底蜕膜间充质干细胞分化为多巴胺能样神经元

Decidua basalis-derived mesenchymal stem cells differentiate into dopamine neuron-like cells

目的探讨人胎盘底蜕膜间充质干细胞体外向多巴胺能样神经元分化的潜能,并优化诱导方案。方法体外分离培养底蜕膜间充质干细胞,用表皮生长因子(EGF)+人碱性成纤维细胞生长因子(bFGF)+B27添加剂和人音猬因子(SHH)+成纤维细胞生长因子8(FGF8)+forskolin+脑源性神经营养因子(BDNF)分两个阶段对其进行诱导;免疫细胞化学先后检测干细胞标记nestin和CD133、成熟神经元标记神经元特异性烯醇化酶(NSE)、神经胶质细胞标记胶质原纤维酸性蛋白(GFAP)、多巴胺能神经元标记酪氨酸羟化酶(TH)的表达;Western blot验证诱导后TH蛋白的表达;高效液相色谱-电化学检测诱导前后多巴胺的分泌。结果经第一阶段诱导后,细胞形成漂浮生长的神经球,nestin和CD133均呈阳性表达;第二阶段诱导后,出现明显的神经元样形态,NSE、GFAP和TH均阳性表达,Western blot也显示TH蛋白的表达,多巴胺分泌量相比诱导前明显增加(P<0.001)。结论底蜕膜间充质干细胞体外可分化为多巴胺能样神经元,可能成为帕金森病干细胞移植治疗新的种子细胞来源。

Objective To explore the differentiation potential of human placental decidua basalis-derived mesenchymal stem cells （DBMSCs） into dopamine neuron-like cells in vitro, and optimize the induction protocols. Methods DBMSCs were isolated and cultured in vitro, and the cells were co-cultured with epidermal growth factor （EGF） ＋ htmmn basic fibmblast growth factor （bFGF） ＋ 1327 and sonic hedgehog （SHH） ＋ fibroblast growth factor 8 （FGF8） ＋ forskolin ＋ brain-derived neurotrophic factor （BDNF） containing medium successively, and the whole induction process was divided into two stages; immunocytochemistry was performed to identify the expression of stem cell markers （nestin and CD133）, mature neuron markers （neuronal-speeitic enolase, NSE ）, astroglial marker （glial fibrillary acidic protein, GFAP）, and dopamine neuron specific marker （tyrosine hydroxylase, TH）; Western blot analysis was performed to confirm the expression of TH protein of differentiated DBMSCs. High performance liquid chronmtography coupled to electrochemical detection was performed to detect the dopamine secretion of pre- and post- differentiated DBMSCs. Results Mter the first stage induction, free-floating neurospheres appeared and were nestin- and CD133-positive staining. After the second stage induction, cells adopted a neural-like morphology and were NSE-, GFAP-, and TH-pesitive staining. Western blot showed the expression of TH protein and dopamine secretion level were significantly increased. Condusion Depending on the differentiation potential towards dopamine neuron- like cells, DBMSCs can serve as a novel seed cell source for transplantation therapy of Parkinson＇s disease.