中国颅骨锁骨发育不良综合征患者的基因突变及相关功能研究
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摘要
颅骨锁骨发育不良(cleidocranial dysplasia,CCD)是一种少见的遗传性疾病,多为常染色体显性遗传,其致病基因定位于染色体6p21,并表明成骨细胞特异性转录因子(RUNX2/CBFA1/PEBP2)的杂合突变、基因插入、缺失等是造成CCD的重要原因。国内有关CCD患者的致病基因突变报道较少,尚未涉及到基因突变对蛋白功能的影响以及相关的细胞学研究。本研究收集5个中国CCD家系,进行基因突变检测及相关功能研究,填补国内有关研究的空白;并利用收集到的标本进行牙齿组织学结构分析以及牙髓细胞培养,探究CCD牙髓细胞的生物学特性及基因表达差异,从一个全新的角度对CCD患者的发病机理进行探讨。本文内容共分为三部分,小结如下:
一、颅骨锁骨发育不良综合征患者的临床检查与分析
本部分主要是CCD相关病例的筛选、诊断,首先是对具有相关临床症状的先证者详细采集病史,明确单发或家族聚集倾向;利用X线检查全面了解患者及其父母的全身骨骼发育情况;详细进行口腔检查,记录恒牙萌出及乳牙迟萌以及埋藏牙、多生牙等典型临床症状;综合上述临床资料,明确诊断。本研究中,五病例均具有明显的临床特征,比较典型的CCD特殊面容,口腔内表现为乳牙滞留,多数恒牙及多生牙埋伏于颌骨中不能萌出(除第4例没有明显多生牙外);均伴有锁骨发育不全,囟门闭合迟缓,身材矮小等症状。并发现一些新的临床畸形表现,如家系3先证者的尺、桡骨向桡侧偏斜致肱尺关节、肱桡关节畸形,肘外侧呈三角变形;家系2先证者的多生牙发生在磨牙区。
利用收集的家系5先证者姐姐(CCD患者)的牙齿标本进行组织学分析,光镜及扫描电镜下观察牙齿磨片,发现恒牙牙冠釉质结构不清晰以及牙本质小管部分闭塞、管周牙本质矿化低,釉牙本质界缺乏典型的连续扇形结构;CCD乳前牙釉质厚度无明显变薄,釉板(或裂隙样)及釉丛结构较多,牙本质小管分布及大小不均,部分闭塞,并可见裂隙;观察CCD恒牙牙根上2/3纵磨片,发现牙骨质较薄,未见明确牙骨质细胞;对滞留乳牙进行观察,亦未发现细胞牙骨质。对CCD患牙以及正常牙的牙釉质以及牙本质分别进行能谱分析,数据用SPSS13.0版本进行处理,两样本均数t检验进行统计分析,结果表明CCD恒牙的牙釉质及牙本质的Ca、P含量均较正常降低,并具有统计学差异(t值分别为29.519、26.998以及9.676、88.982;p值分别为0.001、0.004以及0.011、0.003),CCD乳牙牙釉质的Ca、P含量均降低,并具有统计学差异(t值分别为33.754、23.412;p值分别为0.011、0.019),牙本质各元素的含量有降低,但无统计学差异,可能是样本量较少的原因。组织学分析与能谱分析均表明CCD患牙矿化较差,牙本质亦同样受累。本研究在组织学水平检测了RUNX2基因突变对CCD患者牙齿结构的影响,再次证明RUNX2基因对于牙齿正常发育的重要性。
二、致病基因RUNX2的突变检测及对蛋白亚细胞定位的影响
对五个CCD家系进行RUNX2基因突变分析发现:在五个CCD家系患者中均检测到不同的突变位点,家系中健康成员同一位点均为野生型序列,进一步证明RUNX2基因是CCD患者的致病基因,此基因的单倍体不足是CCD的致病原因。其中家系4的c.475 G＞C错义突变以及家系5的c.1096 G＞T无义突变是本研究检测到的新突变位点;家系2的c.673 C＞T以及家系3的c.1171C＞G突变位点在中国CCD患者中首次检出;家系1的c.674G＞A,R225Q是国外文献报道突变率最高的位点。本研究结果拓展了国内CCD基因层次的研究领域,为国内外CCD致病基因的突变位点数据库增添了新的资料。
为了研究基因突变对蛋白突变体亚细胞定位的影响,通过构建真核表达载体,引入突变位点,转染细胞,观察pEGFP-C1-RUNX2载体转染组细胞的荧光蛋白表达情况,未引入突变位点的野生型RUNX2组仅在胞核内见到绿色荧光表达;引入突变位点c.674G＞A及c.673 C＞T的两组转染细胞,在胞浆及胞核内均可见绿色荧光。pCMV-HA-RUNX2载体转染组细胞,利用荧光免疫组化,引入c.475 G＞C(p G159R)突变组在胞浆胞核内均可见绿色荧光,而野生型仅在胞核内有绿色荧光表达,将转染细胞分离胞浆胞核蛋白进行Western blot检测,可见引入突变组的胞浆胞核成分均具有特异条带,而野生型仅胞核成分检测到特异条带。我们提出G159R对于RUNX2蛋白的核定位具有重要作用,推测G159可能是RUNX2基因新的核定位元件之一,为进一步蛋白结构方面的研究提供一定的实验依据。
三、CCD患者牙髓细胞的生物学特性分析及基因差异研究
