去细胞羊膜负载毛囊干细胞修复裸鼠全层皮肤缺损

Acellular amniotic membrane integrated with hair follicle stem cells in repair of full-thichness skin defects in nude mice

目的利用去细胞羊膜负载毛囊干细胞修复裸鼠全层皮肤缺损。方法体外分离和培养人毛囊干细胞,取经慢病毒介导的绿色荧光蛋白(pGC FU-GFP-Lentiviru)标记后第4代毛囊干细胞接种于去细胞羊膜上;负载后第7天,HE染色光学显微镜观察细胞黏附生长情况。于18只C57BL/6裸鼠背部制作全层皮肤缺损创面,并根据不同的处理方式分为实验组(将负载毛囊干细胞的去细胞羊膜植入裸鼠全层皮肤缺损创面)、去细胞羊膜移植+干细胞注射组(将去细胞羊膜覆盖创面后羊膜下注射5×106毛囊干细胞)和去细胞羊膜移植组(去细胞羊膜单独移植),每组6只。分别于移植后第7、14、21、28天,测量各组创面面积并计算创面收缩率。移植后第28天,荧光显微镜观察创面组织绿色荧光蛋白(GFP)表达,HE染色光学显微镜观察新生皮肤结构特征。结果去细胞羊膜负载毛囊干细胞后第7天,HE染色光学显微镜观察发现,细胞连接成片状覆盖于去细胞羊膜表面。实验组和去细胞羊膜移植+干细胞注射组移植后各时点的创面收缩率均显著小于去细胞羊膜移植组(P<0.05)。移植后第28天,荧光显微镜观察显示实验组创面表皮层GFP表达阳性;HE染色光学显微镜观察发现实验组创面组织表皮层明显增厚,有类似毛囊样结构生成。结论去细胞羊膜负载毛囊干细胞可用于修复裸鼠全层皮肤缺损。

Objective To repair full-thichness skin defects by acellular amniotic membrane integrated with hair follicle stem cells. Methods Human hair follicle stem cells were isolated and cultured in vitro,and those of the fourth generation marked by lentivirus-mediated green fluorescent protein（pGC FU-GFP-Lentiviru） were grafted onto acellular amniotic membrane.Seven days after integration,cell proliferation was observed by light microscopy with HE staining.Wounds of full-thickness skin defects were prepared in 18 C57BL/6 nude mice,and experiment group（wounds of full-thickness skin defects were grafted with acellular amniotic membrane integrated with hair follicle stem cells）,acellular amniotic membrane graft＋stem cell injection group（wounds of full-thickness skin defects were grafted with acellular amniotic membrane,with injection of 5×106 hair follicle stem cells under amniotic membrane） and acellular amniotic membrane graft group（wounds of full-thickness skin defects were grafted with acellular amniotic membrane） were divided,with 6 mice in each group.The areas and contraction rates of wounds in each group were measured 7 d,14 d,21 d and 28 d after grafting.The expression of green fluorescent protein（GFP） of wound tissues was detected by fluorescence microscopy,and the structure of regenerated skins was observed by light microscopy with HE staining. Results Seven days after acellular amniotic membrane integration with hair follicle stem cells,light microscopic observation with HE staining revealed that cells formed in lamellar shape and covered acellular amniotic membrane.The contraction rates of wounds of each time points in experiment group and acellular amniotic membrane graft＋stem cell injection group were significantly lower than that in acellular amniotic membrane graft group（P〈0.05）.Twenty-eight days after grafting,fluorescence microscopic observations revealed that the expression of GFP in cuticular layer of wounds in experiment group was positive,and light microscopic observations with HE staining unveiled that the cuticular layer of wounds in experiment group was significantly thickened,and hair follicle-like structure formed. Conclusion Acellular human amniotic membrane integrated with human hair follicle stem cells can be used in the repair of full-thichness skin defects in nude mice.