【作者】 董广英；

【导师】 吴织芬；

【作者基本信息】 中国人民解放军第四军医大学 ， 口腔临床医学， 2003， 博士

【摘要】 牙周治疗的最终目标是实现牙周组织完全功能性再生，其中包括牙槽骨、牙骨质和牙周膜协调一致地、生理性再生。传统的牙周治疗和引导性组织再生技术虽取得一定的临床效果，但总的看来，尚不能达到牙周组织完全再生的目的。组织工程技术引入牙周病治疗的研究领域，为这一目标的实现带来了新的希望。组织工程学研究的主要内容包括种子细胞的来源、生物载体支架的选择以及细胞／载体支架复合体的构建等三个方面。种子细胞的研究是牙周组织工程的重要内容。牙周膜（PDL）细胞是一组具有多向分化潜能的异质性细胞，其中的未分化间充质细胞可分化为成纤维细胞、成骨细胞或成牙骨质细胞，是牙周组织再生的主要细胞。作为种子细胞，PDL细胞具有一定的局限性，它来源有限，须拔除一定量自体牙才能获得，难以在临床上广泛开展。牙龈成纤维细胞作为牙周组织的组成成分，来源丰富、容易获得、具有很强的生长和自我繁殖能力，在适当的刺激下，可表达成骨细胞表型。人骨形成蛋白2（hBMP₂）是BMP家族中诱骨活性最强的一种，也是唯一能单独诱导成骨的因子，在牙周组织再生和修复中具有重要的作用。本研究利用基因转染技术，将hBMP₂基因转染牙龈成纤维细胞，赋予其成骨细胞特性，探讨其在牙周组织再生中的作用，为将其作为理想的种子细胞应用于牙周组织工程提供实验依据。为此，本研究进行了如下几方面的实验： ①将含有pBK-hBMP₂噬菌粒表达载体的大肠杆菌扩增后，以碱裂解法提取pBK—hBMP₂，并进行提纯和酶切鉴定。为以下实验做好准备。 ②利用脂质体将hBMP₂基因转染至人牙龈成纤维细胞中，经G418筛选后获得了稳定表达hBMP₂基因的细胞克隆；在体外培养扩增后，通过原位杂交、免疫组化、夹心ELISA以及转染细胞培养上清液对MC3T3一E1细胞碱性磷酸 第四军医大学博士学位论文2003－05酶活性和骨钙素合成的影响等检测，从mRNA及蛋白水平证实了稳定转染hBMP。基因的人牙龈成纤维细胞能够表达具有一定生物学活性的hBMP。。转染细胞可持续表达有活性的 hBMP。蛋白达 10代以上。 ③在上述实验的基础上，对稳定表达hBMP。的人牙龈成纤维细胞从形态、增殖特性和细胞周期、碱性磷酸酶活性和骨钙素合成以及体外形成矿化结节的能力等方面，对其生物学特性进行了观察。结果显示，部分转染细胞由梭形转化为多角形：细胞倍增时间和细胞增殖周期无明显改变；与未转染细胞相比，转染细胞碱性磷酸酶活性和骨钙素合成水平明显升高u的．01h 组织化学染色表明转染细胞内碱性磷酸酶含量明显升高，并在矿化液作用下形成矿化结节。结果提示：hBMPz基因转染后人牙龈成纤维细胞生物学特性发生明显改变，表达成骨细胞表型，具有了某些成骨细胞的特点。 ④将 hBMP。基因转染的人牙龈成纤维细胞与藻酸钙凝胶复合后，进行体外培养。利用HE染色技术、免疫组化方法和扫描电镜观察细胞在藻酸钙凝胶中的生物学行为。结果显示，转染细胞在凝胶中呈“悬浮”生长，细胞伸展良好，并分泌细胞外基质，表达hBMP。。表明藻酸钙凝胶与基因转染的牙龈成纤维细胞具有较好的相容性。 ⑤将hBMPz基因转染后的自体牙龈成纤维细胞作为种子细胞，与藻酸钙凝胶复合，植入狗的人工牙周组织缺损区。通过组织学观察和组织学测量，评价基因转染牙龈成纤维细胞对牙周组织再生的影响，并以未转染的自体牙龈成纤维细胞和PDL细胞作对照。结果发现：SW后，基因转染的牙龈成纤维细胞组可见近乎完全的牙周组织再生，未见结合上皮长入、根吸收和骨粘连等不良愈合方式。未转染牙龈成纤维细胞组有少数牙位出现骨粘连和根吸收；基因转染细胞组新生牙槽骨、牙骨质以及牙周结缔组织附着量与PDL细胞组无明显差异（P＞0．05），而比未转染牙龈成纤维细胞组有明显多的牙周组织再生（P＜0．01）。此结果提示，牙龈成纤维细胞经hBMP。基因转染后可替 一4－ 第四军医大学博士学位论文2003．05代PDL细胞作为牙周组织工程的种子细胞。 综上所述，本研究利用PBK—hBMP。表达载体成功地将hBMP。基因转入牙龈成纤维细胞，并使其在细胞内稳定表达；基因转染后，牙龈成纤维细胞可表达成骨细胞表型；此细胞在藻酸钙凝胶中生长良好，其生物学特性不受影响；以藻酸钙凝胶为载体，将基因转染的牙龈成纤维细胞应用于牙周组织工程，可很好地促进实验动物根分叉区人工牙周组织缺损的修复。 本实验结论：外源性的hBMP。基因能够在牙龈成纤维细胞中稳定表达并促进其向成骨细胞转化；基因转染的牙龈成纤维细胞与藻酸钙凝胶具有良好的相容性；二者复合可很好地促进牙周组织再生。因此，可以初步认为hBMP。基因转染牙龈成纤维细胞能作为牙周组织工程的种子细胞。更多还原