本部分从家系5先证者姐姐的下恒牙中分离培养牙髓细胞,与正常牙髓细胞相比,CCD牙髓细胞比正常细胞略显丰满、扁平;描绘生长曲线来评价细胞的生长状态,正常牙髓细胞生长趋势高于CCD细胞,流式数据应用SPSS13.0版本进行处理,用了两样本均数t检验,t值分别为10.14、12.05,两组细胞的G1期及S期具有统计学显著差异,P值分别为0.001以及0.000,提示CCD细胞滞留在G1期的比例显著增多,CCD牙髓细胞从G1期到S期的过渡明显受阻。透射电镜观察发现CCD细胞粗面内质网扩张,胞质内可见大小不等的层板状小体,这是正常牙髓细胞所不具有的特殊表现,粗面内质网扩张并可见部分核糖体颗粒脱落,可能提示细胞蛋白合成能力的改变,较多层板状小体的出现可能与脂类的合成分泌相关。
对CCD牙髓细胞进行矿化液诱导,虽然部分细胞出现细长单极胞浆突起,并呈复层生长,但直至培养7周仍无矿化结节形成,而正常牙髓细胞培养4周后开始有结节形成,表明CCD牙髓细胞的矿化能力减弱,可能提示此细胞向成牙本质样细胞分化能力的减弱,以及CCD患者牙髓组织损伤修复能力减弱。
采用第二代功能分类基因芯片从细胞水平分析基因突变对TGF-β/BMP信号传导通路相关基因表达的影响,发现有18条基因上调,14条基因下调。通过对异常表达基因的分析,提示E 366X RUNX2突变体影响了TGF-β/BMP信号传导通路上的一些相关基因的调控,如Cdc25A、TGFβ2、Smad3、EVI1的表达下调,COL3A1、BMP2、4、7均表达上调,细胞的增殖和分化是由多基因多通路共同调控的,协同作用较强的某些基因将决定细胞最终的趋势和命运,推测TGFβ2、Smad3、EVI1的表达减弱与COL3A1上调表达的协同作用可能阻碍了CCD牙髓细胞向成牙本质细胞的分化趋势;根据R391X突变体的功能研究报道,推测E366X突变极可能影响RUNX2突变体和Smads的相互作用,阻抑BMP对细胞分化的作用,BMP2、4、7的上调表达可能是由于正常的信号通路受到影响,通过其它负反馈机制调控细胞的分化和增殖。
Cleidocranial dysplasia(CCD;MIM # 119600) is a skeletal disorder with autosomal-dominant inheritance.The clinical hallmarks of CCD are clavicle rudiment or absence,delayed closure of cranial fontanels and sutures,Wormian bones,frontal bossing,supernumerary and late erupting teeth,wide pubic symphysis,and other skeletal anomalies.The locus for CCD has been mapped to chromosome 6p21 where the responsible runt-related gene 2(RUNX2) has been located,RUNX2 also refers to as polyomavirus enhancer binding protein 2(PEBP2) or murine leukemia virus enhancer core-binding factor(CBF).So far,there is few investigations about Chinese cases with CCD.The prevalence and range of RUNX2 mutations in Chinese eases with CCD were rarely reported.Here,5 different types of heterozygous mutations in the RUNX2 gene in 5 unrelated CCD cases from China who clearly display variable clinical manifestations were reported,the molecular basis of their dysfunction were studied.The lower permanent teeth from one CCD case were collected and the dental pulp cells were cultured,and characterization and gene expression of the cells were analysed in comparison with corresponding cells from normal control.The study was divided into three parts described as followings.
