【作者】 陈希哲；

【导师】 杨连甲； 刘宝林；

【作者基本信息】 第四军医大学 ， 口腔科学（颌外、病理）， 2001， 博士

【摘要】 组织工程是应用细胞生物学和工程学的原理，对病损组织结构和功能的修复与重建进行研究开发的一门新兴学科。其是继细胞生物学和分子生物学之后，生命科学发展史上的又一里程碑，标志着人类将走出单纯器官移植领域，步入组织、器官再造的新时代。 骨是最早在实验室中获得组织工程化的组织之一。组织工程骨修复亟需研究的内容包括：①能分化为成骨细胞的多能干细胞；②介导多能干细胞游走、分化、增殖的促生长和分化因子；③可吸收的细胞外基质或称支架以支持干细胞在骨缺损区的游走及附着；④在整个新骨形成过程中血管网的构成。基于以上要求，本实验进行了四部分研究：1 按目前学术界普遍认可的方法进行了大鼠骨髓基质细胞的诱导培养，大鼠骨髓基质干细胞在含有地塞米松、左旋抗坏血酸、β—磷酸甘油钠等的培养基中原代培养2周已具明显成骨细胞表型；利用梯密度离心法分离并培养大鼠骨髓内皮细胞，证实了大鼠骨髓内皮细胞具有W—P小体以及其它内皮细胞共有的特征。这两种细胞都属有限细胞系，可传代、冻存及复苏。此部分结果为后续实验打下了基础。2 进行噬菌粒载体pBK-BMP₂的扩增、提纯及酶切鉴定，并利用脂质体将此穿梭质粒转染大鼠骨髓基质干细胞。通过免疫组化、原位杂交及Western Blot印迹杂交检测，从分子水平和蛋白质水平直接或间接地证实经筛选后的细胞克隆能表达、分泌外源性的BMP₂。3 应用纤连蛋白对含孔磷酸钙陶瓷进行表面修饰并证实其有利于成骨细胞的贴附和成骨表型；建立封闭群动物SD大鼠骨组织工程模型并证实其对小型试材骨修复的研究结果更接近临床；将rhBMP₂转染的自体细胞复合同体骨髓基质干细胞并种植于含孔磷酸钙陶瓷上，回植原动物皮下或肌肉内均表现明显的异位成骨能力；这种基因转染自体细胞—陶瓷复合体用于颅骨极量骨缺损的修复可取得与自体成骨细胞移植相同的效果，间接说明了转染细胞能在体表达BMP₂并促使未转染的基质干细胞分化为成骨细胞。4 通过颈总动脉墨汁灌注及透射电镜观察，系统了解并分析自体细胞—陶瓷陈希哲 下四军医大学博士学位论又 一Z一要合汁颅g极量骨缺殒修复过程中血管生成与骨形成及陶瓷降解Z问的X系，1实山向兰ISOtlS提供的含孔磷酸钙陶瓷作为骨组织工程细胞支架有利于血育和骨的丙十，随时问推移陶瓷本导可以降解；种植的成骨细胞与新上的血管内皮别胞关系密切，推测其卜必有某些问于引发的信号转导级联效口。实验设计及技术路线如下图： — — 大鼠左恻殴、温骨骨钮 1 Me＊＊＊－－H＿7w～？ri 一J～＼ F－－s－－－－－－－ WWWk’G一4％：PJ LHerylerx ’tyxtew—“。二｛ilZJZtr－ 成骨纽胞诱导培养 刀卫sC。rhB卜IP。基因转染 ＼＼／ — ———＠p—— 一 细胞一囚瓷 体构鱼 大气颅骨极量骨 修复／、 异位骨形成 回 憾更多还原

【Abstract】 Osteogenic Ability of Autogenous Cells with Gene Transfection and Porous Calcium Phosphate as a Scaffold Tissue engineering is an interdisciplinary science of co-development and combination of modern cell biology, biomaterial science and engineering. The establishment of tissue engineering technique is a breakthrough over conventional organ transplantation to restore and reconstruct the function and structure of tissues lost to the injury and disease. In the light of present knowledge, the formation and repair of bone with tissue engineering repuires study on: 產 source of pluripotential mesenchymal stem cells capable of differentiating into osteoblasts; ゞrowth and differentiation factors that direct these cells to migrate into the osseous defect, to proliferate, and to differentiate into osteoblasts; ゛ bioresorbable matrix or scafold to support the attachment and migration of these cells into the osseous defect; and ゛ngiogenesis and the formation of a vascular network throughout the newly formed bone. The present study is based on these requirements and contains four main parts: 1 To observe the osteoblastic proliferation, differentiation and osteogenetic phenotype of rat bone marrow stromal cells in a a -MEM supplemented with L- ascorbic acid, Na P -glycerophosphate and Dex. It seems that synthesis of mineralized bone-like nodules and collagen in vitro requires the presence of ascorbic acid and P -glycerophosphate in the culture medium. The presence of Dex benefits to cell differentiation but suppresses its proliferation. To obtain endothelial cells from rat bone marrow with discontinuous gradient centrifugation on Percoll. The characteristics of the rat bone marrow endothelial cells in vitro are consistent with those of vascular endothelial cells with the presence of Weibel-Palade bodies. 2 A recombinant human morphogenetic protein 2 (BMP-,) plasmid was duplicated, purificated and transfected into bone marrow stromal cells (MSCs) of rat. It appeared that rhBMP2 mRNA was successfully transcripted and BMP2 expressed in transfected cells from the positive results of in situ hybridization, ?? immunohistochemistrical staining and Western blot. 3 To investigate the effects of fibronectin surface modification on osteoblasts adhesion to porous calcium phosphate bioceramics. it seems that the fibronectin surface coating is beneficial to osteoblast anchorage and its osteogenic phenotype. lb establish a Sprague-Dawley rat model of bone tissue engineering for investigating the ability of heterotopic osteogenesis and critical size calvarial defect repair with autogenous osteoblasts loading on porous calcium phosphate ceramic. It seems that the experimental results got from this closed colony animal model were much close to clinical situation superior to those from other homogenic or immune deficiency animals. To observe heterotopic osteogenesis of autogenous marrow stromal cells with rhBMP2 gene transfection and loading on porous calcium phosphate ceramic scaffolds in a Sprague-Dawley rat model. It seems that autogenous stromal cells with rhBMP2 transfection act as a bioreactor promoting proliferation and differentiation of stem cells when they are replanted into the corresponding animals. Gene engineering and tissue engineering technique was utilized to compose a cell-ceramic hybrid material for repair of a Sprague-Dawley rat model of critical size calvarial defects. It更多还原