【作者】 王忠东；

【导师】 肖明振；

【作者基本信息】 第四军医大学 ， 口腔医学， 2000， 博士

【摘要】 牙髓组织在损伤、感染等外界刺激的情况下，牙髓成纤维细胞可以增殖、分化为成牙本质细胞，分泌修复性牙本质。这是牙髓组具有自身修复潜能的生物学基础。关于牙髓细胞诱导分化机制的研究，是当前牙髓生物学研究的重点内容之一。 近年来，随着现代细胞生物学和分子生物学的深入研究，关于牙髓细胞分化的分子调控机制，人们进行了大量研究。消减杂交技术是80年代初期发展起来的一项寻找细胞差异表达基因的技术。其基本原理是将要比较基因表达差异的双方cDNA进行杂交，然后从杂交混合相中分离出来未杂交部分，再进行分析、鉴定。我们利用牙髓成纤维细胞区别于其它成纤维细胞的特性，即牙髓成纤维细胞具有分化为成牙本质细胞并形成牙本质的能力，而牙龈成纤维细胞者并不具备这种能力，应用基于PCR的改良消减杂交技术—LPS技术克隆人牙髓细胞中的特异表达基因，以期为阐明牙髓细胞增殖、分化的分子调控机制，提供实验依据。实验研究共分3部分：人牙髓细胞和牙龈成纤维细胞体外培养模型的建立 采用组织块培养法建立了人牙髓细胞和牙龈成纤维细胞体外培养模型。人牙髓细胞和牙龈成纤维细胞体外培养的成功率分别为16.7％、40％。原代培养的细胞呈长梭形，胞体丰满、胞浆均匀、核圆、核仁清晰，牙髓、牙龈成纤维细胞在形态上无明显差异。两种细胞的生长曲线相近，均呈“S”形。它们的细胞倍增时间(DT)分别为HDP：52.9h和HGF：47.6h；细胞的贴壁率为HDP：96.5％和HGF：98.6％。经抗波形丝蛋白和角蛋白单抗ABC法染色，均呈现出明显的抗波丝蛋白反应阳性，而抗角蛋白反应阴性，表明两种细胞均为来源于中胚层的成纤维细胞。 2．人牙髓细胞中差异表达基因的克隆、序列测定及分析 在建立人牙髓细胞和人牙龈成纤维细胞体外培养模型的基础上，应用基于PCR的改良消减杂交技术—LPS技术，建立了与人牙髓细胞生长、分化机制相关的cDNA消减文库。从所建立的cDNA消减文库中随机筛选出151个含有差异表达片段的克隆，经双酶切电泳鉴定出长度大于700bp的克隆40个，经进一步反向点杂交，排除了20个假阳性克隆，确定另20是人牙更多还原

【Abstract】 With dental pulp tissue injured, infected by external factors, human dental pulp cells can proliferate, differentiate into odontoblast-like cells and secrete reparative dentin. This is biologically fundamental for the self-reparative potential of dental pulp tissue. Nowadays, the molecular mechanism of proliferation and differentiaton of dental pulp cells has been one of the focuses in the field of dental pulp biology.In the past several years, owing to the further development of modern cellular biology and molecular biology, there has been a great amount of research on molecular mechanism of dental pulp cellular differentiation. Subtractive hybridization is a technique used to clone differentially-expressed genes in cells. According to the principle of this technique, cDNA from different cells are hybridized to isolate unhybridized fragments, and the unhybridized fragments are cloned and analyzed. It has been established that human dental pulp cells are different from other fibroblasts, i.e. the former can differentiate into odontoblast-like cells, whereas human gingival fibroblasts (HGF) can’t. In the present study, based on this principle, differentially-expressed genes were cloned from human dental pulp cells by using improved subtractive hybridization based on PCR (LPS). The objective of the study was to work out the molecular mechanism of the proliferation and differentiation of human dental pulp cells and to provide some data for further experiments and research.The study consists of 3 parts described as below.1. Primary culture of human dental pulp cells and human gingival fibroblastsHuman dental pulp cells and HGF were cultured by tissue explant culture technique. The success rates of the primarily-cultured cells were 16.7% and 40% respectively. The population doubling times of the cultured cells were 52.9h and 47.6h respectively. The cultured cells were shuttle-shaped, plump in form, homogenous in cytoplasma and their nuclei looked clear and round. There was no significant difference between the shapes of更多还原