1.Clinical examination and analysis of patients with cleidocranial dysplasia
5 unrelated families with the clinical diagnosis of CCD were investigated in the present study,and the unaffected parents and siblings were included.There was no parental consanguinity.Radiological examination regarding osseous malformations was carried out over the entire body.A CCD phenotype was defined by the presence of hypoplastic clavicles and delayed closure of the anterior fontanelle in addition to the observation of classic craniofacial features.The skeletal anomalies as well as the oral manifestations of the syndrome were variable within the affected patients,the case 2 had extra teeth in the molar region(at least four molars),which was rarely reported in the literatures.Moreover,both impacted molars and extra molars had abnormal roots with delayed development,and the erupted first molars had relatively normal roots.It is possible that the abnormal root development may be one of the causes of eruption failure of permanent teeth in CCD cases.Interestingly,in case 3, both the ulna and radius leaned to the radialis so that the humeroulnar joints and humeroradial joints were abnormal,and the elbow looked like a triangle,which was rarely reported in the literatures.All the cases in the present study showed malformation of tarsometatarsal joints to a certain extent.Thus we may infer that RUNX2 may have a role in joint formation by affecting some of chondrocyte and osteoblast differentiation pathways.
We have analyzed histologically the structure of teeth obtained from CCD patients.Enamel structure was illegible,dentin tubules appeared obturated, Peritubular dentin had insufficiency mineralization,and typical continuous arc structures were virtually lacking on the enamel-dentinal junction.In deciduous teeth, more enamel lamella and enamel tuft were found,and dentin appeared irregular,size and distribution appeared ununiformed;cellular cementum was virtually lacking in deciduous and permanent teeth.Spectrum analysis of enamel and dentin of teeth from CCD patients and normal subjects showed that the proportion of Ca and P in permanent teeth from CCD was significantly lower than that from normals,so did the enamel of deciduous teeth from CCD patients,however,this did not appear to be the case for dentin of the deciduous teeth.
2.Mutation analysis of RUNX2 and its effect on subcelluar localization of mutants
To identify mutations in the RUNX2 gene in CCD cases,genomic DNA.from 5 CCD cases and their families were analysed,totally different mutations for each CCD case were detected.Novel c.475 G＞C(p G159R) missense and c.1096 G＞T nonsense mutations were included,and their healthy parents didn't carry the same mutation.Although the other three mutations were reported in the literatures,R225W and R391X mutations were reported in Chinese cases with CCD for the first time.
