P53和Parkin共同调节线粒体自噬对BMSCs修复早期ONFH的作用及机制

The survival and stemness of bone marrow mesenchymal stem cells (BMSCs) in the area of osteonecrosis of the femoral head (ONFH) are the keys to determine the efficacy of transplantation. However, oxidative stress in the osteonecrosis area lead to stress-induced apoptosis and senescence of transplanted BMSCs. It remains to be determined how to improve the survival and stemness of BMSCs. Studies have indicated that the main and important reason for the stress-induced apoptosis and senescence of transplanted BMSCs was the accumulation of damaged mitochondria in cells. Our previous study indicated that the upregulation of parkin and downregulation of P53 in BMSCs can enhance the mitophagy, and resist the stress-induced apoptosis and senescence of BMSCs. Based on these, our hypothesis is that P53 and parkin could regulate mitophagy through the PINKl-parkin pathway, resist the stress-induced apoptosis and senescence of BMSCs, and improve the efficacy of BMSCs transplantation. In the study, gene transfection and other techniques were used to determine whether P53 and parkin could regulate mitophagy through the above pathway, and then intervene the stress-induced apoptosis and senescence of BMSCs. The P53 and parkin genes were transfected into BMSCs to repair early steroid-induced ONFH models, then the repaired effect was evaluated by Micro-CT and immunology. In order to explore new ideas for resisting the stress-induced apoptosis and senescence of BMSCs and improving the effect of transplantation.

骨髓间充质干细胞（BMSCs）在股骨头坏死（ONFH）区的存活和干性是决定移植疗效的关键。但是，骨坏死区氧化应激导致移植的BMSCs应激性凋亡和衰老。如何提高BMSCs的存活和干性是尚待解决的难题。研究揭示受损线粒体积累是BMSCs应激性凋亡和衰老的重要原因。我们在BMSCs中上调Parkin并下调P53，可增强线粒体自噬，抵抗BMSCs应激性凋亡和衰老。据此我们推测：P53和Parkin可通过PINKl-Parkin途径调节线粒体自噬，进而抵抗BMSCs应激性凋亡和衰老，提高BMSCs移植疗效。本项目拟采用基因转染等技术，研究P53和Parkin是否通过上述途径调节线粒体自噬，进而干预BMSCs应激性凋亡和衰老；并将P53和Parkin基因转染BMSCs修复早期激素性ONFH模型，利用Micro-CT和免疫学等技术，评估修复效果；为抵抗BMSCs应激性凋亡和衰老、提高移植疗效探求新思路。

骨髓间充质干细胞（BMSCs）在早期激素性股骨头坏死（ONFH）的治疗中发挥重要的作用，但股骨头坏死区氧化应激导致移植的BMSCs出现应激性凋亡和衰老，从而降低了BMSCs对早期激素性ONFH的修复作用。如何提高BMSCs的存活和干性是亟待解决的难题。研究表明，受损线粒体的积累是导致BMSCs应激性凋亡和衰老的重要原因。为此，本项目主要研究P53和Parkin共同调节线粒体选择性自噬对BMSCs应激性凋亡和衰老的作用及机制；P53和Parkin共同调节线粒体选择性自噬对BMSCs移植修复早期激素性ONFH的作用；以及本项目延伸的两个子课题：（1）PARK7增强BMSCs抗氧化应激对BMSCs移植修复早期激素性ONFH的作用及机制；（2）NMNAT3通过改善线粒体功能抵抗BMSCs应激性凋亡的作用及机制。最终，得出以下重要结果：（1）在上调Parkin的同时下调P53的表达，可显著增强线粒体选择性自噬，清除细胞内积累的受损线粒体，能有效抵抗BMSCs应激性凋亡和衰老。（2）P53可与Parkin相互作用，抑制Parkin的线粒体易位和VDAC1的泛素化水平，导致线粒体自噬水平下降，细胞内受损线粒体含量增加，而下调P53的表达，可逆转上述结果；证明PINKl-Parkin途径是P53和Parkin共同调节线粒体选择性自噬的信号通路。（3）通过调控Parkin和P53的表达，增强BMSCs线粒体自噬，抵抗BMSCs应激性凋亡和衰老，可有效提高BMSCs对早期激素性ONFH的修复效果。（4）PARK7可通过调节Nrf2和ERK1/2信号通路增强BMSCs的抗氧化应激能力，提高BMSCs在早期激素性ONFH中的治疗效果。（5）NMNAT3可通过NAD+-Sirt3信号轴改善线粒体功能，进而抵抗BMSCs应激性凋亡。本项目探索了多种抵抗BMSCs应激性凋亡和衰老的干预方法，并为其提供了潜在的药物筛选靶点；为提高BMSCs移植修复早期激素性ONFH探索了新的思路。