DLX2基因促进脐带干细胞成骨分化

目的：研究Distal-less homeobox 2(DLX2)对脐带干细胞成骨定向分化能力的影响。方法成骨分化诱导培养基诱导脐带干细胞体外成骨分化；逆转录病毒转染构建过表达DLX2的脐带干细胞,进行 DLX2获得性功能研究；碱性磷酸酶活性实验检测成骨早期分化指标-碱性磷酸酶活性；茜素红染色及钙离子定量分析检测干细胞体外矿化能力；实时荧光定量反转录PCR( real time reverse transcription PCR,qRT-PCR)检测DLX2及成骨分化相关基因-Sp7转录因子、骨涎蛋白和骨钙素的表达。结果 qRT-PCR结果显示,DLX2在牙周膜干细胞的表达明显高于非牙源性干细胞-脐带干细胞、骨髓干细胞、脂肪干细胞。碱性磷酸酶活性结果显示过表达DLX2明显增强脐带干细胞的碱性磷酸酶活性；茜素红染色及钙离子定量分析结果显示过表达DLX2明显增强脐带干细胞体外矿化能力；qRT-PCR结果显示过表达DLX2明显促进Sp7转录因子、骨涎蛋白和骨钙素的表达。结论 DLX2具有促进脐带干细胞体外成骨分化的潜能。

Dlx2在胚胎间充质细胞成骨分化中的作用

目的 研究Distal-less homeobox 2(Dlx2)在胚胎间充质细胞成骨分化中的作用.方法 Bmp4诱导小鼠胚胎间充质细胞系C3H10T1/2细胞体外成骨分化.逆转录病毒转染构建过表达及基因敲除的Dlx2细胞稳定转染细胞,进行Dlx2获得性及丧失性功能研究.碱性磷酸酶活性实验检测成骨早期分化指标-碱性磷酸酶活性,实时荧光定量反转录PCR(real time reverse transcription PCR,qRT-PCR)检测成骨分化相关基因-Sp7转录因子、碱性磷酸酶和骨钙素的表达.结果 结果显示过表达Dlx2明显增强胚胎间充质细胞C3H10T1/2的碱性磷酸酶活性.qRT-PCR结果显示过表达Dlx2明显促进Sp7转录因子、碱性磷酸酶和骨钙素的表达.基因敲除Dlx2明显抑制C3H10T1/2细胞的碱性磷酸酶活性,qRT-PCR结果显示基因敲除Dlx2明显抑制Sp7转录因子、碱性磷酸酶和骨钙素的表达.结论 Dlx2具有促进胚胎间充质细胞体外成骨分化的功能.

Dlx2基因过表达与前成骨细胞系MC3T3-E1成骨分化过程中的细胞凋亡和周期调控

背景:Dlx2 基因在颅神经嵴细胞迁徙进入第一鳃弓过程和颅颌面骨骼发育中起重要作用,但Dlx2 基因对成骨细胞分化过程中细胞凋亡和细胞周期调控的影响尚未见报道.目的:观察Dlx2 基因过表达对前成骨细胞系MC3T3-E1 成骨分化过程中细胞凋亡和细胞周期调控的影响.方法:构建反转录病毒pMSCV-puro-Dlx2 并转染矿化诱导液培养下的MC3T3-E1 细胞,构建稳定过表达Dlx2 基因的细胞系MC3T3-E1-Dlx2.RT-PCR 和Western blot 验证Dlx2 基因过表达细胞系的建立.Annexin V/PI 双染色后流式细胞分选检测细胞凋亡,PI/RNase 双染色后流式细胞分选检测细胞周期变化.结果与结论:实验成功构建稳定过表达Dlx2 基因的细胞系MC3T3-E1-Dlx2.发现Dlx2 基因过表达促进细胞凋亡(P < 0.05),同时阻滞细胞周期于G1/G0 期(P < 0.05),减低细胞增殖性,促进细胞分化行使功能.

The first branchial arch malformation(FBAM)is a rare congenital defect associated with anomalous development of the first and second branchial arches. Cause of FBAM still remains unknown, and is thought in most cases to be multifactorial, involving beth genetic and enviromental factors. Dlx2 as a member of the Dlx homeobox gene family,plays a crucial role in the development of the first branchial arch. The tissues regulated mainly by Dlx2 are coincident with the tissues mainly involved in FBAM. Dlx2 over-expression generated by electroporation transfection can disturb the migration and differentiation of cranial neural crest cells(CNCCs), which migrate to the branchial arches and in turn give rise to much of the facial skeleton and connective tissues. Furthermore, Dlx2 over-expression can be found in the first branchial arch spontaneous mutant mice. So we hypothesize that Dlx2 over-expression mutation causes FBAM due to an increase in cell-cell adhesion and inhibiting the migration of CNCC to the first branchial arch in the early stage, or migrating to an incorrect position and can't differentiate into normal tissues. What an exact role of Dlx2 over-expression in FBAM remains to be investigated and DIx2 over-expression transgenic mouse will be a nice model for further research in FBAM. Supported by Key Basic Research Project of Science and Technology Commission of Shanghai Municipality (08JC1417800, 10JC1408700), Shanghai Loading Academic Discipline Project(S30206) and Creative Team Project of Shanghai Universities.

Dlx2条件性激活转基因小鼠模型的建立

目的:建立Dlx2条件性激活转基因小鼠,为实现Dlx2基因在不同组织特异性激活奠定基础.方法:构建Dlx2条件性激活真核表达质粒iZEG-Dlx2并线性化,通过显微注射,获得Dlx2条件性过表达转基因小鼠,应用PCR鉴定其基因型.结果:共获得8只转基因阳性首建鼠,阳性率为11.59％.结论:运用转基因技术,可成功获得Dlx2条件性过表达转基因小鼠,为在体内研究Dlx2基因的功能奠定基础.

在大脑胚胎脑皮质神经细胞Dlx2基因表达的影响

目的:观察KCl刺激培养的大鼠E17脑皮质神经细胞后不同时间点Dlx2、Arx、Rb1基因的表达,探讨Dlx2、Arx、Rb1的表达的变化.方法:原代细胞培养获取胎龄E17大鼠脑皮质神经细胞,采用反转录-聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)技术检测Dlx2、Arx、Rb1在经KCl刺激0.5～24 h后的表达变化情况.结果:RT-PCR结果显示KCl诱导组不同时间点神经细胞中Rb1 mRNA表达在0.5～6h时呈现递增的趋势,而在12 h mRNA表达明显大幅度降低,24h又出现上升;Dlx2随着KCl的诱导表现出表达的一个长时程增强的过程,其mRNA表达随着时间的变化而增多,并在24 h达到表达的高峰;Arx mRNA的表达则出现增强趋势,持续24h,但此时略有下降.结论:经KCl刺激后Dlx2 mRNA表达升高对胚胎期神经细胞的存活功能可能具有重要作用.