To detect the effect of mutations on nuclear localization of RUNX2 protein, fusion proteins were constructed between green fluorescent protein and RUNX2.The constructs were transiently transfected into mouse fibroblast NIH 3T3 cells.The wild-type RUNX2 protein was detected exclusively in the nucleus.However,R225Q and R225W mutants showed dual localization to both the cytoplasm and the nucleus. Immunofluorescent staining and western blotting showed that wild-type RUNX2 protein was localized exclusively in the nucleus,however;the mutant protein was found in both the nucleus and the cytoplasm,which demonstrated that transport of the RUNX2 mutant into the nucleus was disturbed by the G159R mutation.These results indicate that G159 is very important to promote RUNX2 nuclear-localization, therefore,G159 may be one of new elements of RUNX2 gene nuclear localization signals.
3.Characterization and gene expression analysis of dental pulp cells of CCD patient
To identify morphological and molecular alteration associated with CCD dental tissues,human permanent dental pulp cell cultures were established from age-and sex-matched CCD patient and normal subject.Dental pulp cells were compared for general morphology,proliferation rate,and gene expression profiles microarray technology.CCD pulp cells were in some sort flatter than the normal ones,however, the normal pulp proliferation rates were greater at time points tested than the cells from CCD patient.The flow cytometric analysis showed that the progression from the G1 to S phase in the CCD pulp cell cycle was impeded.The ultrastructure of CCD pulp cells showed that the rough endoplasmic reticulum was expended,and more lamellar body was found in cytoplasma.Partial ribosomes fell off the rough endoplasmic reticulum,and this change might impede the ability to synthesize protein. The CCD pulp cells were induced with mineralization inducer,and no mineralized nodule was found until seven weeks passed,although partial cells showed single pole process of cytoplasma and overlapping growth.The weakening of mineralization ability showed that its potential ability of differentiation might weaken.
The 96 genes of Human TGF-β/BMP signaling pathway were analysed by PCR array,and 18 genes displayed significant up-regulated at least two-fold in expression levels,and 14 genes down-regulated.Cdc25A,TGFβ2,Smad3 and EVI1 were down-regulated,and COL3A1,BMP2,4,7 were up-regulated.The proliferation and differentiation were modulated by multi-gene,some genes cooperated with each other and determine the final fate of cells.It was inferred that the potential differentiation of CCD pulp cells into odontoblasts was impeded to some extent by the cooperation between down-regulation of TGFβ2,Smad3 and EVI1 and up-regulation of COL3A1. According to the disfunction of R391X reported in the literatures,E366X mutation may have an effect on the interaction of RUNX2 mutant and Smads,and impede the function of BMP to cell differentiation.The up-regulation expression of BMP2,4,7 might be caused by other negative feedback because of breakoff of the normal signaling pathways.
