人牙髓细胞中差异表达基因的克隆及DPDP-2和DPDP-3基因序列分析
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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
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 fibroblasts
Human 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
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
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 fibroblasts
Human 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
引文
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1．高玉好。牙髓细胞培养研究进展。国外医学口腔分册，1991，18(2)：96
2. Nakashima M. Establishment of primary cultures of pulp cells from bovine permanent incisors. Arch Oral Biol, 1991,36(9) :655
3．吴军正，司徒镇强，陈建元，等．体外培养的人牙龈牙周膜牙髓纤维细胞生长特点．实用口腔医学杂志，1993，6(4)：227
4. D Souza RD, Bachman T, Baumgardner KR, et al. Characterization of cellular responses involved in reparative dentinogenesis in rat molars. J Dent Res. 1995 ;74(2):702-9
5. Thesleff I, Vaahtokari A, Kettunen P, et al. Epithelial-mesenchymal signaling during tooth development. Connect Tissue Res 1995;32(1):9-15
6．陈健．人牙髓细胞Smad2基因MH2结构域cDNA克隆及其在大肠杆菌中的表达．第四军医大学硕士学位论文。西安：1999
7. Bronckers A, Lyaruu DM, Woltgens JHM, et al. lmmunohistochemistry of extracellular matrix proteins during various stages of dentinogenesis. Connect Tissue Res 1989,22:65
8. noki R, Kudo T, Olgart FM. Dynamic aspects of dental pulpmolecular biology, pharmacology and pathophysiology. London Hapman and Hall Press, 1990,29
9. Kasugai S, Adachi M, Ogura H. Establishment and characterization of a clonal cell line(RPC-C2A) from dental pulp of the rat incisor. Arch Oral Biol,1988,33(12):877
1．王忠东，文玲英。牙髓自身修复潜能的研究进展．国外医学口腔医学分册，1996，23(2)：65
2. McClelland M, Mathieu DF, Welsh J. RNA fingerprinting and differential display using arbitrarily primed PCR. Trends-Genet. 1995; 11(6): 242-6
3. Zhao Z, Huang X, Li N, et al. Direct cloning of cell differential expression genes with full-length by a new strategy based on the multiple rounds of 'long distance' polymerase chain reaction and magnetic beads mediated subtraction. J Biotechnol. 1999;73(1):35-41
4．F．奥斯伯，R．E．金斯顿，J．G．塞德曼，等。颜子颖，王海林，译。精编 分子生物学实验指南，北京：科学出版社，1998
5. Wieland I, Bolger G, Asouline G, et al. Amethodfor difference cloning:gene amplification following subtractive hybridization. Proc Natl Acad Sci U S A. 1990;87(7):2720-4.
6. Chenchik A, Moqadam F, Siebert P. Ane wmethod for full-length cDNA cloning by PCR. In a Laboratory Guide to RNA: Isolation, Analysis, and Synthesis. Ed. Krieg, P. A. (Wiley-Liss, Inc. ), 273-321
7．J Sambrook．EF Fritsob and T Maniatjs．(《分子克隆实验指南》第二版。金冬雁，黎孟枫等译．北京：科学出版社出版．1992年．
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