【Abstract】 Periodontal regeneration is expected to reconstitute the injured tissues of the periodontal attachment apparatus physiologically and functionally, including cementum, periodontal ligament and alveolar bone. Traditional periodontal therapy and guided tissue regeneration failed to fullfil the goal. Tissue engineering has been introduced into the field of periodontology, which contribute a new vista to periodontal regeneration. But there are some difficulties in finding proper seeded cells. Owing to its heterogeneity, periodontal ligament (PDL) cell is reagrded as the main source of cell to regenerate periodontal tissues and expected to be the seeded cells in previous studies,, because of their potential of differentiation to fibroblasts, osteoblasts, or cementoblasts. However, there are also some disadvantages for PDL cell to be seeded cells, for example , extraction must be carried out to obtain PDL cell, which is often refused by patients in clinic.Gingival fibroblast is also the constituent of periodontal tissues, and easier to obtain clinically than PDL cell, gingival fibroblast have strong proliferation potential and even show some characters of osteoblasts when they are induced by some bone-inducing factors such as human bone morphogenetic protein-2(hBMP2) . hBMP2 is a kind of strong bone-inducing factor and plays a crucial role in periodontal regeneration. So, combining GINGIVAL FIBROBLAST with hBMP2 may be used in periodontal tissue engineering to fulfill the periodontal therapy goal.This study tries to reform gingival fibroblast to be the seeded cells in periodontal tissue engineering by transfecting hBMP2 gene into gingival fibroblast. Several experiments as below are carried on in this dissertation:1. The phagemid expression vector for hBMP2was amplified in E. coli., then, the plasmid was extracted, purified, and identified. All of these were the preparation for following experiments.2. hBMP2 gene was transfected into gingival fibroblast by using LipofectAMINE. Positive clones were selected with G-418. The expression of hBMP2 protein in gingival fibroblast transfected were determined by in situ hybridization, immunohistochemistry and sandwich-in ELISA. We also proved the hBMP2 protein has biological activity by investigating ALP activity and osteocalcin (OC) quantity in MC3T3-E1 cells treated with the conditioned media from the gene-transfected gingival fibroblast cultures. The results showed that hBMP2 protein was successfully expressed in gingival fibroblast transfected with hBMP2 gene.3 Observed the biological behaviours of gingival fibroblast transfected with hBMP2 gene stably, it was found that the shape of parts of gingival fibroblast changed from fusiform to polygonal; compared with parental cells, there were no obvious difference in doubling time and proliferative ability ; but enhanced both ALP activity and OC quantity of the hBMP2 gene transfected cells (P < 0.01); the ALP synthesis was upregulated in the hBMP2 gene transfected cells byhistochemistry staining, furthermore, mineral nodules developed in mineralization culture medium after gene transfection. Our results showed that the hBMP2 gene transfected cells expressed osteoblastic phenotype.4 We investigated the biological behaviors of hBMP2-transfected gingival fibroblast cultured in calcium alginate gel. The results showed that the cells spread very well in calcium alginate gel, secreted extracellular matrix, and expressed hBMP2 protein.5 To evaluate their effects in periodontal tissue regeneration, we compounded the hBMP2-transfected gingival fibroblast to calcium alginate gel and implanted them to artificial furcation defects of dogs. The periodontal apparatus attachments regenerated within 8 weeks. Root resorption and ankylosis were not found in experiment group. In experimental group, the quantities of new formed bone, cementum and connective tissue were more significantly than them in negative control group which implanted gingival fibroblast (P<0.01), but had no obvious differen更多还原