一、颅骨锁骨发育不良综合征患者的临床检查与分析
本部分主要是CCD相关病例的筛选、诊断,首先是对具有相关临床症状的先证者详细采集病史,明确单发或家族聚集倾向;利用X线检查全面了解患者及其父母的全身骨骼发育情况;详细进行口腔检查,记录恒牙萌出及乳牙迟萌以及埋藏牙、多生牙等典型临床症状;综合上述临床资料,明确诊断。本研究中,五病例均具有明显的临床特征,比较典型的CCD特殊面容,口腔内表现为乳牙滞留,多数恒牙及多生牙埋伏于颌骨中不能萌出(除第4例没有明显多生牙外);均伴有锁骨发育不全,囟门闭合迟缓,身材矮小等症状。并发现一些新的临床畸形表现,如家系3先证者的尺、桡骨向桡侧偏斜致肱尺关节、肱桡关节畸形,肘外侧呈三角变形;家系2先证者的多生牙发生在磨牙区。
利用收集的家系5先证者姐姐(CCD患者)的牙齿标本进行组织学分析,光镜及扫描电镜下观察牙齿磨片,发现恒牙牙冠釉质结构不清晰以及牙本质小管部分闭塞、管周牙本质矿化低,釉牙本质界缺乏典型的连续扇形结构;CCD乳前牙釉质厚度无明显变薄,釉板(或裂隙样)及釉丛结构较多,牙本质小管分布及大小不均,部分闭塞,并可见裂隙;观察CCD恒牙牙根上2/3纵磨片,发现牙骨质较薄,未见明确牙骨质细胞;对滞留乳牙进行观察,亦未发现细胞牙骨质。对CCD患牙以及正常牙的牙釉质以及牙本质分别进行能谱分析,数据用SPSS13.0版本进行处理,两样本均数t检验进行统计分析,结果表明CCD恒牙的牙釉质及牙本质的Ca、P含量均较正常降低,并具有统计学差异(t值分别为29.519、26.998以及9.676、88.982;p值分别为0.001、0.004以及0.011、0.003),CCD乳牙牙釉质的Ca、P含量均降低,并具有统计学差异(t值分别为33.754、23.412;p值分别为0.011、0.019),牙本质各元素的含量有降低,但无统计学差异,可能是样本量较少的原因。组织学分析与能谱分析均表明CCD患牙矿化较差,牙本质亦同样受累。本研究在组织学水平检测了RUNX2基因突变对CCD患者牙齿结构的影响,再次证明RUNX2基因对于牙齿正常发育的重要性。
二、致病基因RUNX2的突变检测及对蛋白亚细胞定位的影响
对五个CCD家系进行RUNX2基因突变分析发现:在五个CCD家系患者中均检测到不同的突变位点,家系中健康成员同一位点均为野生型序列,进一步证明RUNX2基因是CCD患者的致病基因,此基因的单倍体不足是CCD的致病原因。其中家系4的c.475 G＞C错义突变以及家系5的c.1096 G＞T无义突变是本研究检测到的新突变位点;家系2的c.673 C＞T以及家系3的c.1171C＞G突变位点在中国CCD患者中首次检出;家系1的c.674G＞A,R225Q是国外文献报道突变率最高的位点。本研究结果拓展了国内CCD基因层次的研究领域,为国内外CCD致病基因的突变位点数据库增添了新的资料。
为了研究基因突变对蛋白突变体亚细胞定位的影响,通过构建真核表达载体,引入突变位点,转染细胞,观察pEGFP-C1-RUNX2载体转染组细胞的荧光蛋白表达情况,未引入突变位点的野生型RUNX2组仅在胞核内见到绿色荧光表达;引入突变位点c.674G＞A及c.673 C＞T的两组转染细胞,在胞浆及胞核内均可见绿色荧光。pCMV-HA-RUNX2载体转染组细胞,利用荧光免疫组化,引入c.475 G＞C(p G159R)突变组在胞浆胞核内均可见绿色荧光,而野生型仅在胞核内有绿色荧光表达,将转染细胞分离胞浆胞核蛋白进行Western blot检测,可见引入突变组的胞浆胞核成分均具有特异条带,而野生型仅胞核成分检测到特异条带。我们提出G159R对于RUNX2蛋白的核定位具有重要作用,推测G159可能是RUNX2基因新的核定位元件之一,为进一步蛋白结构方面的研究提供一定的实验依据。
三、CCD患者牙髓细胞的生物学特性分析及基因差异研究
本部分从家系5先证者姐姐的下恒牙中分离培养牙髓细胞,与正常牙髓细胞相比,CCD牙髓细胞比正常细胞略显丰满、扁平;描绘生长曲线来评价细胞的生长状态,正常牙髓细胞生长趋势高于CCD细胞,流式数据应用SPSS13.0版本进行处理,用了两样本均数t检验,t值分别为10.14、12.05,两组细胞的G1期及S期具有统计学显著差异,P值分别为0.001以及0.000,提示CCD细胞滞留在G1期的比例显著增多,CCD牙髓细胞从G1期到S期的过渡明显受阻。透射电镜观察发现CCD细胞粗面内质网扩张,胞质内可见大小不等的层板状小体,这是正常牙髓细胞所不具有的特殊表现,粗面内质网扩张并可见部分核糖体颗粒脱落,可能提示细胞蛋白合成能力的改变,较多层板状小体的出现可能与脂类的合成分泌相关。
对CCD牙髓细胞进行矿化液诱导,虽然部分细胞出现细长单极胞浆突起,并呈复层生长,但直至培养7周仍无矿化结节形成,而正常牙髓细胞培养4周后开始有结节形成,表明CCD牙髓细胞的矿化能力减弱,可能提示此细胞向成牙本质样细胞分化能力的减弱,以及CCD患者牙髓组织损伤修复能力减弱。
采用第二代功能分类基因芯片从细胞水平分析基因突变对TGF-β/BMP信号传导通路相关基因表达的影响,发现有18条基因上调,14条基因下调。通过对异常表达基因的分析,提示E 366X RUNX2突变体影响了TGF-β/BMP信号传导通路上的一些相关基因的调控,如Cdc25A、TGFβ2、Smad3、EVI1的表达下调,COL3A1、BMP2、4、7均表达上调,细胞的增殖和分化是由多基因多通路共同调控的,协同作用较强的某些基因将决定细胞最终的趋势和命运,推测TGFβ2、Smad3、EVI1的表达减弱与COL3A1上调表达的协同作用可能阻碍了CCD牙髓细胞向成牙本质细胞的分化趋势;根据R391X突变体的功能研究报道,推测E366X突变极可能影响RUNX2突变体和Smads的相互作用,阻抑BMP对细胞分化的作用,BMP2、4、7的上调表达可能是由于正常的信号通路受到影响,通过其它负反馈机制调控细胞的分化和增殖。
