Qi Chun-hua, Huang Guo-ying, Zhou Guo-min
Pediatric Heart Center, Children's Hospital of Fudan University, Shanghai 200032, China.
Zhonghua Yi Xue Za Zhi. 2007 Jun 26;87(24):1709-12.
To investigate the changes in the expression of cardiac transcription factors in the cardiac outflow tract (OFT) tissues in the connexin43 knockout homozygotes (Cx43 KO), connexin43 heterozygotes, and connexin43 wild-type mice (Cx43 WT).
The cDNA was retrotranscribed from the RNA extracted from the OFT tissues of 6 Cx43 KO, 6 Cx43 WT, and 6 Cx43 heterozygotes genotyped by PCR method on the embryonic day (ED) 13.5 and 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 detect the expression of genes. The mRNA expression levels of 3 cardiac transcription factors: Sox11, Foxp1, and Tbx20 were measured by real time quantitative RT-PCR.
The ratios of the expression of the 6 genes, all cardiac transcription factors: Gata4, Mef2C, Sox4, Sox11, Foxp1, and Tbx20 between the Cx43 KO and Cx43 WT groups were 1:1.41, 1:2.30, 1:3.25, 1:0.71, 1:0.66, and 1:0.54. The expression levels of Sox11 and Foxp1 on ED13.5 in the Cx43 K group were 4.76 +/- 0.19 and 5.08 +/- 0.28 respectively, both significantly lower than those of the Cx43 WT group (5.34 +/- 0.25 and 5.64 +/- 0.15 respectively, both P < 0.01), and expression level of Tbx20 on ED 13.5 in the Cx43 K group was 7.18 +/- 0.16, not significantly different from that of the Cx43 WT group (7.47 +/- 0.27, P > 0.05). The expression levels of the genes Sox11, Foxp1, Tbx20 on ED 14,5 were 4.71 +/- 0.27, 5.25 +/- 0.31, and 7.05 +/- 0.17 respectively, all significantly lower than those of the Cx43 WT group (5.00 +/- 0.19, 5.77 +/- 0.16,) and 7.43 +/- 0.25, all P < 0.05). The results of the expression of these genes by real time PCR analysis showed an excellent concordance with those indicated by the microarray analysis.
The cardiac transcription factors such as Sox11, Foxp1, and Tbx20 that are differently expressed in the Cx43 KO OFT tissue may be involved in the pathogenesis of the OFT defects.
研究连接蛋白43基因敲除纯合子（Cx43 KO）、连接蛋白43基因杂合子及连接蛋白43基因野生型小鼠（Cx43 WT）心脏流出道（OFT）组织中心脏转录因子表达的变化。
通过PCR方法对胚胎期（ED）13.5天和ED 14.5天的6只Cx43 KO、6只Cx43 WT和6只Cx43杂合子小鼠的OFT组织提取的RNA进行反转录得到cDNA。将cDNA转录得到的生物素标记的cRNA片段化作为探针。探针与Affymetrix Mouse Genome 430 2.0芯片进行杂交。使用基因芯片扫描仪筛选杂交信号并检测基因表达。通过实时定量RT-PCR检测3种心脏转录因子Sox11、Foxp1和Tbx20的mRNA表达水平。
Cx43 KO组与Cx43 WT组之间6种基因（均为心脏转录因子）Gata4、Mef2C、Sox4、Sox11、Foxp1和Tbx20的表达比值分别为1:1.41、1:2.30、1:3.25、1:0.71、1:0.66和1:0.54。Cx43 K组在ED13.5时Sox11和Foxp1的表达水平分别为4.76±0.19和5.08±0.28，均显著低于Cx43 WT组（分别为5.34±0.25和5.64±0.15，均P<0.01），Cx43 K组在ED 13.5时Tbx20的表达水平为7.18±0.16，与Cx43 WT组（7.47±0.27，P>0.05）无显著差异。ED 14.5时Sox11、Foxp1、Tbx20基因的表达水平分别为4.71±0.27、5.25±0.31和7.05±0.17，均显著低于Cx43 WT组（分别为5.00±0.19、5.77±0.16和7.43±0.25，均P<0.05）。实时PCR分析这些基因的表达结果与芯片分析结果高度一致。
在Cx43 KO OFT组织中差异表达的心脏转录因子如Sox11、Foxp1和Tbx20可能参与了OFT缺陷的发病机制。
