IPO8转运RUNX2入核调控成骨分化机制研究

The aims of this study is to demonstrate the mechanism of RUNX2 through IPO8 nucleocytoplasmic transport pathways regulating osteoblast differentiation in vitro induced from human mesenchymal stem cells (hMSCs).At the different period of hMSCs directly committed into osteoblast co-immunoprecipitation,RNA interference,western blot,and immunofluorescence will be expression between nuclear and cytosol and to observe tendency of RUNX2 nuclear import. We then observe corresponding chang of osteoblastic phenotype after IPO8 silencing.Base on above studying, we will further perform truncated and mutant analysis to confirm the effective site of RUNX2 through IPO8 nuclear import. We will therefore systematically clarify the mechanism of RUNX2 through IPO8 nuclear import regulating differentiation of human osteoblast. This project will offer valuable information on treatment of the osteoporosis and further understand how RUNX2 regulates osteoblast differentiation induced from hMSCs.

本项目以核质转运为视角，沿着IPO8转运RUNX2入核调控人类成骨细胞分化的思路，朝着在核质转运水平上系统阐述RUNX2调控人类成骨细胞分化的机制开展工作。研究以人类骨髓间充质干细胞成骨定向分化的不同时期为对象，采用免疫共沉淀、RNA干扰、免疫印迹和免疫荧光等技术，分析RUNX2和IPO8的相互作用、检测核内外的RUNX2蛋白表达差异和观察RUNX2核转位的变化趋势，同时在沉默IPO8后研究成骨细胞表型特征的相应改变，验证IPO8转运RUNX2入核和成骨分化各阶段调控作用；在此基础上分别对IPO8进行系列截短和突变分析，确定IPO8转运RUNX2入核的有效部位，从而系统阐明IPO8转运RUNX2入核调控人类成骨细胞分化的机制，明确这一机制具有重要的理论意义及临床应用价值，为更深刻认识和有效治疗骨质疏松这类骨的退行性疾病提供理论依据。

我们前期研究发现，核转运蛋白IPO8在成骨细胞分化过程中可能发挥重要的调节作用，但机制尚不清楚。本项目沿着IPO8转运RUNX2入核调控人类成骨细胞分化的思路阐述人类成骨细胞分化的机制开展工作。采用基因克隆、RNA干扰、免疫印迹、免疫荧光和转录组测序等技术，分析（1）IPO8基因与RUNX2基因表达在成骨分化过程中的相关性；（2）RUNX2对IPO8基因转录的调控作用；（3）IPO8基因沉默对hMSC转录组及micro RNA组表达的影响。结果显示：（1）IPO8分子与RUNX2分子在成骨分化过程中的表达具有显著相关性；（2）RUNX2在转录水平对IPO8基因转录的调控作用；（3）未发现IPO8转运RUNX2分子入核的证据；（4）发现部分受IPO8直接或间接调控的mRNA及miRNA。研究结果初步阐明IPO8与RUNX2相互调控对人类成骨细胞分化的影响，明确这一机制为进一步认识和有效治疗骨质疏松这类疾病提供理论依据。