staining and scanning electron microscope（SEM）respectively.ADSCs were seeded on epithelial side of HAAM at the density of 2×105/cm2,co-cultured,and observed by SEM at different time.MTT test was used to detect viability of cells that seeded on HAAM,the group without HAAM was used as control.Thirty SD rats were made models of full-thickness skin wound and randomly divided into three groups（A,B,and C）.Wound was repaired with HAAM/ADSCs composites in group A,with HAAM in group B,and with gauze as control in group C.The rats underwent postoperative assessment of wound healing rate and histological observation at the 1st,2nd,and 4th weeks.Results HE staining showed that the 3rd passage ADSCs was spindle-shaped with an ovoid nucleus which located in the middle of cell;the immunocytochemistry staining showed positive result for CD44 and CD49d and negative result for CD34.There were no residues of cells in the HAAM by HE staining.SEM showed that there were different structures at the two sides of HAAM;one side had compact reticular structure and the other side had fibrous structure.After 3 days of co-culture,ADSCs showed good growth on HAAM;the cells were closely packed onto the HAAM,attached firmly and proliferated to confluence on the stromal surface of HAAM.MTT test showed that the cells on the HAAM grew well and had strong proliferation vitality.There was no

关 键 词：组织工程皮肤 人脱细胞羊膜 脂肪源性干细胞 皮肤缺损 创面愈合 大鼠 

分 类 号：R318.2[医药卫生—生物医学工程] R681.3[医药卫生—基础医学]