腺相关病毒介导的tet-on -DKK1-shRNA靶向治疗激素性骨质疏松的实验研究

The main reason of osteopenia in glucocorticoid-induced osteoporosis is lipoblasts increase at the cost of decreasing osteogenesis induced by glucocorticoid. It is considered up-regulated DKK-1 induced by glucocorticoid could block Wnt classic pathway that promotes osteogenesis and inhibits adipogenesis of bone marrow stem cells. In this research, we planned to construct tetracycline up-regulated Tet-on gene expression system and make use of its regulate factor－vibramycin to compose bone matrix-vibramycin slow-release system which could facilitate targeted activation of Tet-on gene expression system injected and expression of Dkk1-siRNA via adenoassociated virus .In this way, Wnt classic pathway was activated for osteoporosis therapy. Additionally, we examined expression of key transcription factors and specific genes in osteogenesis and adipogenesis process induced by glucocorticoid without DKK-1 expression using transgenic technique. Furthermore, We hope to explore molecular mechanism of DKK-1 regulation at transcription level toward differentiation to osteogenesis and adipogenesis , key signals or molecular pathways in process of specific differentiation of BMSCs,which also provide theoretical support for treating glucocorticoid-induced osteoporosis as well as other osteopenia diseases.

激素性骨质疏松骨量减少的重要原因之一,是激素导致的以牺牲成骨为代价的脂肪细胞增多。研究发现，激素可通过上调Dkk-1表达从而阻断Wnt经典通路的抑制髓内干细胞成骨分化，促进其成脂分化的作用。本项目拟构建四环素正向调节的Tet-on基因表达系统，利用其调控因子强力霉素的亲骨特性,预先使强力霉素成沉积在骨基质中，组成骨基质－强力霉素缓释系统，从而使注射入体内的Tet-on基因表达系统在骨组织中靶向启动和表达骨内靶向表达腺相关病毒介导的Dkk1-siRNA，从而激活经典Wnt通路对骨质疏松的治疗作用。同时通过转基因技术，研究DDK1表达缺失后对激素诱导下BMSCs成骨－成脂分化过程中关键转录因子和特异性功能基因的表达进行检测，探索DDK1调控BMSCs成脂、成骨分化转录水平的分子机制，为阐明BMSCs向特定谱系分化的关键信号和分子通路，为激素性骨质疏松症及其他骨量丢失疾病的临床治疗提供理论依据。

激素性骨质疏松骨量减少的重要原因之一,是激素导致的以牺牲成骨为代价的脂肪细胞增多。前期研究发现,激素可通过上调 DKK-1 表达从而阻断 Wnt 经典通路的抑制髓内干细胞成骨分化,促进其成脂分化的作用。本项目通过病毒感染技术,对DKK1胞内水平进行上/下调控，进而研究DKK1表达水平变化对激素诱导下 BMSCs 成骨/成脂分化过程中关键转录因子和特异性功能基因的表达的影响调控作用,探索 DKK1 调控 BMSCs 成脂/成骨分化转录水平的分子机制,为阐明 BMSCs 定向分化的关键信号通路, 为激素性骨质疏松症及其他骨量丢失疾病的临床治疗提供理论依据。 通过本实验研究结果,初步得出结论:激素的干预可以明显升高BMSCs内 DKK1的表达水平。而通过增加b-catenin的磷酸化即降解，DKK1可以中和Wnt/b-catenin通路对PPAR-γ的抑制作用。进而引起BMSCs的成骨分化抑制，及向成脂分化过程的转变。