Cleidocranial dysplasia(CCD;MIM # 119600) is a skeletal disorder with autosomal-dominant inheritance.The clinical hallmarks of CCD are clavicle rudiment or absence,delayed closure of cranial fontanels and sutures,Wormian bones,frontal bossing,supernumerary and late erupting teeth,wide pubic symphysis,and other skeletal anomalies.The locus for CCD has been mapped to chromosome 6p21 where the responsible runt-related gene 2(RUNX2) has been located,RUNX2 also refers to as polyomavirus enhancer binding protein 2(PEBP2) or murine leukemia virus enhancer core-binding factor(CBF).So far,there is few investigations about Chinese cases with CCD.The prevalence and range of RUNX2 mutations in Chinese eases with CCD were rarely reported.Here,5 different types of heterozygous mutations in the RUNX2 gene in 5 unrelated CCD cases from China who clearly display variable clinical manifestations were reported,the molecular basis of their dysfunction were studied.The lower permanent teeth from one CCD case were collected and the dental pulp cells were cultured,and characterization and gene expression of the cells were analysed in comparison with corresponding cells from normal control.The study was divided into three parts described as followings.
1.Clinical examination and analysis of patients with cleidocranial dysplasia
5 unrelated families with the clinical diagnosis of CCD were investigated in the present study,and the unaffected parents and siblings were included.There was no parental consanguinity.Radiological examination regarding osseous malformations was carried out over the entire body.A CCD phenotype was defined by the presence of hypoplastic clavicles and delayed closure of the anterior fontanelle in addition to the observation of classic craniofacial features.The skeletal anomalies as well as the oral manifestations of the syndrome were variable within the affected patients,the case 2 had extra teeth in the molar region(at least four molars),which was rarely reported in the literatures.Moreover,both impacted molars and extra molars had abnormal roots with delayed development,and the erupted first molars had relatively normal roots.It is possible that the abnormal root development may be one of the causes of eruption failure of permanent teeth in CCD cases.Interestingly,in case 3, both the ulna and radius leaned to the radialis so that the humeroulnar joints and humeroradial joints were abnormal,and the elbow looked like a triangle,which was rarely reported in the literatures.All the cases in the present study showed malformation of tarsometatarsal joints to a certain extent.Thus we may infer that RUNX2 may have a role in joint formation by affecting some of chondrocyte and osteoblast differentiation pathways.
