携带RUNX2基因突变的人牙囊细胞的分离培养及鉴定

目的：研究携带RUNX2基因突变位点的人牙囊细胞的分离、鉴定及意义。方法：收集颅骨锁骨发育不良（cleidocranial dysplasia,CCD）患者,鉴定其致病基因RUNX2的突变位点,通过离体培养CCD患者的未萌牙牙囊细胞,检测其携带的基因突变位点,鉴定RUNX2＋/m牙囊细胞。结果：全血基因组测序结果发现,患者RUNX2基因第2外显子插入TG突变,该突变型为c.309_310insTG,成功分离培养及鉴定RUNX2＋/m牙囊细胞,并发现RUNX2基因突变减少了牙囊细胞中此蛋白的表达水平。结论：成功分离培养携带RUNX2基因突变位点的人牙囊细胞,为进一步研究RUNX2基因在牙囊及牙齿萌出中的作用提供实验模型。     

颅骨锁骨发育不良综合征患者RUNX2基因突变检测

目的鉴定4个颅骨锁骨发育不良（cleidocranial dysplasia,CCD）家系RUNX2基因突变点。方法采用先证者查证法,对各家系成员进行全身健康状况及口腔专科检查,明确CCD诊断;抽取先证者及其父母外周静脉血,提取基因组DNA,PCR扩增RUNX2基因并测序,BLAST同源分析。结果在4个CCD家系患者中均检测到RUNX2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,进一步证实RUNX2基因是CCD患者的致病基因。其中家系4的c.475G〉C错义突变是此次检测到的新突变位点;家系2的c.673C〉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。结论本研究拓展了国内外CCD基因层次的研究领域,证实RUNX2基因的单倍体不足是CCD的致病原因。     

颅骨锁骨发育不良综合征患者的RUNX2基因突变检测分析

目的:检测鉴定我国一个颅骨锁骨发育不良综合征(CCD)家系RUNX2基因突变情况.方法:采用先证者查证法,对家系各成员进行全身健康状况及口腔专科检查,进行CCD诊断;抽取先证者及其父母外周静脉血,提取基因组DNA, PCR扩增RUNX2基因并测序,BLAST同源分析,同时检测100名健康人的相同位点,排除多态位点的可能.结果:先证者具有典型的CCD临床特征,其母亲亦为CCD患者,父亲则无相应临床表现;将先证者的RUNX2基因测序结果进行Blastn比较分析,在Exon 2上发现了一个A→G突变;实际测序图谱显示双峰结构(G、A),对其CCD母亲的基因检测表明,此突变来自母系染色体该基因478位点的基因突变;密码子AAC→GAC可能部分引起第160位氨基酸的改变,天冬酰胺(Asn,N)变成天冬氨酸(Asp,D);该突变型为478 A>G,N160D.家系健康成员同一位点显示G的单峰,即与野生型序列相同.结论:检测到的478 A＞G,N160D为新的基因突变位点,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.     

一颅锁骨发育不全综合征家族的RUNX2基因分析研究

目的 检测一个颅锁骨发育不全综合征（CCD）家系RUNX2基因突变情况。方法 采用先证者查证法,对CCD家系各成员进行全身健康状况及口腔专科检查,拍摄X线片;抽取先证者及其父母、姐姐外周静脉血,提取基因组DNA,聚合酶链反应（PCR）扩增RUNX2基因并测序,将先证者及其父母、姐姐RUNX2基因测序结果进行Blastn比较分析。结果 在先证者RUNX2基因的外显子2上发现了一个C→T突变,此突变来自母系染色体该基因568位点的基因突变;密码子CGG→TGG引起RUNX2编码的转录因子第190位保守的精氨酸变成色氨酸,突变型为c.568C>T。结论 c.568C>T突变是导致该家系发病的分子基础。     

颅锁骨发育不全综合征患者下颌骨喙突异常2例

颅锁骨发育不全综合征(cleidocranial dysplasia, CCD)是一种罕见遗传病,临床表现常为颅骨、锁骨、牙齿发育不全,较少出现下颌骨喙突异常,本文报道2例CCD患者喙突伸长,且1例患者存在RUNX2基因突变(NM＿001024630:exon5:c.586＿587del AG),为新突变。     

家族性锁骨颅骨发育不全的基因突变检测

目的 研究家族性锁骨颅骨发育不全家系的Cbfal基因突变。方法 对临床收集到的一个牙列异常、锁骨颅骨发育不全家系行外周血基因组DNA的提取，利用聚合酶链式反应，DNA直接测序检测突变。结果在该家系中每例患者均存在Cbfal基因外显子3中的杂合性突变，即cDNA674位G〉A的单碱基突变，从而使255位的精氨酸替换为谷氨酰胺（Pa55Q），改变了runt结构域的氨基酸序列。结论Cbfal基因的突变是引起此家系锁骨颅骨发育不全的致病原因。突变检测的结果可用于该家系后代的产前诊断。这也是在中国人家族性锁骨颅骨发育不全患者中首次检测到的基因突变。     

颅骨锁骨发育不全1例及基因检测分析

颅骨锁骨发育不全是一种罕见的常染色体显性遗传,主要影响全身骨骼和牙齿发育,发病率约为1∶1 000 000。本文对1例颅骨锁骨发育不全病例进行报道及文献回顾,基因检测证实RUNX2 6p21.1 NM_001024630.3 Exon4 c.534dupA p.（Val179fs）移码突变,为一个新的突变位点。     

