Snail基因修饰对骨髓基质干细胞骨架结构稳定作用及促迁移和对无血清培养诱导细胞凋亡的保护作用研究

The Advantages for Snail Expression to Promote Cell Migration and Induce Actin Reorganization and to Protect Against the Serum-deprivation-triggered Apoptosis of Bone Marrow Stem Cells

摘要:

转录因子Snail是调控肿瘤细胞迁徙转移的重要调控分子，基于干细胞与肿瘤细胞分子机制的重叠性，提出通过借鉴肿瘤细胞迁移的相关机制以用于提高骨髓基质干细胞向缺氧受损组织迁移能力的假设和研究思路，探讨Snail基因在人骨髓基质干细胞（MSCs）中的转染和表达情况，及转染后对基质干细胞促迁移作用、骨架结构的稳定作用及对无血清培养诱导细胞凋亡的保护作用。密度梯度离心法及细胞体外培养分离纯化人骨髓MSCs，脂质体法将重组表达载体pCAGGSneo-Snail-HA转染MSCs，G418筛选稳定表达，流式细胞仪检测MSCs表面抗原，采用免疫荧光染色技术检测转染后MSCs报告基因HA及目的基因Snail表达，Transwell细胞迁移实验和Western-blot评估细胞迁移能力和检测有关细胞信号转导通路分子水平变化，荧光染色分析细胞骨架，Sub-G₁凋亡峰流式细胞仪检测细胞凋亡率并评估细胞抗凋亡能力。经流式细胞仪选择检测分离纯化扩增MSCs表面分子特点为CD34(-)/CD29(+)，Snail及报告基因在转染后MSCs呈阳性表达，Snail质粒转染MSCs(MSCs-Sna)较对照空质粒转染MSCs(MSCs-neo)细胞迁移率增加(P<0.05)，PI-3K信号通路特异性抑制剂Wortmannin能显著抑制此迁移率的增加，无血清培养72h后，MSCs-Sna凋亡率较MSCs-neo低(P<0.05)。经Snail基因转染，MSCs迁移能力、骨架结构的稳定性及在无血清培养环境中抗凋亡能力增加。

Abstract:

The Snail transcription factor has been described as a strong repressor of E-cadherin and its stable expression induces epithelial-mesenchymal transitions responsible for the acquisition of motile and invasive properties during tumor progression. A fascinating analogy that has been raised is the seemingly similar and shared characteristics of stem cells and tumorigenic cells，which prompted us to investigate whether the mechanisms of the acquisition of invasiveness during tumor progression are also involved in bone marrow stem cells(MSCs). In this study，we examined whether Snail gene expression acts in the mobility，cytoskeleton and anti-apoptosis of MSCs. Cell Transmigration Assay and Western Blotting were performed to evaluate the cell migratory capability and the related Signaling pathways in MSCs transfected with the Snail expression vector of pCAGGSneo-Snail-HA (MSCs-Sna)，compared with MSCs(MSCs-neo) transducted with the control vector(pCAGGSneo). Actin cytoskeleton by Immunofluorescence and Sub-G1 detection by a FACScan flow cytometer were performed to analyze the cytoskeleton and anti-apoptotic capability of MSCs-Sna. Compared with MSCs-neo，MSCs-Sna show significantly more migration in the transwell migration system (P<0.05). And suppression of PI-3K activation by the specific PI-3K inhibitor，Wortmannin，brought on a reduction in Snail-mediated MSCs migration. In addition，we provide evidences that high expression of Snail inhibited the serum-deprivation triggered apoptosis and cytoskeleton changement of MSCs. These data suggest the possibility of facilitating MSCs migration to injured tissue and subsequent survival and maintenance in the local microenvironment after their transplantation，by investigating and increasing the advantage factors such as Snail high expression in MSCs.