We have analyzed histologically the structure of teeth obtained from CCD patients.Enamel structure was illegible,dentin tubules appeared obturated, Peritubular dentin had insufficiency mineralization,and typical continuous arc structures were virtually lacking on the enamel-dentinal junction.In deciduous teeth, more enamel lamella and enamel tuft were found,and dentin appeared irregular,size and distribution appeared ununiformed;cellular cementum was virtually lacking in deciduous and permanent teeth.Spectrum analysis of enamel and dentin of teeth from CCD patients and normal subjects showed that the proportion of Ca and P in permanent teeth from CCD was significantly lower than that from normals,so did the enamel of deciduous teeth from CCD patients,however,this did not appear to be the case for dentin of the deciduous teeth.
2.Mutation analysis of RUNX2 and its effect on subcelluar localization of mutants
To identify mutations in the RUNX2 gene in CCD cases,genomic DNA.from 5 CCD cases and their families were analysed,totally different mutations for each CCD case were detected.Novel c.475 G＞C(p G159R) missense and c.1096 G＞T nonsense mutations were included,and their healthy parents didn't carry the same mutation.Although the other three mutations were reported in the literatures,R225W and R391X mutations were reported in Chinese cases with CCD for the first time.
To detect the effect of mutations on nuclear localization of RUNX2 protein, fusion proteins were constructed between green fluorescent protein and RUNX2.The constructs were transiently transfected into mouse fibroblast NIH 3T3 cells.The wild-type RUNX2 protein was detected exclusively in the nucleus.However,R225Q and R225W mutants showed dual localization to both the cytoplasm and the nucleus. Immunofluorescent staining and western blotting showed that wild-type RUNX2 protein was localized exclusively in the nucleus,however;the mutant protein was found in both the nucleus and the cytoplasm,which demonstrated that transport of the RUNX2 mutant into the nucleus was disturbed by the G159R mutation.These results indicate that G159 is very important to promote RUNX2 nuclear-localization, therefore,G159 may be one of new elements of RUNX2 gene nuclear localization signals.
3.Characterization and gene expression analysis of dental pulp cells of CCD patient
To identify morphological and molecular alteration associated with CCD dental tissues,human permanent dental pulp cell cultures were established from age-and sex-matched CCD patient and normal subject.Dental pulp cells were compared for general morphology,proliferation rate,and gene expression profiles microarray technology.CCD pulp cells were in some sort flatter than the normal ones,however, the normal pulp proliferation rates were greater at time points tested than the cells from CCD patient.The flow cytometric analysis showed that the progression from the G1 to S phase in the CCD pulp cell cycle was impeded.The ultrastructure of CCD pulp cells showed that the rough endoplasmic reticulum was expended,and more lamellar body was found in cytoplasma.Partial ribosomes fell off the rough endoplasmic reticulum,and this change might impede the ability to synthesize protein. The CCD pulp cells were induced with mineralization inducer,and no mineralized nodule was found until seven weeks passed,although partial cells showed single pole process of cytoplasma and overlapping growth.The weakening of mineralization ability showed that its potential ability of differentiation might weaken.
The 96 genes of Human TGF-β/BMP signaling pathway were analysed by PCR array,and 18 genes displayed significant up-regulated at least two-fold in expression levels,and 14 genes down-regulated.Cdc25A,TGFβ2,Smad3 and EVI1 were down-regulated,and COL3A1,BMP2,4,7 were up-regulated.The proliferation and differentiation were modulated by multi-gene,some genes cooperated with each other and determine the final fate of cells.It was inferred that the potential differentiation of CCD pulp cells into odontoblasts was impeded to some extent by the cooperation between down-regulation of TGFβ2,Smad3 and EVI1 and up-regulation of COL3A1. According to the disfunction of R391X reported in the literatures,E366X mutation may have an effect on the interaction of RUNX2 mutant and Smads,and impede the function of BMP to cell differentiation.The up-regulation expression of BMP2,4,7 might be caused by other negative feedback because of breakoff of the normal signaling pathways.
引文
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