SDF-1/CXCR4-JNK通路在调节组织工程骨移植后宿主MSC动员过程中作用机制的研究

Tissue-engineered bone (TEB) is one of promising methods for repairing huge bone defect. TEB could recruit mesenchymal stem cells (MSC) to repair bone defect by seeding cells' osteoinductive,in which MSC mobilization and migration are involved.The mechanism of MSC migration is clear, however, MSC mobilization is unknown yet. Our pre-experiment showed that expression of SDF-1/CXCR4-JNK pathway was elevated in MSC from the graft after TEB transplantation. And the activity of protease was also increased just as uPA.At the same time, SDF-1 antibody could decrease the number of MSC derived from the host in the graft. During mobilization of haemopoietic stem cells, the effect of SDF-1/CXCR4-JNK pathway is similar to these. The hypothesis was logically set up that SDF-1/CXCR4-JNK pathway meidates MSC mobilization by increase activity of protease.According to this hypothesis, preparation of parabiotic mouse model between GFP positive mice and GFP negative mice, by which the tissue origin of mobilized MSC would be determined.We will detecte the rate of CXCR4 positive MSC in the different tissue with flow cytometry after TEB was transplanted into the model of SDF-1 conditional knockout mice. After inhibition of CXCR4 and/or JNK,the activity of protease and number of CXCR4 positive MSC would be further analysed. The effect of protease on extracellular matrix and adhesion molecule was detected during this process. The effect of SDF-1/CXCR4-JNK pathway on MSC mobilization would be evaluated on the basis of these experiments.This study will make the mechanism of TEB osteogenesis clearer, supply a guide for elevating osteogenic potency of TEB, and explore a new strategy of application of TEB.

组织工程骨（TEB）是修复大段骨缺损最有希望的策略之一，可通过种子细胞的骨诱导特性募集宿主间充质干细胞（MSC）修复缺损，其中MSC动员的机制仍不明。前期研究发现TEB移植后SDF-1/CXCR4-JNK通路在移植区MSC中表达增高，uPA等蛋白酶活性增强，SDF-1抗体可减少移植物内迁移来的MSC数量，结合此通路在造血干细胞动员中的作用，提出"SDF-1/CXCR4-JNK通路增强蛋白酶活性，调节MSC动员"假说。拟建立GFP+ - GFP-连体小鼠模型，观察动员MSC的来源。在SDF-1条件基因敲除小鼠模型移植TEB，检测MSC动员过程中在不同组织内的数量变化；应用CXCR4和/或JNK抑制剂，再次对比蛋白酶活性和MSC数量变化；观察蛋白酶对细胞外基质和粘附分子的作用；综合评价此通路在MSC动员中的效应。本研究将进一步阐明TEB的成骨机制，为提高TEB的成骨效能提供理论指导和实验依据。

组织工程骨（TEB）是临床上修复大段骨缺损最有希望的策略之一，可通过移植创伤和骨诱导特性募集宿主间充质干细胞（MSC）修复缺损，其中MSC动员的机制仍不明。本研究通过小鼠股骨缺损模型和联体模型以SDF-1/CXCR4-JNK通路为主线，观察组织工程骨移植后在移植区、外周血和骨髓中的MSC的表达变化，再辅以SDF-1条件基因敲除小鼠模型，验证SDF-1在组织工程骨移植后启动的MSC的迁移中的作用。本研究建立了小鼠双侧股骨缺损模型，为后面组织工程骨成骨评价建立模型。探索了脐带获取大量间充质干细胞（MSC）的方法，为大量构建组织工程骨提供了充分的细胞来源。通过建立GFP+—GFP-连体的血液相通的模型，观察到组织工程骨移植后MSC由骨髓到移植区的动员、迁移变化规律。通过ELISA检测组织工程骨移植前与后不同时像点骨髓、外周血和移植组织内SDF-1的浓度，发现了SDF-1在组织工程骨移植后，移植区和外周血的SDF-1浓度升高，而骨髓内的浓度则降低，提示MSC可能是沿着SDF-1的浓度梯度进行迁移。应用CXCR4拮抗剂和JNK抑制剂，可明显减少组织工程骨移植区内MSC的比例，相应的外周血内MSC的比例也有减少，而骨髓内的MSC数量略有增加，提示CXCR4和JNK可能参与组织工程骨移植后MSC的迁移调控。WB检测结果显示，SDF-1可促进CXCR4的表达，而阻断CXCR4抑制了P-JNK的表达，进而P-JNK的表达高低与N-cadherin的表达没有相关性。应用PCR和ELISA法检测组织工程骨移植前与后骨髓、外周血和移植组织内MMP-2、MMP-9等蛋白酶的活性，发现MMP-9与SDF-1/CXCR4-JNK通路可能并无确切的关系，而MMP-2可能参与了早期的调控。采用SDF-1条件基因敲除小鼠和拮抗剂发现SDF-1/CXCR4-JNK通路在组织工程骨移植后MSC动员中发挥了重要作用。组织工程骨移植后体内，移植区SDF-1来源于周围宿主细胞的分泌。采用SDF-1条件基因敲除小鼠和野生型小鼠分别给予CXCR4抗体和JNK抑制剂，发现SDF-1/CXCR4-JNK对组织工程骨组织的成骨质量具有重要的影响。本研究对组织工程骨移植后宿主SDF-1/CXCR4-JNK通路的变化进行的观察和研究，发现其在成骨中的重要作用，为提高组织工程骨的成骨效能提供理论指导和探索新策略。