过表达Notch1胞内域对c-Kit+骨髓间充质干细胞分化的影响

摘要

BACKGROUND:Activation of Notch signaling plays a critical role in stem cel differentiation, and this effect seems to be cel-type dependent. Little is reported on the role of activation of Notch1 signaling in the differentiation of c-Kit+ bone marrow mesenchymal stem cels. OBJECTIVE:To analyze the influence of activation of Notch1 signaling on the differentiation of c-Kit+ bone marrow mesenchymal stem cels. METHODS:The Notch1 intracelular domain (N1-ICD) was obtained from the cDNA library by PCR and cloned intoBamHI/AgeI digested adenoviral GV314 plasmid to construct N1-ICD overexpressing shuttle plasmid, and the positive clones were verified by sequencing. N1-ICD shuttle plasmid and helper plasmids pBHGloxΔE1,3 Cre were used to co-transfect HEK293T cels to obtain N1-ICD overexpressing adenoviral particles (N1-ICD-Ad). The c-Kit+ subpopulation were isolated from bone marrow mesenchymal stem cels of the Sprague-Dawley rat femurviamagnetic activated cel sorting. After transfection of the c-Kit+ BMSCs with N1-ICD-Ad adenovirus, we assessed the activation of Notch1 signaling and differentiation in c-Kit+ bone marrow mesenchymal stem cels by quantitative RT-PCR and immunofluorescent staining. RESULTS AND CONCLUSION:N1-ICD coding sequence was successfuly generated from the cDNA library, and then was cloned into the linearized adenoviral vectors GV314. The resistant clones were verified by sequencing. With the assistance of packaging plasmids, recombinant N1-ICD-Ad adenovirus plasmids were successful packaged in HEK293T cels, and its title was 2×1012 PFU/L. c-Kit+ bone marrow mesenchymal stem cels with the purity of 91.6% were successfuly isolated from the bone marrow mesenchymal stem cels of the Sprague-Dawley rat femur. Compared with the blank and negative controls, N1-ICD-Ad infection in the c-Kit+ bone marrow mesenchymal stem cels led to substantial accumulation of N1-ICD in the cytoplasm and nuclei, significantly unregulated expressions of Hes1 (a downstream gene of Notch) and cardiomyocyte differentiation genes Nkx2.5 and cTnT, significantly increased the expression of von Wilebrand factor, an endothelial cel differentiation gene, and mildly increased the expression of smooth muscle22α, a smooth muscle cel differentiation gene. These experimental results indicate that the activation of Notch1 signaling contributes to multi-lineages differentiation of c-Kit+ bone marrow mesenchymal stem cels, and the construction of N1-ICD overexpressing adenoviral vector makes the foundation for further research on the role of Notch1 signaling in stem cel biology.%背景：Notch信号活化对干细胞分化有重要的影响，其效应具有细胞特异性，但有关Notch信号与c-Kit+骨髓间充质干细胞分化的相关报道较少。目的：分析Notch1信号活化对c-Kit+骨髓间充质干细胞分化的影响。方法：应用PCR从cDNA文库中调取Notch1受体胞内域(N1-ICD)序列，定向克隆入腺病毒穿梭质粒GV314构建重组质粒，与腺病毒辅助包装质粒pBHGlox∆E1,3 Cre在HEK293T细胞进行同源重组，获得N1-ICD过表达腺病毒颗粒(N1-ICD-Ad)。分离SD大鼠股骨骨髓间充质干细胞，应用磁激活细胞分选法分选出c-Kit+亚群并用流式鉴定其纯度。将N1-ICD-Ad腺病毒感染c-Kit+骨髓间充质干细胞，8 d后定量RT-PCR或免疫荧光分析c-Kit+骨髓间充质干细胞中Notch1信号的活化及细胞的分化。结果与结论：(1)从cDNA文库中成功获得N1-ICD序列，将此序列成功克隆入线性化GV314载体，抗性克隆经测序证实无误；(2)在辅助包装质粒pBHGloxΔE1,3 Cre的协助下，成功获得滴度为2×1012 PFU/L重组腺病毒颗粒N1-ICD-Ad；(3)成功从SD大鼠骨髓间充质干细胞获得c-Kit+细胞亚群，纯度达91.6%。包装的腺病毒能有效感染c-Kit+骨髓间充质干细胞；(4)与空白对照组和阴性对照病毒组相比， N1-ICD-Ad 感染 c-Kit+骨髓间充质干细胞后 N1-ICD 在胞质与胞核聚集，显著上调了 Notch 下游基因Hes1的表达，显著诱导心肌细胞分化基因Nkx2.5和cTnT的表达，显著上调内皮细胞分化基因vWF的表达，轻微上调平滑肌细胞分化基因SM22α的表达。(5)实验结果提示Notch1信号的活化有助于c-Kit+骨髓间充质干细胞的多向分化，N1-ICD 过表达载体构建及腺病毒的包装并转染成功，为进一步研究Notch1信号活化对于干细胞生物学功能的影响奠定了基础。