过表达Notch1胞内域对c-Kit^+骨髓间充质干细胞分化的影响  

Effects of over-expression of Notch1 intracellular domain on the differentiation of c-Kit^+ bone marrow mesenchymal stem cells

作　　者：哈艳平 王振良[1] 雷洪[1] 丁然然[1] 蒋晓帆[1] 王可可[1] 申志华[1] 揭伟[1] 

机构地区：[1]广东医学院病理学系,广东省湛江市524023

出　　处：《中国组织工程研究》2016年第6期785-792,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81170121;81541004);广东医学院优硕培育基金(YS2015004;YS2015005)~~

摘　　要：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c DNA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p BHGlox?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c DNA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p BHGloxΔE1,3 Cre的协助下,成功获得滴度为2×10^(12)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c Tn T的表达,显著上调内皮细胞分化基因v WF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。BACKGROUND： Activation of Notch signaling plays a critical role in stem cell differentiation, and this effect seems to be cell-type dependent. Little is reported on the role of activation of Notch1 signaling in the differentiation of c-Kit^＋ bone marrow mesenchymal stem cells. OBJECTIVE： To analyze the influence of activation of Notch1 signaling on the differentiation of c-Kit^＋ bone marrow mesenchymal stem cells. METHODS： The Notch1 intracellular domain（N1-ICD） was obtained from the cDNA library by PCR and cloned into Bam HI/Age I 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 p BHGloxΔE1,3 Cre were used to co-transfect HEK293 T cells to obtain N1-ICD overexpressing adenoviral particles（N1-ICD-Ad）. The c-Kit^＋ subpopulation were isolated from bone marrow mesenchymal stem cells of the Sprague-Dawley rat femur via magnetic activated cell 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 cells by quantitative RT-PCR and immunofluorescent staining. RESULTS AND CONCLUSION： N1-ICD coding sequence was successfully generated from the c DNA 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 HEK293 T cells, and its title was 2×10^12 PFU/L. c-Kit^＋ bone marrow mesenchymal stem cells with the purity of 91.6% were successfully isolated from the bone marrow mesenchymal stem cells 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 cells led to substantial accumulation of N1-ICD in the cytoplasm and nuclei, significantly unregulated expressions of Hes1（a do

关 键 词：干细胞 骨髓干细胞 细胞分化 NOTCH信号 腺病毒载体 病毒包装 骨髓间充质干细胞 C-KIT 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]