人羊膜间充质干细胞来源的雪旺细胞样细胞移植在皮瓣神经再生中的作用  

The role of Schwann cells-like cells derived from human amniotic membrane mesenchymal stem cells transplantation in flap nerves regeneration

作　　者：龚飞宇[1] 魏在荣[1] 金文虎[1] 李海[1] 邓呈亮[1] 吴必华[1] 聂开瑜[1] 

机构地区：[1]遵义医学院附属医院烧伤整形外科,贵州遵义563000

出　　处：《中国修复重建外科杂志》2018年第1期80-90,共11页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81360295);贵州省科技计划项目(黔科合支撑[2017]2877)~~

摘　　要：目的通过将人羊膜间充质干细胞(human amniotic membrane mesenchymal stem cells,hAMSCs)在体外诱导分化为雪旺细胞样细胞(Schwann cells-like cells,SCs-like cells),分别观察两种细胞移植对皮瓣神经再生的作用。方法采用胰蛋白酶/胶原酶二步消化法分离提取hAMSCs并传代,流式细胞术和免疫荧光法鉴定hAMSCs。取第3代培养6 d的hAMSCs体外诱导分化为SCs-like cells,诱导分化19 d后,分别采用免疫荧光法、Western blot及实时荧光定量PCR(real time fluorescent quantitative PCR,qPCR)检测SCs-like cells和第3代培养6 d的hAMSCs中S-100、p75和神经胶质酸性蛋白(glial fibrillary acidic protein,GFAP)的表达;ELISA检测第3代培养6 d的hAMSCs以及加入诱导液1、2、3诱导后19 d内上清液中BDNF和NGF表达量。另取SD雌性大鼠75只,建立大鼠腹壁失神经支配穿支皮瓣模型,分为3组(n=25),A、B、C组分别于大鼠皮瓣标记处皮下多点注射培养6 d处于增殖期的第3代hAMSCs 1×10~6个、SCs-like cells 1×10~6个和等体积PBS,分别于移植后2、5、7、9、14 d采用免疫荧光法观察神经丝重链多肽抗体(neurofilament heavy polypeptide antibody,NF-01)阳性表达的神经再生情况。结果经流式细胞术和免疫荧光法鉴定提示培养的细胞为hAMSCs。免疫荧光法、Western blot及qPCR检测结果显示,SCs-like cells的S-100、p75和GFAP阳性细胞百分比、蛋白表达量及基因相对表达量均明显高于hAMSCs,差异均有统计学意义(P<0.05)。ELISA检测示:第3代培养6 d的hAMSCs中加入诱导液1后,BDNF和NGF表达量明显降低,随着诱导液2及诱导液3的依次添加,BDNF和NGF表达量逐渐升高,且浓度明显高于第3代培养6 d的hAMSCs,各时间点间差异均有统计学意义(P<0.05)。免疫荧光法观察示,移植后5~14 d B组再生神经纤维数高于A组和C组。结论 hAMSCs在体外经适当的化学因子调节后可诱导分化为SCslike cells,在诱导分化过程中释放了大量促进神经生长的�Objective Inducing human amniotic membrane mesenchymal stem cells（hAMSCs） to Schwann cells-like cells（SCs-like cells） in vitro, and to evaluate the efficacy of transplantation of hAMSCs and SCs-like cells on nerves regeneration of the rat flaps. Methods hAMSCs were isolated from placenta via two-step digestion and cultured by using trypsin and collagenase, then identified them by flow cytometry assay and immunofluorescence staining. The 3 rd generation of hAMSCs cultured for 6 days were induced to SCs-like cells in vitro; at 19 days after induction, the levels of S-100, p75, and glial fibrillary acidic protein（GFAP） were detected by immunofluorescence staining, Western blot, and real-time fluorescence quantitative PCR（qPCR）. The levels of brain-derived neurotrophic factor（BDNF） and nerve growth factor（NGF） were measured by ELISA in the supernatant of the 3 rd generation of hAMSCs cultured for 6 days and the hAMSCs induced within 19 days. In addition, 75 female Sprague Dawley rats were taken to establish the rat denervated perforator flap model of the abdominal wall, and were divided into 3 groups（n=25）. The 3 rd generation of hAMSCs（1×10~6 cells） in the proliferation period of culturing for 6 days, the SCs-like cells（1×10~6 cells）, and equal volume PBS were injected subcutaneously in the skin flap of the rat in groups A, B, and C, respectively. At 2, 5, 7, 9, and 14 days after transplantation, 5 rats in each group were killed to harvest the flap frozen sections and observe the positive expression of neurofilament heavy polypeptide antibody（NF-01） by immunofluorescence staining. Results The cells were identified as hAMSCs by flow cytometry assay and immunofluorescence staining. The results of immunofluorescence staining,Western blot, qPCR showed that the percentage of positive cells, protein expression, and gene relative expression of S-100,p75, and GFAP in SCs-like cells group were significantly higher than those in hAMSCs group（P0.05）. The results of ELISA demonstrat

关 键 词：人羊膜间充质干细胞 雪旺细胞样细胞 诱导分化 神经再生 皮瓣 

分 类 号：R622[医药卫生—整形外科]