Cx43基因敲除胎鼠心脏近端流出道隔心肌化过程中的关键基因

Key genes in the myocardialization of proximal cardiac outflow tract septum in Cx43 knockout mice

目的研究Cx43基因纯合敲除(Cx43-/-)小鼠胚胎心脏近端流出道组织中基因表达谱的改变,筛选可能导致Cx43-/-小鼠流出道梗阻的关键基因。方法以胎龄(embryonic day,ED)14.5的Cx43-/-和野生型(Cx43+/+)鼠胚心脏近端流出道部分为研究对象,分别提取总RNA,逆转录成cDNA,并在体外转录为cRNA,同时进行生物素标记及片段化,再与Affymetrix-430 2.0基因芯片进行杂交。杂交信号经扫描后,应用相关生物信息软件分析基因表达情况。结果与Cx43+/+组相比,Cx43-/-组中表达上调2倍以上的基因共有143个,表达下调2倍以上的基因有235个。其中表达差异的基因参与转录调控、细胞周期、细胞黏附、细胞活动和细胞骨架的信号通路等主要生理过程。进一步筛查表达差异1.5倍以上的基因发现,与圆锥动脉干畸形相关的TGFβ/BMP信号通路上的多个基因以及Ssr1、Ptk2、Bmp6等基因在Cx43-/-组有明显变化。对这些基因进行荧光定量PCR验证,结果与基因芯片一致(P<0.05)。结论利用基因芯片技术初步筛选出与Cx43-/-鼠胚心脏近端流出道发育有关的多个基因,并经荧光定量PCR验证。其中TGFβ/BMP信号通路上的多个基因以及Ssr1、Ptk2、Bmp6等基因可能与Cx43-/-小鼠流出道梗阻的发生有关。

Objective To investigate the changes of gene expression in the cardiac outflow tract（OFT） in Cx43 knockout（Cx43-/-） mouse embryos,and to elucidate the genes involved in the myocardialization of proximal cardiae outflow tract septum. Methods The cDNA was retrotranscripted from RNA,which extracted from OFT tissues of both Cx43-/-and Cx43 wild type（Cx43＋/＋） mouse embryos on embryonic day（ED） 14.5.The biotin-labeled cRNA derived from the transcription of cDNA was fragmented as probes.The probes were hybridized with Affymetrix Mouse Genome 430 2.0 Array.Gene Array Scanner was used to screen the signals of hybridization,and the expression of genes was detected. Results Among the differentially expressed genes,there were 143 upregulated and 235 downregulated in Cx43 knockout OFT tissue compared with those in Cx43＋/＋ heart.Functions of proteins encoded by the altered genes encompassed all functional categories,with the largest percentage in genes involved in signaling pathways such as regulation of transcription,cell cycle,etc.Among the differentially expressed genes in the Cx43-/-heart,some were related with TGFβ/BMP signaling pathway,and Ssr1,Ptk2 and Bmp6 were related with conotruncal defects.These genes were verified by Real-time PCR,and the result was consistent with that of microarray（P〈0.05）.Conclusions Scaned by Gene Array and verified by Real-time PCR,genes related with TGFβ/BMP signaling pathway,and Ssr1,Ptk2 and Bmp6 were differentially expressed in ED 14.5 Cx43-/-OFT tissue,which may be involved in the myocardialization of proximal cardiac outflow tract septum.