腺病毒介导的心肌营养素-1对大鼠骨髓间充质干细胞神经分化的影响

Effect of cardiotrophin -1 mediated by recombinant adenovirus on the neural differentiation of bone marrow mesenchymal stem cells of rats

目的探讨腺病毒（Adv）介导的心肌营养素-1（CT-1）对大鼠骨髓问充质干细胞（rBMSCs）向神经方向分化的影响。方法全骨髓贴壁筛选法分离培养rBMSCs，流式细胞术鉴定细胞表面标记性蛋白。将已纯化细胞分为4组：对照组、重组腺病毒增强型绿色荧光蛋白（Adv-EGFP）＋诱导州组（空病毒组）、Adv-EGFP-CT-1＋诱导剂组（CT-1组）及诱导剂组，分别向各转染组中加入对应的腺病毒液。转染后2d，除对照组外，其他各组均予神经分化诱导处理，于诱导后5h、3d和7d观察各组细胞形态，细胞免疫荧光检测各组巢蛋白（Nestin）、神经元特异性核抗原（NeuN）及神经胶质纤维酸性蛋白（GFAP）的表达。结果诱导处理后，除对照组外，其他各组均显示类神经细胞样形态改变，其中CT-1转染组变化最明显。诱导后5h Nestin检出阳性率最高，其中CT-1组阳性率[（86．31±4．27）％]均高于其他各组，差异均有统计学意义（P均〈0．001），随后逐渐下降，CT-1组较其他各组变化明显，差异均有统计学意义（P均〈0．001）；各时间点空病毒组与诱导剂组Nestin的阳性率差异无统计学意义（P均〉0．05）。诱导后5h，CT-1组NeuN、GFAP即有少量表达，随后NeuN、GFAP阳性率逐渐升高，至第7天时其阳性率[（64．41±3．65）％、（47．14±4．29）％]最高，其中，CT-1组各时间点NeuN、GFAP阳性率均高于其他各组，差异均有统计学意义（P均〈0．001）；各时间点空病毒组与诱导刑组NeuN、GFAP的阳性率差异无统计学意义（P均〉0．05）。结论CT-1可促进rBMSCs向神经方向分化。

Objective To investigate the effect of cardiotrophin - 1 （CT - 1 ） mediated by recombinant adeno- virus （Adv） on the neural differentiation of bone marrow mesenchymal stem cells （rBMSCs） of rats. Methods The rBMSCs were isolated and cuhured by attachment method. The surface marker protein was identified by flow cytometry. The rBMSCs were divided into 4 groups ： control group, AdV enhanced green fluorescence protein （ Adv - EGFP） ＋ in- duction group （ empty virus group）, CT - 1 group and induction group. The ceils were transfected by Adv in the corre- sponding multiplicity of infections. Each group was induced by neural induction medium except for control group. The cells morphological changes were observed by microscope and the expressions of Nestin, neuronal nuclei （NeuN） and glial fibrillary acidic protein（GFAP） were detected respectively by cellular immunofluorescence at 5 h,3 d and 7 d af- ter induction. Results After induction treatment, neuron - like cells morphological changes were observed in all the groups except for control group, among which the most obvious change was found in CT - 1 group. The positive rate of Nestin was the highest at 5 h after induction. Positive rate of CT - 1 group [ （86.31 ± 4.27 ）% ] was higher than any other groups,the differences were statistically significant （all P 〈 0. 001 ）;after which its positive rate gradually de- clined, the positive rate of CT - 1 group changed more obviously than other groups, and the differences were statistically significant （ all P 〈 0.001 ）. The difference in Nestin positive rate between the empty virus group and induction group was not statistically significant （ all P 〉 0.05 ） at various time points. The NeuN and GFAP could be observed in CT - 1 group at 5 h after induction. Then the positive rate of NeuN and GFAP increased gradually, and climbed to the highest point [ （64.41 ±3.65）%, （47. 14 ±4.29）% ] on 7 d after induction. Positive rate of NeuN and GFAP in CT- 1 group at various time points were higher than that of other groups, and the differences were statistically significant （ all P 〈0.001 ）. The difference of NeuN and GFAP positive rate between the empty virus group and induction group was not statistically significant （all P 〉 0.05）. Conclusion CT - 1 could promote the neural differentiation of rBMSCs.