HIF-1/p53调节乏氧张力下BMSC增殖和成骨分化的特点及与miRNA相关机制研究

The microenvironment that bone marrow derived mesenchymal stem cells (BMSCs) stay is under an oxygen concentration of 2%~7%. BMSCs face severer hypoxia stress when pathological risks present. The stress can recruit local and circulating precursor cells (especially BMSCs) for following reparation. However, the properties of multiplication and osteogenic differentiation that precursor cells possess under severe hypoxia is unclear. Hypoxia inducible factor 1 (HIF-1) is a critical transcription factor which can regulate biological behavior of BMSCs under hypoxia. The effect of hypoxia on BMSCs is dependent on the severity and duration of hypoxia. Moreover, the function of p53 to regulate osteogenic differentiation of BMSCs under hypoxia is closely related to HIF-1. In current research, it aims to explore the characteristics of multiplication and osteogenic differentiation of human BMSCs, and reveal intrinsic relationship between HIF-1 and p53 in controlling multiplication and osteogenic differentiation of BMSCs, as well as differential expressions of microRNA. Furthermore, the expressions of HIF-1 and p53 are regulated through the technique of small interference RNA or enhancer transfection. These reconstructed BMSCs are employed for the treatment of animal model with osteonecrosis of the femoral head, and control groups are set for comparison. The combination of in vitro and in vivo studies is to reveal the individual and reciprocal function of HIF-1/p53 and networks with miRNA, and to better employ BMSCs for cytotherapy in the near future.

骨髓间充质干细胞(BMSC)生存于2%~7%低氧微环境,当病理因素存在时,BMSC 会面临更窘迫的乏氧应激。应激因素常具有募集局部和外周循环中前体细胞(BMSC等)的功能，但募集来的前体细胞如何在此环境中增殖和成骨分化所知甚少。乏氧诱导因子1(HIF-1)是调控乏氧下BMSC细胞周期重要的转录因子；乏氧对 BMSC 增殖的影响与其张力大小有关，并且与乏氧维系时间有关。同时，p53 调控 BMSC 乏氧张力下的分化行为与HIF-1之间关系密切，miRNA在参与构建p53/HIF-1调控网络发挥了重要作用。本研究拟探索乏氧张力下BMSC增殖和成骨分化特点,miRNA差异表达及HIF-1/p53在调控BMSC增殖、分化中的机制，并通过调节HIF-1/p53在BMSC的表达、设计基于基因修饰BMSC治疗骨坏死的动物模型，为阐释BMSC的细胞生物学特点和基于BMSC的临床治疗奠定相关的理论基础。

尽管股骨头坏死的发病机制尚不完全清楚，但其终末病理表现为股骨头内血供的减少，股骨头内骨和血管相关细胞均面临乏氧应激。乏氧在股骨头坏死的发生和进展中发挥了重要作用。乏氧应激首先会诱导细胞表达乏氧诱导因子(HIF)上调，进一步通过下游血管内皮细胞生长因子(VEGF)，使得微环境血管生成能力增强。. 我们发现在骨髓间充质干细胞(MSCs)内过表达HIF-1α能促进细胞向成骨方向分化，从而增强细胞的成骨能力;其在细胞内的过表达能促进血管内皮细胞分泌VEGF明显增加。我们通过慢病毒载体将HIF-1α基因转导入MSCs，从而使其在MSCs内过表达，在体外评估其成骨分化能力和成血管能力，并使用这种HIF-1α过表达的MSCs治疗兔的激素性股骨头坏死模型。结果证实HIF-1α转染的MSCs在体外成骨分化增强，并分泌更多的VEGF。同时，我们发现HIF-1α过表达的MSCs被移植入骨坏死区域后能继续存活并分化成成骨细胞，且骨坏死区的新生骨组织和血管明显多于单纯的MSCs治疗组。HIF-1α基因转染的MSCs移植能有效的治疗早期兔激素性股骨头坏死模型，为早期股骨头坏死的治疗提供了新的思路。. 然而，虽然通过慢病毒将HIF-1α基因转导入MSCs，能够使其在MSCs内过表达，但是HIF-1α基因转导入MSCs后将永久性的在细胞内表达。且慢病毒载体潜在的风险，如引起肿瘤、排除反应等，极大的阻碍了它的临床应用。二甲氧乙二酰甘氨酸（DMOG）是一种小分子化合物，是脯氨酸羟化酶的抑制剂。它能在常氧的情况下抑制HIF-PH的作用，上调细胞内HIF-1α基因的表达。我们发现DMOG在体外能够剂量依赖性的增强HIF-1α的表达，且使MSCs表达RUNX-2、OCN、ALP等成骨标志物增多，分泌的VEGF也比正常的MSCs增多，同时DMOG处理过的MSCs更加明显的促进大鼠的颅骨缺损模型的骨再生。我们通过使用DMOG处理的MSCs和β-TCP构建的组织工程骨移植修复大鼠的颅骨缺损，发现DMOG处理的MSCs比正常MSCs更加明显的促进组织工程骨在体内的血管化，从而改善组织工程骨的骨修复能力。因此，我们认为DMOG用于提高MSCs的骨组织修复能力具有广阔的研究和应用前景。