MIF调控lncRNA-Meg3优化BMSCs负载微冰胶微创修复激素性股骨头坏死的机制研究

Bone mesenchymal stem cells (BMSCs) based therapies have had positive outcomes in treatment of Early steroid-induced avascular necrosis of femoral head (ESANFH). However, lots of BMSCs went apoptosis, reducing their ability to contribute to ESANFH injury repair. Long noncoding RNAs (lncRNAs) regulate diverse biological processes. Latest research found that lncRNA-Meg3 took part in cellular apoptosis though modulating cellular oxidative stress, and can be modulated. Apoptosis induced by hypoxia/serum deprivation is closely related with cellular oxidative stress in BMSCs. Macrophage migration inhibitory factor (MIF) can modulate oxidative stress to resist cellular apoptosis in many kinds of cells. In our previous experiments, we found that the expression of lncRNA-Meg3 was increased under hypoxia/serum deprivation condition when compared with normal, while the expression of lncRNA-Meg3 was inhibited by exogenous MIF. Here, we suggest that exogenous MIF may ameliorate the BMSCs apoptosis induced by hypoxia/serum deprivation through modulating lncRNA-Meg3, Previous inventing novel injectable microcryogels delivery, which have confirmewd its excellent characteristics of minimally invasive injectable, provided mechanically strength and avoided cell leaking will be loaded and improve effect of therapy of ESANFH in vivo. In vitro, we culture rabbit BMSCs, and research Wnt/β-catenin signaling pathway function in MIF conferring resistance apoptosis through modulating lncRNA-Meg3. The purpose is to provide a new idea and therapeutic implications of minimally invasive treatment for ESANFH.

骨髓间充质干细胞（BMSCs）在激素性股骨头坏死（ESANFH）治疗中发挥重要潜能，但移植后大量细胞凋亡极大限制了治疗效果。长链非编码RNA（lncRNAs）具有广泛的生物学功能，新近研究发现lncRNA-Meg3通过调节氧化应激参与凋亡，且具有可调控性。缺血/缺氧诱导BMSCs凋亡与氧化应激关系密切。巨噬细胞迁移抑制因子（MIF）通过调节氧化应激反应，发挥抗凋亡作用。前期预实验提示：缺血/缺氧条件下，BMSCs中lncRNA-Meg3表达明显升高，可被MIF所抑制。因此，我们推测MIF可调控lncRNA-Meg3降低BMSCs凋亡，并且联合新型PEG微冰胶，利用其微创可注射性、优异的力学性质和防细胞渗漏特点，进行兔ESANFH模型体内移植治疗。同时体外探究Wnt/β-catenin信号通路在MIF调节lncRNA-Meg3抗凋亡中的关键作用，为ESANFH损伤提供微创治疗新理念和新策略。

骨髓间充质干细胞（BMSCs）在激素性股骨头坏死（ESANFH）治疗中发挥重要潜能，但移植后大量细胞凋亡极大限制了治疗效果。长链非编码RNA（lncRNAs）具有广泛的生物学功能，新近研究发现lncRNA-Meg3通过调节氧化应激参与凋亡，且具有可调控性。缺血/缺氧诱导BMSCs凋亡与氧化应激关系密切。巨噬细胞迁移抑制因子（MIF）通过调节氧化应激反应，发挥抗凋亡作用。本课题从体外实验证实了在缺血缺氧环境下移植干细胞的凋亡及氧化应激现象，在二维和三维层次上发现MIF对BMSCs的保护作用，并且通过转基因技术研究其内在机理，最后通过体内动物实验，应用骨组织工程手段（微冰胶+MIF+BMSCs）评估新型可注射微冰胶装载MIF预处理BMSCs后在ESANFH生物治疗领域的应用潜能。研究结果发现在缺血缺氧环境下LncRNA-MEG3表达量及氧化应激指标ROS明显增高，其内源性MIF浓度轻度升高。而给予MIF后发现MEG3浓度明显降低，其凋亡CASP3、Bax、Bcl-2基因含量升高，氧化应激指标含量降低，给予三维负载后发现更具有抗凋亡作用。通过进一步的转基因技术发现，沉默MEG3后可以抑制p53基因，其下游信号通路WNT/β-catenin表达被激活，氧化应激指标含量降低，细胞凋亡数量明显减少，过表达MEG3后具有相反作用。体内实验研究证实与传统单纯BMSCs移植对比，微冰胶+MIF+BMSCs更能够延缓ESANFH的病程，通过免疫组化及组织病理学结果证实治疗组（微冰胶+BMSCs+MIF）空骨陷窝、骨小梁结构较单纯BMSCs组明显改善，优于造模组，免疫组化染色显示Dkk-1、PPARγ、GSK-3β成骨基因在治疗组明显增高，与病理结果类似；Micro-CT显示微冰胶+MIF+BMSCs组能够显著促进骨小梁结构形成及促进软骨下小血管形成，以上结果显示“外源性MIF可通过调控 lncRNA-Meg3，联合生物材料降低移植后凋亡，提高对ESANFH 的治疗作用”的假说，通过降低移植细胞凋亡、早期及时的微创注射方法、防止细胞泄露等措施，为临床治疗ESANFH实施微创化的生物学治疗提供一种新的治疗方案及可行性奠定理论依据。