人脐带间充质干细胞向神经细胞分化的实验研究

Differentiation of human umbilical cord mesenchymal stem cell to neural cells

目的研究人脐带分离的间充质干细胞(UC-MSC)在体外分离、培养、扩增和鉴定以及向神经干细胞和神经元细胞分化的可能性,为临床治疗糖尿病视网膜病变(DR)神经损伤提供理论基础。方法选用足月妊娠剖宫产健康胎儿的脐带,剔除动静脉后采用贴壁法分离得到UC-MSC,选用20%FBS血清培养液进行原代培养,经传代培养、流式细胞检测后,用神经分化培养基诱导UC-MSC分别向神经干细胞和神经元细胞分化,在荧光显微镜下观察细胞形态,并以免疫荧光和RT-PCR方法进行鉴定。结果 UC-MSC呈贴壁生长,倒置显微镜可见细胞呈形态相对均一的梭形细胞,呈放射状排列生长或旋涡状生长。流式细胞检测表明这些细胞表达CD105(SH2)、CD73(SH3)及CD90等MSC标志物;不表达CD34、CD45、CD11b和CD19。经神经干细胞分化诱导后,细胞开始聚集形成大小不等的细胞簇形成悬浮的细胞团;RT-PCR及免疫荧光检测表明,其NSE和Neuro D1阳性细胞分别达(87.3±14.7)%、(72.6±11.8)%。经神经元细胞分化诱导后,细胞呈神经元样的突起,细胞胞体饱满、折光性明显变强;RT-PCR及免疫荧光检测表明,其MAP2和NF-M阳性细胞分别达(43.1±10.3)%、(69.4±19.5)%。结论在一定的诱导条件下,UC-MSC能向神经细胞分化,可能作为DR神经损伤的细胞移植治疗来源。

Objective To explore a method to isolate and culture umbilical cord mesenchymal stem cells （ UC-MSC） and to induce them into neural differentiation.To provide a basis for the clinical treatment of nerve damages in dia-betic retinopathy （DR）.Methods The umbilical cords （UC） were collected from women who received C-section and gave birth to full-term healthy neonates.Written consents and approval of the Clinic Ethnics Committee were obtained prior to the study.UC-MSC were isolated by adherent culture in the medium contains 20%fetal bovine serum （ FBS） , and then maintained in the medium contains 10%FBS.The cellular morphology was studied using fluorescence microscope.Surface markers were examined with flow cytometry.MSC was then induced to differentiate towards neuron stem cells and neurons. Neural markers were detected by immunocytochemistry and RT-PCR.Results MSC grew adherently and cells were rela-tively uniform in spindle shape arranged in spiral or radial formation.FACS revealed that these cells expressed common markers of MSCs, such as CD105 （SH2）, CD73 （SH3） and CD90.After induction, cells begin to aggregate to form clus-ters.Cells grew neuron-like protrusions, became plump, and refracted more strongly.RT-PCR and immunofluorescence showed that the NSE and NeuroD1-positive cells reached 87.3 ±14.7%and 72.6 ±11.8% respectively.MAP2 and NF-M-positive cells reached 43.1 ±10.3%and 69.4 ±19.5% respectively.Conclusion MSCs from human umbilical cord showed an important neural differentiation potential and may represent a novel candidate for the therapy for DR nerve dam-age.