骨组织工程技术构建骨髓造血微环境的相关研究

Our previous study indicates that osteogenically-induced human bone marrow mesenchymal stem cells (BMSCs) possess the capacity to form new bone and organize hematopoiesis when transplanted subcutaneously into immunocompromised mice using beta-tricalcium phosphate (β-TCP) as scaffolds, but the non-induced BMSCs do not have such similar capabilities. Moreover, it is found that adipose-derived stem cells (ASCs) and umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) could be used as seed cells to repair bone defects, but they can not form new bone and bone marrow at ectopic sites using tissue engineering approaches. The objective of this study is to investigate the possibility of sorting and selecting these adult stem cells with sepcific osteogenic and hematopoietic potential. Firstly, with single-cell-cloning culture and osteogenic-induction identification, BMSCs which can form clones would be divided into two types: with or without the potential of osteogenic differentiation. By comparing the difference of surface protein expression between these two types of cells with gene analysis, certain surface proteins related to the osteogenic potential would be seleted. Then, such specific molecular markers could be used to sort ASCs and UCB-MSCs using flow cytometric analysis and immunological bead sorting.The expression of such related membrane proteins of osteogenic differentiated ASCs and UCB-MSCs is investigated at mRNA and protein levels using real-time PCR, Western blot and ELISA measurements. The relationship between specific membrane proteins and signal transducer pathways will be investigated, and RNAi techniques will be applied to confirm the function of these membrane proteins. The results of this project are expected to provide basis for tissue engineering hematopoietic microenvironment using different types of adult stem cells.

我们的前期研究发现成骨诱导分化的骨髓间充质干细胞（BMSCs）异位构建的组织工程骨伴有骨髓再生及造血功能，而未诱导分化的BMSCs则无此现象发生。进一步实验发现，脂肪源性干细胞（ASCs）及脐带血间充质干细胞（UCB-MSCs）可以作为骨组织工程种子细胞，但无异位成骨及再生骨髓的功能。本项目拟通过单细胞克隆培养、成骨定向诱导鉴定与基因组学的方法，将BMSCs分为具有成骨分化潜能和非成骨分化潜能的两类细胞克隆群，全面比较两者膜表面蛋白表达谱的差异，结合裸鼠皮下回植的体内实验结果，筛选确定与成骨/骨髓分化潜能相关的膜蛋白。在此基础上，通过流式细胞及免疫磁珠分选ASCs与UCB-MSCs的成骨细胞亚群，检验成骨诱导分化效果。开展特异性蛋白因子抑制实验，确定膜蛋白表达与细胞信号通路的相关性，并应用RNA干扰技术验证膜蛋白调控体内造血的功能，为建立稳定有效的骨组织工程构建造血微环境技术提供实验依据。

成体干细胞（骨髓间充质干细胞BMSCs；脂肪源性干细胞ASCs；脐带血间充质干细胞UCB-MSCs；肌肉来源干细胞MDSCs等）均为由多种细胞亚群组成的混合细胞群体，其亚群分型、分子标志与定向分化潜能等研究已成为组织工程与再生医学领域的热点内容。本课题立项以来，重点围绕成体干细胞特异性成骨潜能特性，开展了相关系列研究。运用组织工程技术体内模拟再造骨髓组织，证实早期（8周内）形成组织工程骨的成骨细胞来源于移植供体，之后供体细胞凋亡并为募集而来的宿主细胞替代；而新形成的骨髓造血细胞均来自宿主，并可在体内存在18个月以上。课题组在不使用免疫抑制剂的情况下，将成骨分化的犬同种异体ASCs复合珊瑚材料植入到颅骨标准缺损区，术后未发现CD4/CD8比值及其免疫相关细胞因子发生显著改变。通过GFP标记技术证实有新生骨组织形成并保持稳定6个月以上，明确了ASCs在组织工程骨组织形成过程中的转归。在人UCB-MSCs体外免疫原性研究方面，以混合淋巴细胞反应模拟体内免疫微环境条件，发现成骨诱导分化细胞HLA-II类分子的阳性表达率升高，对淋巴细胞增殖的抑制作用明显减弱，在干扰素-γ作用后反有刺激淋巴细胞增殖的效果。课题组观察了自体ASCs移植对双侧卵巢切除术后骨质疏松兔的影响，发现自体细胞移植治疗12周后，兔股骨远端骨小梁密度、数量、厚度及结构模型指数均明显高于对照组，证实自体ASCs移植对兔骨质疏松有一定的疗效。牛血清白蛋白（BSA）是胎牛血清中含量最高的蛋白成分，残余量过高会导致免疫排斥反应，进而影响组织工程骨植入人体的安全性。本课题研究表明：酶联免疫法适用于组织工程骨中BSA 残余量的检测；多孔支架材料较细胞更易吸附BSA，植入前的清洗程序对BSA残余量有显著影响。在优化组织工程骨体内外成骨条件的基础上，课题组证实ASCs条件培养液可通过旁分泌生长因子的方式促进兔眼表角膜化学损伤，探讨了人眼眶ASCs体外增殖与分化能力随年龄增长的变化规律，并首次从人眼轮匝肌中分离培养获得MDSCs，其具有贴壁培养、体外扩增和多向诱导分化潜能。