骨髓间充质干细胞失巢凋亡下存活及迁移机制

The potential applications of bone marrow mesenchymal stem cells (BMSCs) have been widely advocated, however, many barriers hinder their clinical utilization. Anoikis triggers programmed cell death, leading to a marked decrease in the efficiency of cell trafficking and engraftment. Anoikis induced massive cell death has emerged as the major challenge in the application of BMSCs. How some of BMSCs can overcome this adversity and migrate towards distant destinations remains largely unexplored. My study aims to explore the survival and migratory mechanism of BMSCs under anoikis stress. Our pilot study suggested that the surviving BMSCs could circumvent anoikis stress by forming self-supporting cellular aggregates. The survival mechanism could be partially explained by the increase in anti-apoptosis effect via up-regulated Bcl-2 expression and autophagy activation. Importantly, compared to adherent BMSCs, anoikised-BMSCs had better migratory response to stromal cell derived factor-1 (SDF-1). We hypothesize that this enhanced migratory response can assist BMSCs homing to the targets. We will further explore the effects of autophagy on BMSCs survival and the role of SDF-1/CXCR4 (SDF-1’s receptor) axis in regulating BMSCs migration in vitro and in vivo using transwell migration assay and Graft versus Host Disease (GvHD) mouse model, respectively. Furthermore, the expression level of CXCR4 on anoikised-BMSCs will be detected. This study will delineate the survival and migratory characteristics of BMSCs under anoikis stress and the potential mechanism responsible for overcoming this adversity. Our findings will optimize the clinical application of BMSCs.

失巢凋亡极大阻碍了骨髓间充质干细胞(BMSCs)向损伤组织定向迁移及植入，阐明BMSCs失巢状态下存活及迁移机制是推动其临床应用的迫切需要。我们前期研究发现: 1)BMSCs在失巢状态下可形成自供给聚合团以促进细胞存活；2)失巢可上调抗凋亡蛋白Bcl-2表达并可激活自噬；3)与粘附状态培养BMSCs相比，失巢BMSCs显示出对趋化因子SDF-1更强的趋化迁移能力。据此推测，BMSCs通过激活自噬以促进存活，并通过提高对SDF-1的迁移反应以克服失巢凋亡完成归巢。本研究拟通过1)阻断自噬功能后观察细胞活性以揭示自噬在抗失巢凋亡中的调控作用；2)在抑制SDF-1/CXCR4(SDF-1受体)信号通路后，利用体外迁移实验与GvHD小鼠模型体内实验证实失巢BMSCs迁移依赖于SDF-1/CXCR4信号轴；3)检测细胞表面CXCR4水平以阐述其迁移反应增强机制。研究结果有助于推动BMSCs临床应用。

骨髓间充质干细胞（Bone marrow mesenchymal stem cells, BMSCs）不仅具有自我更新及多能分化潜能，更凭借其特有的造血调控、免疫调节能力，显示出广阔的临床应用前景。尽管存在巨大应用潜力，BMSCs由研究领域向临床应用转化中面临的最大挑战是其归巢机制尚未明确，特别是由于BMSCs为粘附细胞，其向靶组织迁移过程中不可避免地需脱离细胞外基质支持而进入程序性死亡，此过程称为“失巢凋亡”。失巢凋亡极大阻碍了其向靶组织的定向迁移及植入，已成为BMSCs临床应用中遇到的最大瓶颈。在失巢状态下，部分BMSCs如何克服困境存活并最终迁移至损伤组织发挥生物学效应尚未可知。因此，研究BMSCs失巢状态下迁移及存活机制，已成为推动其临床应用进程的迫切需要。. 本研究发现BMSCs在悬浮状态下形态由纺锤形而逐渐形成“球样”自供给聚合团。通过Boyden Chamber体外迁移实验，我们发现BMSCs在失巢凋亡下不仅具有向趋化因子SDF-1的迁移能力，而且较正常粘附状态培养BMSCs显示出更强趋化反应。更为重要的是，此迁移反应可被CXCR4特异性拮抗剂（AMD3100）阻断，从而提示增强的趋化反应为CXCR4依赖。我们通过建立GvHD小鼠模型，显示在阻断CXCR4后，BMSCs向损伤组织的迁移与植入被显著抑制，从而进一步证实了失巢BMSCs归巢依赖于SDF-1/CXCR4信号通路。不仅如此，通过流式细胞检测技术，我们发现失巢BMSCs细胞表面CXCR4表达水平高于粘附状态BMSCs，提示失巢BMSCs可通过上调CXCR4表达从而利用对趋化因子SDF-1迁移反应的提高从而克服失巢凋亡到达损伤组织发挥生物学效应。尽管失巢凋亡导致细胞进入程序性凋亡，但部分BMSCs可通过上调抗凋亡蛋白Bcl-2表达及激活自噬功能调控细胞存活。. 综上所述，BMSCs在失巢状态下显示出独特的迁移及存活特征。失巢BMSCs悬浮状态下形成“球样”聚合团，通过提高对趋化因子SDF-1的迁移反应以克服失巢压力完成定向迁移。通过体外及体内实验证实此迁移过程依赖于SDF-1/CXCR4信号通路。此外，BMSCs可利用上调抗凋亡蛋白Bcl-2表达及自噬机制调控细胞存活能力从而克服失巢凋亡。本研究结果为研究BMSCs迁移机制提供了新思路，有助于推动BMSCs发挥更大临床应用价值。