论文题目：遗传性乳光牙本质致病基因的鉴定和DSPP基因敲除小鼠的建立
作者简介：张晓海
，男,1973年07月出生，1999年09月师从于中国协和医科大学沈岩教授，于2002年07月获博士学位。
摘
要
遗传性乳光牙本质（dentinogenesis imperfecta type II, DGI-II）是一种常染色体显性遗传病。发病率为1/6,000-8,000. 患者的乳牙和恒牙都受到影响。受累的牙齿呈乳白色光泽，髓室和根管变窄甚至完全闭锁。患者牙齿的牙釉质很容易从牙本质上脱落，使本来就发育不良的牙本质过度磨损，导致患者牙齿显著变短。结果，遗传性乳光牙本质患者不得不接受广泛的牙齿护理和昂贵的治疗。
牙本质的无机成分主要为羟基磷灰石晶体，有机成分主要为胶原纤维，非胶原蛋白质包括分泌型焦磷酸蛋白（secreted phosphoprotein 1, SPP1），牙本质基质蛋白1（dentin matrix protein 1, DMP1），牙本质涎蛋白（dentin sialoprotein, DSP）、牙本质磷蛋白（dentin phosphoprotein, DPP）和骨唾液酸蛋白（bone sialoprotein, IBSP）等。其中DPP约占非胶原蛋白质的50%，它是一个高度磷酸化的蛋白质，被认为在羟基磷灰石晶体的形成中有重要作用。DSP和DPP是由一个基因DSPP编码的。一般认为DSPP基因的表达具有牙齿特异性，它只在成牙本质细胞中表达和成釉质细胞中短暂表达。
遗传性乳光牙本质的致病基因已被定位在染色体4q21上的2 MB的范围内，在此范围内有一个与组织矿化过程有关的基因簇。SPP1、DMP1、DSPP和IBSP都位于此基因簇中。但是，SPP1，DMP1和IBSP都被排除了作为DGI-II的侯选基因。
天津医科大学口腔医院在临床中发现一个遗传性乳光牙本质家系。我们选取染色体4q21上的8个短串联重复序列多态性标记(short tandem polymorthisms, STRPs)，经荧光标记PCR，等位片段分析，用Linkage软件包MLINK软件对所采用的STRPs与此DGI-II家系致病位点进行2点连锁分析。连锁分析结果支持此家系的致病基因位点位于染色体4q21上。接着我们选取DSPP基因做突变分析，通过直接测序法和限制性内切酶法发现DSPP基因第3658位发生了突变C→T。这个突变使编码Gln45的密码子变为终止密码子。这个突变在正常个体中不存在。这一突变预计会使DSPP基因翻译提前终止，突变的DSPP的翻译产物除去信号肽后为仅29个氨基酸的短肽，而且根本没有产物DPP，从而严重影响DSPP基因的功能。
由于牙齿取材困难，DSPP基因又只在牙齿发育的特定阶段表达，为了更好的研究DSPP基因的功能，我们想建立动物模型来模拟DGI-II。我们构建了基因敲除载体，通过电穿孔法把线形化的基因敲除载体导入胚胎干细胞中，用Southern blot筛选neo基因插入到小鼠Dspp基因第三外显子中的同源重组克隆。选取4个阳性克隆做显微注射。注射了ES细胞的胚泡被植入到代孕母鼠中，现在已经获得了嵌合小鼠。我们希望Dspp基因敲除小鼠动物模型可以促进对遗传性乳光牙本质致病机理的研究。
关键词：遗传性乳光牙本质，牙本质涎磷蛋白，连锁分析，基因敲除
Abstract
Dentinogenesis imperfecta Shields type II (DGI-II, MIM 125490) is an autosomal dominant disorder in which both primary and permanent teeth are affected. It occurs with an incidence of 1:8000 live births. The teeth are amber and opalescent, with pulp chamber obliterated by abnormal dentin. The enamel, although unaffected, tends to fracture, which makes dentin undergo rapid attrition leading to a marked shortening of the teeth.
The DGI-II locus was refined to 2 Mb interval on chromosome 4q21, in which a group of genes expressed in teeth that might be involved in dentin mineralization. Among these genes, dentin sialophosphoprotein (DSPP) is mainly expressed in odontoblasts and transiently in preameloblasts. Dentin sialoprotein and dentin phosphoprotein (DPP) are cleavage products expressed from a single transcript encoded by DSPP.
We studied a Chinese family with dentinogenesis imperfecta Shields type II. All of the affected individuals showed discoloration and severe attrition of their teeth, with obliterated pulp chamber. Eight STRPs flanking the DGI-II locus were used to perform genetic linkage analysis in 13 individuals from this family. The 2-point linkage analysis with MLINK software support this locus located in chromosome 4q21.
By direct sequencing PCR products which cover exons 1-4 of DSPP and their flanking sequences, we found a C→T transition at nt 3658. This mutation creates a stop codon in exon 3 (Gln45stop), and disrupts a RsaI site, which was used subsequently to identify mutation carriers in the family. We found the mutation to be heterozygous in all 10 affected members in this family, but not in the 3 unaffected. Furthermore, the Gln45stop mutation was not present in 102 control chromosomes. This premature termination caused by Gln45stop may result in the absence of DPP and greatly shortened DSP (only 29 amino acids excluding signal peptide).
To gain a better understanding of Dspp function in tooth development and in DGI-II, we targeted the Dspp locus with the neo insertion in exon3 of the Dspp gene. For this purpose, we constructed two gene targeting vectors and introduced them in ES cells by elcetroporation. After selection with G418 for 8-10 days, resistant ES colonies were picked. These colonies were screened for homologous recombianation by mini-Southern strategies. Blastocysts were injected with four positive ES colonies and transferred in the uteri of pseudopregnat recipient female mice. Now chimerical mice have been obtained. This mouse model may accelerate our understanding of the Dspp gene function.
Key word: dentinogenesis imperfecta type (DGI-II), dentin sialophosphoprotein (DSPP), linkage analyis, knock out