RUNX2突变牙髓细胞转化生长因子-β相关信号通路基因表达的分析

目的利用第二代功能分类基因芯片检测颅骨锁骨发育不良患者成骨细胞特异性转录因子(runt-related gene 2,RUNX2)基因突变的牙髓细胞在转化生长因子-β(transforming growth factor-β,TGF-β)-骨形成蛋白(bone morphogenetic protein,BMP)信号传导通路上的基因表达差异。方法利用本课题组前期成功分离培养的携带RUNX2致病基因突变的牙髓细胞,通过第二代TGF-β/BMP信号传导通路功能分类基因芯片,进行实时定量PCR基因芯片,检测携带突变的颅骨锁骨发育不良患者牙髓细胞与正常牙髓细胞的基因表达差异,进行数据分析。结果基因芯片的实时定量PCR结果分析表明,在TGF-β/BMP信号传导通路上,RUNX2基因突变的牙髓细胞有18条基因表达上调,14条基因表达下调。结论 RUNX2基因可能通过调节TGF-β/BMP信号传导通路相关基因表达而影响牙髓细胞生物学特性。     

MicroRNAs和RUNX2基因网对人牙囊细胞的作用及其机制的研究

本研究目的是探讨RUNX2和miRNAs之间的串扰是否参与由牙囊细胞(DFCs)调节的牙齿萌出过程及其可能的分子机制。我们从锁骨颅骨发育不良患者收集血样和牙囊,并对其RUNX2基因突变进行了分析,然后分离鉴定出RUNX2(+/m)DFCs,并对其特点进行评估。通过微阵列芯片可对RUNX2(+/m)DFCs和RUNX2(+/+)DFCs中miRNAs的表达差异进行测定,而通过miRGen可预测靶标基因。我们     

Dysostosis cleidocranialis

Background

Cleidocranial dysplasia (CCD) is a rare dysplasia of bony and dental tissue. Characteristic are typical craniofacial and dental findings including morphological anomalies. CCD is possibly the only general syndrome that can be diagnosed based on the dental findings alone. CCD correlates with mutations in the RUNX2 gene.

Purpose

The present interdisciplinary study correlates phenotypic findings with genetic variations in the corresponding gene.

Patients and methods

The coding sequence of the RUNX2 gene from 31 CCD patients from 20 families was analyzed using molecular genetic methods including polymerase chain reaction and direct sequencing. The craniofacial and dental findings of each patient were evaluated according to a standardized scoring scheme and tested with homogeneity analysis for general phenotypic findings.

Results

Several mutations of the RUNX2 gene were identified. Depending on the mutation type, they showed different distribution patterns within the gene coinciding with the functional domains of the gene product. With homogeneity analysis of the phenotype cardinal (especially dental findings) and minor findings (pneumatization disturbances, Wormian bones) were identified. In combination with the genetic data, the statistical analysis showed that loss-of-function mutations of the RUNX2 gene result in a milder markedness of the CCD phenotype than gain-of-function or decrease-of-function mutations.

Conclusions

We found that type and location of a specific mutation within the RUNX2 gene might have an impact on the expressivity of CCD. Due to the limited sampling size this hypothesis must be verified by investigations in larger patient groups.     

Identification of a familial cleidocranial dysplasia with a novel RUNX2 mutation and establishment of patient-derived induced pluripotent stem cells

Cleidocranial dysplasia (CCD) is an autosomal dominant hereditary disease associated with the gene RUNX2. Disease-specific induced pluripotent stem cells (iPSCs) have emerged as a useful resource to further study human hereditary diseases such as CCD. In this study, we identified a novel CCD-specific RUNX2 mutation and established iPSCs with this mutation. Biopsies were obtained from familial CCD patients and mutation analyses were performed through Sanger sequencing and next generation sequencing. CCD-specific human iPSCs (CCD-hiPSCs) were established and maintained under completely defined serum, feeder, and integration-free condition using a non-integrating replication-defective Sendai virus vector. We identified the novel mutation RUNX2_c.371C>G and successfully established CCD-hiPSCs. The CCD-hiPSCs inherited the same mutation, possessed pluripotency, and showed the ability to differentiate the three germ layers. We concluded that RUNX2_c.371C>G was likely pathogenic because our results, derived from next generation sequencing, are supported by actual clinical evidence, familial tracing, and genetic data. Thus, we concluded that hiPSCs with a novel CCD-specific RUNX2 mutation are viable as a resource for future studies on CCD.

     

表皮生长因子对牙囊细胞增殖活性的影响

目的：研究表皮生长因子（EGF）对体外培养的大鼠牙囊细胞增殖活性的影响。方法：原代培养Wistar大鼠牙囊细胞,选择生长良好的第3代细胞,分别加入不同浓度的EGF（0.5、1、5、10、50 ng/mL）与牙囊细胞共孵育5 d,MTT法对比观察不同浓度的EGF对牙囊细胞增殖活性的影响。数据采用SPSS 13.0软件包进行方差分析。结果：在EGF浓度为5～10 ng/mL时,牙囊细胞的增殖活性显著增高（P〈0.05）,10 ng/mL时达到最高（P〈0.01）。结论：EGF可作用于牙囊细胞,合适浓度的EGF能显著增加牙囊细胞的增殖活性。