Ju Xiaojun, Pan Feng, Bai Shuling, Tian Xiaohong, Tong Hao, Wang Jun
Department of Anatomy, China Medical University, Shenyang Liaoning, 110001, P.R. China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2010 Feb;24(2):143-9.
Human acellular amniotic membrane (HAAM) contains collagens, glycoproteins, protein-polysaccharide, 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.
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 x 10(5)/cm2, cocultured, 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.
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 significant difference between ADSCs cultured in the HAAM and control group (P > 0.05). One, 2, 4 weeks after graft, there were significant differences in wound healing rate between group A and groups B, C (P < 0.05), between group B and group C (P < 0.05). HE staining showed that wound healed faster in group A than in groups B, C. Cytokeratin 19 (CK19) immunohistochemical staining showed that there were more CK19 positive cells in group A than in groups B, C.
The graft of HAAM with ADSCs plays an effective role in promoting the repair of full-thickness skin wound.
人脱细胞羊膜（HAAM）含有胶原蛋白、糖蛋白、蛋白多糖、整合素及板层结构，可为细胞增殖和分化提供丰富营养。探讨以HAAM复合脂肪干细胞（ADSCs）作为良好的组织工程皮肤替代物修复皮肤缺损的可能性。
取30只健康4月龄、体重250 - 300 g的SD大鼠（雌雄不限）腹股沟脂肪获取原代ADSCs，进行体外培养和纯化。采用免疫细胞化学染色法检测第3代ADSCs的CD44、CD49d及CD34。HAAM经物理及胰蛋白酶处理后，分别行HE染色及扫描电镜（SEM）观察。将ADSCs以2×10(5)/cm2的密度接种于HAAM上皮侧，进行共培养，并于不同时间行SEM观察。采用MTT法检测接种于HAAM上细胞的活力，以未接种HAAM的细胞为对照。将30只SD大鼠制作成全层皮肤缺损模型，随机分为3组（A、B、C组）。A组用HAAM/ADSCs复合物修复创面，B组用HAAM修复，C组用纱布作为对照。术后于第1、2、4周对大鼠创面愈合率进行评估并进行组织学观察。
HE染色显示第3代ADSCs呈梭形，细胞核呈椭圆形，位于细胞中央；免疫细胞化学染色显示CD44、CD49d阳性，CD34阴性。HE染色显示HAAM内无细胞残留。SEM显示HAAM两侧结构不同：一侧为致密网状结构，另一侧为纤维结构。共培养3天后，ADSCs在HAAM上生长良好；细胞紧密贴附于HAAM上，牢固黏附并在HAAM基质表面增殖至汇合。MTT法检测显示HAAM上的细胞生长良好，增殖活力强。HAAM培养的ADSCs与对照组比较差异无统计学意义（P > 0.05）。移植后1、2、4周，A组与B组、C组创面愈合率比较差异有统计学意义（P < 0.05），B组与C组比较差异有统计学意义（P < 0.05）。HE染色显示A组创面愈合较B组、C组快；细胞角蛋白19（CK19）免疫组化染色显示A组CK19阳性细胞多于B组、C组。
HAAM复合ADSCs移植对促进全层皮肤创面修复有显著作用。
