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

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.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 3rd 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 3rd 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 3rd 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.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 ( P<0.05). The results of ELISA demonstrated that the expression of BDNF and NGF was significantly decreased after added induced liquid 1, and the level of BDNF and NGF increased gradually with the induction of liquids 2 and 3, and the concentration of BDNF and NGF was significantly higher than that of hAMSCs group ( P<0.05). Immunofluorescence staining showed that the number of regenerated nerve fibers in group B was higher than that in groups A and C after 5-14 days of transplantation.The hAMSCs can be induced into SCs-like cells with the proper chemical factor regulation in vitro, and a large number of promoting nerve growth factor were released during the process of differentiation, and nerve regeneration in flaps being transplanted the SCs-like cells was better than that in flaps being transplanted the hAMSCs, which through a large number of BDNF and NGF were released.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。.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。.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。.hAMSCs 在体外经适当的化学因子调节后可诱导分化为 SCs-like cells，在诱导分化过程中释放了大量促进神经生长的因子；且 SCs-like cells 移植后通过释放大量 BDNF 和 NGF，在神经再生数量方面高于 hAMSCs 移植。.