人尿源干细胞的分离培养及体外成神经细胞分化研究

Isolation, culture and neural differentiation of human urine-derived stem cells

摘要:

目的 从正常成人尿液中分离提取间充质干细胞(MSC)来源的人尿源干细胞(hUSC),并研究比较其在不同诱导液中的体外成神经分化能力。方法 提取正常成人来源hUSC,经体外培养、扩增,流式细胞术鉴定,用4种不同配方的成神经诱导液分别诱导hUSC向神经细胞分化,在诱导至第14天时,光镜下观察细胞形态改变, 并用逆转录－聚合酶链反应(RT-PCR)方法鉴定Nestin、S100、β3-tublin和NF-200基因表达。采用SPSS 13.0统计软件对数据进行分析。组间比较采用t检验和方差分析。结果 从正常成人提取的hUSC成功表达MSC表面标志物(CD27⁺、CD44⁺、CD73⁺、CD90⁺、CD31-、CD34-、CD45-、HLA-DR-)。hUSC在B、C、D成神经诱导液中诱导培养14 d后,细胞胞体回缩饱满、折光度变强,呈现神经元样突起。相比A成神经诱导液,B、C和D成神经诱导液中细胞Nestin、S100表达水平增高,B和D成神经诱导液中细胞β3-tublin、NF-200表达水平降低。其中C成神经诱导液中细胞Nestin表达水平高于B和D成神经诱导液中的细胞,B成神经诱导液中细胞S100表达水平高于C和D成神经诱导液中细胞,差异均具有统计学意义(P＜0.05)。结论 hUSC具备MSC特性；B、C、D成神经诱导液均有助于hUSC向神经类细胞方向分化,提示在优化诱导条件下,hUSC能向神经干细胞(NSC)及胶质细胞亚群分化。

Abstract:

Objective To isolate human urinary stem cells (hUSC) with mesenchymal stem cells (MSC) characteristics from urine samples of healthy individuals, and determine the neurogenic differentiation capabilities of the cells in different neurogenic induction medium. Methods hUSC were isolated from the urine sample of normal individuals, and then cultured, passaged and identified with flow cytometry. Then, after 14 days′ induction with 4 kinds of neurogenic induction medium, cell morphology was observed under a light microscope, and the mRNA levels of Nestin, S100, β3-tublin and NF-200 were detected by RT-PCR. SPSS statistics 13.0 was used to perform the statistical analysis. Student′s t test and Chi-square test were employed for comparison among groups. Results The obtained cells derived from the urine samples of healthy adults could successfully express surface markers of MSC (CD27⁺, CD44⁺, CD73⁺, CD90⁺, CD31-, CD34-, CD45-, HLA-DR-). After 14 days′ induction with neural indution medium B, C and D, the hUSC showed spherical cell bodies with enhanced refraction and neuron-like processes. RT-PCR indicated compared with the medium A, the mRNA levels of Nestin and S100 were increased medium B, C, D induction, and those of β3-tublin and NF-200 were decreased after medium B and D induction. What′s more, medium C induced the highest Nestin level than medium B and D, while medium B induction obtained the highest level of S100 than medium C and D (P<0.05). Conclusion hUSC possess the characteristics of MSC. Medium B, C and D contribute to the differentiation of hUSC into neuron cells. Our results suggest that hUSC can be differentiated into neural stem cells and glial subpopulations under optimized induction conditions.