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目的通过检测感染携带肝细胞生长因子(HGF)腺病毒的脐带间充质干细胞(hUC-MSC)内酪氨酸羟化酶(TH)、多巴胺转运体(DAT)和多巴胺(DA)的表达水平及动态变化,探讨hUC-MSC在HGF过表达的情况下是否能向多巴胺能神经元分化,为HGF基因修饰hUC-MSC治疗帕金森病提供细胞基础。方法采用组织块法分离培养hUC-MSC,流式细胞仪检测细胞是否表达间充质干细胞分子表面标志CD29、CD44和CD105;不同感染复数(0、50、100、150、200、400)的携带绿色荧光蛋白的腺病毒(Ad-GFP)感染hUC-MSC后,流式细胞仪检测感染效率,并确定最佳感染复数;运用携带HGF的腺病毒感染hUC-MSC后,细胞免疫酶化学方法检测TH和DAT的表达情况。结果采用组织块法可从脐带中成功分离出(表达间充质干细胞分子表面标志CD29、CD44和CD105)hUC-MSC;Ad-GFP可高效感染hUC-MSC,随着感染复数的升高,感染效率可达99.99%;感染携带HGF的腺病毒(Ad-HGF)后,hUC-MSC可逐渐表达TH和DAT,同时细胞形态也逐渐由纺锤状向多角形和不规则样转变。结论腺病毒可高效感染hUC-MSC;感染Ad-HGF后,hUC-MSC可逐渐向多巴胺能样神经元分化。
Objective To detect the expression levels of tyrosine hydroxylase (TH), dopamine transporter (DAT) and dopamine (DA) in umbilical cord mesenchymal stem cells (hUC-MSCs) infected with hepatocyte growth factor (HGF) To investigate whether hUC-MSCs can differentiate into dopaminergic neurons in the presence of HGF overexpression, and to provide a cellular basis for HGF gene modified hUC-MSCs to treat Parkinson’s disease. Methods Human umbilical vein endothelial cells (hUC-MSCs) were isolated and cultured by tissue block method. Flow cytometry was used to detect the expression of mesenchymal stem cells (CD29, CD44 and CD105) After infection of hUC-MSC with green fluorescent protein (Ad-GFP), the infection efficiency was determined by flow cytometry and the optimal number of infection was determined. After hUC-MSCs were infected with HGF-carrying adenovirus, the immunocytochemical method TH and DAT expression. Results The hUC-MSCs were successfully isolated from the umbilical cord (tissue surface markers of CD29, CD44 and CD105) by using the tissue block method. Ad-GFP could efficiently infect hUC-MSCs. As the infection multiplicity increased, Efficiency of up to 99.99%. After infected with adenovirus carrying HGF (Ad-HGF), hUC-MSC gradually expressed TH and DAT, meanwhile the cell morphology gradually changed from spindle to polygons and irregular samples. Conclusions Adenovirus can efficiently infect hUC-MSCs. After Ad-HGF infection, hUC-MSCs can gradually differentiate into dopaminergic neurons.