【作者】 艾林；

【导师】 肖明振； 王捍国；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2008， 博士

【摘要】 MEPE,又名成骨细胞/骨细胞因子45(Osteoblast/Osteocyte Factor 45;OF45)或是Osteoregulin。2000年,Rowe在筛选肿瘤性低血磷性骨软化症(oncogenic hypophosphatemic osteomalacia,OHO)患者肿瘤组织cDNA文库中表达上调的基因时,发现了MEPE。RT-PCR检测发现,正常组织中,MEPE在骨髓和大脑中表达,肺、肾和胎盘中的表达水平很低。同年,Petersen等在大鼠骨髓源性的成骨细胞中克隆到MEPE基因,Northern blot和免疫组织化学研究显示:MEPE仅在骨组织中表达,特别是骨小梁和皮质骨中的骨细胞呈高表达。2003年,Gowen等克隆了小鼠MEPE基因,发现其上游存在三个蛋氨酸密码子,可以选择性转录起始,最大可编码441个氨基酸残基的蛋白质。Northern和Western blot显示MEPE在骨组织和成牙本质细胞中表达。目前在口腔医学领域MEPE的研究还是处于初期,对于MEPE的功能我们还知之甚少。前期研究提示MEPE可能参与牙本质形成。本课题通过MEPE基因克隆、原核以及真核蛋白表达等方法,在mRNA和蛋白质的水平上,从MEPE的表达、在牙齿细胞中功能以及在羟基磷灰石晶体形成和生长中作用等几个方面,全面深入地研究MEPE参与牙本质基质形成和矿化的过程、作用和机理等。这将阐明MEPE在牙本质牙髓复合体形成和损伤修复中的机制,丰富牙髓生物学和牙齿发育生物学,并为临床上生活牙髓的保存治疗和组织工程化牙本质修复牙体组织缺损提供理论基础本研究分为以下5个部分内容:一、MEPE基因克隆和序列分析利用PCR技术,设计一对引物从人脑cDNA文库中扩增出MEPE编码区基因片段(约1.5kb),将扩增的基因片段插入pGEM-T Easy克隆载体,限制性酶切进行鉴定并进行核苷酸序列测定。结果表明:克隆到人MEPE蛋白编码区基因片段,序列与GenBank中收录的人MEPE cDNA序列一致。二、MEPE基因在原核和真核细胞中的表达将MEPE编码区全长片段亚克隆至表达载体pGEX-4T-1中,大肠杆菌中诱导表达,纯化后获得人MEPE蛋白。以重组、纯化的MEPE为抗原,混入完全/不完全弗氏佐剂,免疫新西兰大白兔,制备MEPE多克隆抗体。分别将MEPE基因亚克隆入非融合和融合真核表达载体pcDNA3.1和pEGFP-C3中,脂质体介导将载体转染哺乳动物细胞COS-7,通过免疫组织化学、Western Blot等方法检测MEPE蛋白在哺乳动物细胞中的表达。发现表达的绿色荧光蛋白融合蛋白所发出的荧光位于细胞浆中,胞核中无荧光,与免疫组化结果一致,说明MEPE位于细胞浆中。Western Blot检测显示在COS-7细胞中表达的MEPE的分子量为61kDa,与理论值相符。三、MEPE蛋白的组织表达特性研究用制备的抗人MEPE蛋白抗体对狗牙胚组织切片进行免疫组化染色,发现成牙本质细胞染色阳性,前期牙本质染色阳性,牙髓细胞染色阴性。四、MEPE蛋白对牙髓细胞和牙周膜细胞的生物学作用采用四唑盐比色法观察了MEPE蛋白对人牙周膜细胞、牙髓细胞增殖的影响,结果发现MEPE蛋白对牙周膜细胞增殖无促进作用、对牙髓细胞的增殖有促进作用。酶动力学方法观察了MEPE蛋白对人牙周膜细胞、牙髓细胞ALP活性的影响。研究结果表明,MEPE蛋白可提高牙髓细胞、牙周膜细胞的ALP活性。Von kossa染色结果显示MEPE蛋白可促进牙髓细胞的体外矿化。五、采用稳态琼脂糖凝胶的HA生长系统,研究MEPE蛋白质在体外矿化中的作用本实验首次将不同浓度的MEPE蛋白质加入HA晶体生长系统的平衡透析池,测定凝胶中磷酸盐和钙离子浓度、以及HA晶体的大小和完整性,证明MEPE蛋白质在体外可以促进HA晶体形成、生长。更多还原

