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目的探讨不同方法诱导大鼠骨髓基质干细胞(BMSC)分化为肠神经细胞的可能性,并探索适宜的诱导方法。方法体外培养并鉴定BMSC,流式细胞法检测CD90和CD45。传代至第6代后行神经诱导分化。先以10ng/ml碱性成纤维生长因子(bFGF)预诱导24h,然后分2组：胶质细胞源神经营养因子(GDNF)组以10ng/ml GDNF,在胎肠培养基(FGCM)条件下诱导10d；维甲酸组以0．5 mmol/L维甲酸、10μmol/L ZnSO_4和FGCM诱导10d。免疫荧光法检测神经特异性烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)、蛋白基因产物(PGP9．5)、一氧化氮合酶(nNOS)和血管活性肠肽(VIP)的表达。所得数据行t检验。结果流式细胞检测BMSC呈高表达,其标志为CD90,而不表达造血细胞标志CD45。诱导10d后,GDNF组和维甲酸组均有部分细胞在形态上表现出神经元样改变,免疫荧光染色NSE、NF、PGP9．5、nNOS、VIP阳性,GFAP阴性。GDNF组NF、PGP9．5、nNOS、VIP阳性比例显著高于维甲酸组(75．6％±8．4％比48．5±7．5％；57．7％±6．5％比35．7％±7．2％；46．6％±5．4％比30．5％±6．6％；72．3％±6．7％比40．4％±7．4％；P＜0．01)。结论在体外BMSC可被诱导分化为肠神经样细胞并表达肠神经递质,GDNF在胎肠培养基条件下诱导肠神经样细胞比例高于维甲酸。
Objective To explore the possibility of different methods of inducing bone marrow stromal stem cells (BMSCs) into enteric neurons and to explore appropriate induction methods. Methods BMSCs were cultured and identified in vitro. CD90 and CD45 were detected by flow cytometry. From passage 6 to passage 4, neural differentiation was induced. The cells were pre-induced with 10 ng / ml basic fibroblast growth factor (bFGF) for 24 h, then divided into 2 groups: GDNF group (10 ng / ml GDNF) Induced for 10 days. The retinoic acid group was induced with 0.5 mmol / L retinoic acid, 10 μmol / L ZnSO 4 and FGCM for 10 days. Immunofluorescence was used to detect neuroendocrine enolase (NSE), neurofilament (NF), glial fibrillary acidic protein (GFAP), protein gene product (PGP9.5), nitric oxide synthase (nNOS) and vasoactive Intestinal peptide (VIP) expression. The data obtained t test. Results Flow cytometry showed high expression of BMSC, which was marked by CD90 but not by hematopoietic cell marker CD45. After induction for 10 days, some of the cells in the GDNF group and the retinoic acid group showed neuron-like morphological changes. Immunofluorescence staining showed NSE, NF, PGP9.5, nNOS, VIP positive and GFAP negative. The positive rates of NF, PGP9.5, nNOS and VIP in GDNF group were significantly higher than those in retinoic acid group (75.6% ± 8.4% vs 48.5 ± 7.5%; 57.7% ± 6.5% vs 35 .7% ± 7.2%; 46.6% ± 5.4% vs. 30.5% ± 6.6%; 72.3% ± 6.7% vs. 40.4% ± 7.4%; P <0.01). Conclusions BMSCs can be induced to differentiate into enteric neuron-like cells and express enteric neurotransmitters in vitro. GDNF induces more enteric neuron-like cells than retinoic acid in fetal gut culture medium.