间充质干细胞移植改善胶原缺陷性异常成骨的机制研究

Osteogenesis imperfecta(OI)is a genetic bone disorder with the feature of osteopenia, frequent fracture of bone and abnormality of figure caused mutation of type I collagen. OI could not be cured during these days, and medcine and surgery could not treat part of symptoms. Transplantation of mesenchymal stem cells could synthesize normal type I collagen and dramatically enhance the bone mass, decrease the risk of frature and achieve almost normal development in patients and mouse model. As the engraftment rate was lower than 5%， the mechanism of significant correction of abnormal osteogenesis with limited engrafted MSCs is unknown. Our previous study showed decreased number and maturation of osteoclast in mouse transplanted with bone marrow-derived mesenchymal stem cells, demonstrating depressed bone absorbation. It was found osteoblastic lineage is under continous stimulation, however, only a proportion of cells attain the mature osteoblst stage. Indeed, immature osteoblsts exhibit a stronger potential to support osteoclast formation and differentiation with expression of a higher Rankl/Opg ratio. Type I collagen could connect with integrin, which locates on membrane of osteoblast and promote osteoblst maturation. Present research is composed of CXCR4 and/or BMP-2 transfectionof MSCs, irradation of oim mouse(an OI model) and targeted MSCs transplantation. Engraftment, osteopoietic chimerism, fracture and bone mineral density would be oberserved and compared with control. Influence on comitment and mature of osteoclast and osteoblast was obeserved. Information about the mechanism of transplatation of mesenchymal stem cells for OI will be provide.

成骨不全症（OI）是I型胶原缺失或异常导致的，以骨脆弱、反复骨折和矮小畸形为特征的遗传性疾病。该病目前无法治愈。间充质干细胞移植患者或模型小鼠的移植率低于5%，移植后可分泌正常I型胶原，并在一段时间内显著提高骨量、降低骨折率并改善生长发育。少量移植细胞如何取得显著疗效的机制尚不明确。既往研究发现OI小鼠中成骨细胞成熟抑制、RANKL/OPG比值升高，进而促进破骨细胞生成和分化成熟。我们前期研究发现细胞移植后模型小鼠体内破骨细胞数量减少、成熟抑制，说明细胞移植后抑制了骨吸收。移植细胞主要带入正常I型胶原，而I型胶原可以结合成骨细胞表面的整合素，促进成骨细胞成熟。因而我们拟对干细胞移植后oim小鼠体内的成骨细胞、破骨细胞数量、分化及成熟情况及相关调节因子进行观察，结合体外细胞培养观察。验证干细胞是否通过I型胶原促进自体成骨细胞成熟、降低RANKL/OPG比值抑制破骨细胞活动来纠正异常成骨。

.成骨不全症（OI）是I型胶原基因突变导致的遗传病，临床表现为骨脆性增加、反复骨折、发育迟缓及畸形矮小。干细胞移植治疗成骨不全症儿童已有个案，疗效显著但仅能维持2-4年。干细胞移植可以改善成骨不全症小鼠模型（oim小鼠）的骨强度、减少骨折次数并改善生长发育。oim小鼠接受干细胞移植治疗时，移植率仅为2-5%但疗效显著。本研究针对这一现象，推测干细胞移植可能并非通过外源性干细胞成骨改善骨质量，而是通过某种机制纠正了内在的异常骨代谢。在本研究中，我们采用过表达归巢受体CXCR4提高骨髓间充质干细胞(MSCs)定向骨组织的移植率，观察移植率是否与疗效有关，证实研究假设并初步了解干细胞治疗OI的主要机制。.在本研究中，我们建立了两个oim小鼠MSCs移植治疗组（MSCs-CXCR4+组、MSCs组）。MSCs-CXCR4+组移植率是MSCs组的2.7倍（MSCs-CXCR4+移植率13.2% VS 普通MSCs移植率4.9%）。与未接受治疗的oim小鼠相比，2个治疗组骨折小鼠数量减少大于75%，总骨折数减少大于90%，下肢长骨生物力学参数、形态学、组织学参数均有显著改善，差异有统计学意义；但两个治疗组之间无显著差异。上述结果均证实了本研究假设：干细胞移植率与临床症状、骨骼参数的改善无显著量效关系。.oim小鼠血清OCN、NTx及TRACP5b均高水平表达，提示骨转化活跃。移植治疗后血清OCN水平升高6%，NTx及TRACP5b分别下降19%及14%，说明干细胞移植后成骨细胞分化成熟，破骨细胞骨数量级吸收能力均显著被抑制。.体外试验中oim小鼠成骨细胞培养、成骨-破骨体外共培养体系中，经I型胶原处理的成骨细胞分化增强、成骨活跃，破骨细胞分化、吸收能力显著减弱，而这些效应可以被整合素a2b1受体抗体所拮抗。.上述发现证实，MSCs归巢到骨组织的数量不是决定其临床疗效的主要因素，因而研究如何延长干细胞的作用周期或找到合理的定期移植方案可能更有价值。根据移植后的骨代谢情况变化，推测干细胞治疗OI的主要机制很可能是：外源性MSCs合成正常I型胶原，促进oim小鼠成骨细胞分化成熟，降低RANKL/OPG比值、减弱破骨活动，纠正了oim小鼠异常活跃的骨转化。提示也可从引入正常I型胶原的角度，探索OI的治疗方法。进一步研究明确I型胶原在成骨/破骨调节中的作用机制，可为临床治疗OI提供思路