【Abstract】 Recently, a new bone matrix protein cDNA has been cloned from human, rat, and mouse by independent groups . The human clone, termed MEPE(matrix extracellular phosphoglycoprotein), was isolated from a human oncogenic hypophosphatemic osteomalacia tumor(OHO) cDNA library with polyclonal antibodies that neutralized OHO tumor secreted phosphate uptake-inhibiting factor . MEPE , BSP (Bone sialoprotein), OPN (osteopontin, but sometimes known as SPP1 and Eta-1), DMP I(dentin matrix protein I), DSPP(dentin sialophosphoprotein) are the products of five genes clustered along human chromosome 4q21 between EST markers D4S2785(WI-6336) and D4S2844. The majority of each protein is encoded by the last one or two exons and contains the integrin-binding RGD tripeptide. Another point of similarity of all these genes is that all introns always interrupt between codons (type 0), thus leaving open the possibility of splicing together any two exons without causing frame shifts. We have named this family of proteins the SIBLING family for Small Integrin-Binding LIgand, N-linked Glycoprotein based not on current theories of their functions (which are poorly understood) but based on the simple biochemical and genetic features shared by all members. The human MEPE has 1989 bp cDNA clone and encodes a predicted 525-amino-acid protein rich in Asp, Ser, and Glu residues (26%) containing a 17-amino-acid signal peptide. MEPE contains two N-glycosylation motifs (NNSTandNNSR),a glycosaminoglycan attachment site (SGDG), an RGD cell attachment motif, several predicted phosphorylation motifs,and N-myristoylation sites.In dental tissue, MEPE is expressed in odontoblasts during odontogenesis, Liu has found that Dentonin(a 23-amino-acid peptide derived from MEPE) can promote DPSC proliferation, taking a potential role in pulp repair. That study suggestted that Dentonin affects primarily the innitial cascade of events leading to pulp healing.In this study, we have cloned MEPE cDNA by RT-PCR, expressed MEPE in E.coli and COS-7 cells, purified the recombinant protein, prepared anti-MEPE antibodies. Then we proceeded western blot and immunohistochemistry. Finally, a Steady-State Agarose Gels HA Growth System will be used to study the effects of MEPE to HA.The matrix extracellular phosphoglycoprotein (MEPE) gene is highly expressed in tumors that cause onco-genic hypophosphatemic osteomalacia (OHO). MEPE is also known as one of the bone-tooth matrix proteins and is associated with bone and teeth mineralization. We developed a rabbit polyclonal antibody directed against recombinant human MEPE after cloning its cDNA from the cDNA library of a human brain cDNA library the predicted secreted MEPE protein has a calculated molecular mass of 56 kDa. Like human MEPE, Fourteen of the serine residues in Mepe are potential phosphorylation sites for protein kinase C, casein kinase II, and cAMP-dependent protein kinase. As reported for human MEPE, a specific feature of the MEPE protein is the occurrence in the C-terminus of a serine-rich sequence, DDSSESSDSGSSSES (residues 417–430), that displays homology to repeat motifs found in dentin phosphoprotein, DPP/DSPP (SDSSDSSDSSSSSDSS), DMP1 (SSRRRDDSSESSDSGSSSESDG), osteopontin (DDSHQSDESHHSDESD) and the parent protein, dentin sialophosphoprotein,.. Using this anti-body, we analyzed the distribution of MEPE in dog dental germ tissure by immunohistochemistry. In this specimens, MEPE was predominantly expressed by odontoblasts cell and predentin not by dental pulp cells. Furthermore we use a steady-state agarose gel system, MEPE were incorporated at 5-25μg/ml into steady-state agarose gels and incubated for 5 days, MEPE caused significantly higher levels of calcium phosphate accumulation than control gels didthe results reported here suggest that MEPE can induce HA and we propose that this protein has a potential effect on dental rehabilitation. .更多还原