人脐血间充质干细胞诱导分化为神经细胞的研究

STUDY ON INDUCED DIFFERENTIATION OF HUMAN UMBILICAL CORD BLOOD MESENCHYMAL STEM CELLS INTO NEURONS

目的探讨体外诱导人脐血间充质干细胞(MSCs)向神经细胞分化的条件,为治疗中枢神经系统损伤提供实用的干细胞来源。方法体外分离、纯化、扩增脐血MSCs,流式细胞仪检测细胞表面标志。采用脑源性神经营养因子BDNF 10ng/ml+维甲酸RA0.5μM+碱性成纤维生长因子bFGF 20ng/ml协同诱导脐血MSCs定向分化。免疫荧光染色检测诱导后细胞的星形胶质细胞特异标志GFAP及神经元特异标志MAP2的表达情况。建立大鼠脊髓横断损伤模型,将BrdU标记的诱导后的细胞移植入损伤的脊髓中,采用BBB运动功能评分标准在术后24h及1、2、3、4、5周各时间点对大鼠进行运动功能评分。用组织学和免疫组化方法检测移植到大鼠脊髓中的BrdU阳性细胞的存活、迁移、分化情况。结果脐血MSCs体外培养三代后,细胞表面CD11b、CD34、CD45和CD44表达阴性。诱导分化7d后,大部分细胞的形态类似神经元,免疫荧光染色检测MAP2阳性细胞占大多数,明显多于GFAP阳性细胞。5周后,细胞移植组大鼠的后肢运动功能恢复情况较对照组好。免疫组织化学结果显示植入的细胞可长时间在宿主脊髓中存活,并向损伤处两端迁移。结论人脐血MSCs于体外在特定的条件下可以诱导分化为神经元样细胞。移植脐血MSCs诱导后的神经细胞可在损伤的脊髓中存活、迁移,并能促进脊髓损伤后行为和功能恢复。

Objective To explore the optimal conditions of inducing mesenchymal stem cells （MSCs） derived from human umbilical cord blood （HUCB） to differentiate into neurons in vitro, in order to provide a kind of valuable stem cell resource for healing central nervous injury. Methods Isolation, culture and amplification of UCB- derived MSCs were done in vitro. MSCs were displayed with antibodies and analyzed with flow cytometry. Then MSCs were induced with DMEM/F12 containing 20% FBS, BDNF 10ng/ml ＋ RA 0. 5μM ＋ bFGF 20ng/ml. After seven days＇culture, the expressions of GFAP and neuron marker MAP2 in the induced cells were detected by immunofluorescence staining. The spinal cord transection rat models were prepared. The inducecl MSCs were labelled by BrdU and then transplanted into the impaired spinal cord. Hindlimb motor function was assessed by using the Basso-Beattie-Bresnahan （BBB） locomotor rating scale once a week. The survival and differentiation of transplanted ceils in rat spinal cord in vivo were evaluated by histology and immunohistochemistry. Results It was found by flow cytometry that the adherent cells were CDllb-CD34^-CD45^-CD44^-at the third passage detected. After induction, most of these ceils exhibited the typical neuron-like phenotype. The differentiation ratio of glial ceils was lower than that of neurons. Five weeks after the transplantation, the locomotor function recovery of rats was significantly improved in the experimental group than that in the control group. The transplanted ceils were found surviving and extending in the surrounding spinal cord tissue for a long time in the transplantation group. Conclusion UCB-derived MSCs can be induced to differentiate into neural ceils in vitro. Our findings showed that transplantation of induced MSCs into the injured rat spinal cord can survive, migrate to the lesion site, and improve the neurological function recovery in rats.