CT-1对脐血间充质干细胞神经分化作用

The effect of Adv-CT-1 on the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells

目的探讨心肌营养素1(CT-1)在诱导脐血间充质干细胞(hUCB-MSCs)向神经细胞分化中的作用。方法培养hUCB-MSCs,流式细胞术检测表面标志物CD34、CD29、CD44及CD105;将携带CT-1的腺病毒(Adv-CT-1)感染hUCB-MSCs,在不同时间点用免疫荧光染色检测CT-1表达率,摸索最佳感染复数(MOI);将细胞分为4组:(1)对照组;(2)神经诱导剂组(NIM);(3) CT-1组(Adv-EGFP-CT-1+NIM);(4)空病毒组(Adv-EGFP+NIM)。于6 h和4 d检测各组神经相关蛋白标志物(nestin、NeuN、GFAP)表达。结果 hUCB-MSCs高表达CD29、CD44、CD105,不表达CD34; MOI最佳转染复数为100PFU/细胞。诱导后6 h,CT-1组呈现类神经细胞样形态改变,Nestin阳性率最高(86. 82%±4. 29%),与其他3组相比,差异有统计学意义(P <0. 05);至4 d时,各组Nestin表达明显减少;诱导后6 h,CT-1组NeuN、GFAP均有少量表达(14. 26%±2. 20%、16. 96%±4. 41%),随后逐渐升高,4 d时阳性率分别达(48. 33%±2. 53%)、(65. 83%±5. 17%); CT-1组阳性率均高于其他各组(P <0. 05)。结论CT-1可促进hUCB-MSCs向神经细胞分化。

Objective To investigate the differentiation potential of human umbilical cord blood-derived mesenchymal stem cells( h UCB-MSCs) into neuron-like cells by recombinant adenovirus cardiotrophin-1( Adv-CT-1). Methods By density gradient centrifugation and adherent culture,MSCs were harvested from h UCB. The surface antigen phenotype such as CD29、CD44、CD105 and CD34 were detected by flow cytometry. h UCB-MSCs were transfected with Adv-CT-1 at different multiplicity of infection( MOI) ranging from 50、100、200,to 300 PFU/cell. The transfection efficiency was assessed by fluorescence microscope at different time. The cell morphological changes were observed by microscope and the expression of Nestin,Neu N and GFAP were measured by cellular immunofluorescence at 6 h and 4 d after induction among control group,EGFP group,CT-1 group and neural induction medium( NIM) group respectively. Results By flow cytometry,hUCB-MSCs were found positive for CD29,CD44 and CD105,but failed to express CD34. The transfection efficiency was much higher( 87. 60% ± 1. 36%) when MOI 100 PFU/Cells at 72 h after transfection. The effect of Adv-CT-1 on the neural differentiation of hUCB-MSCs: About 6 h after induction,the cells treated with NIM and Adv-CT-1 exhibited stronger expression neural markers Nestin than cells treated with NIM and without Adv-CT-1 or control group or EGFP group. CT-1 group exhibited stronger expression neural markers Nestin( 86. 82% ± 4. 29%) than other groups. To 4 d after induction,the positive of Neu N and GFAP was gradually increasing to 48. 33% ± 2. 53%,65. 83% ± 5. 17% respectively in CT-1 group,the difference were statistically significant with other three groups( P < 0. 05). Conclusion The h UCB-MSCs transfected with CT-1 may be induced to differentiate to neuron-like cells.