摘要
目的研究连接蛋白43(Cx43)基因敲除小鼠胚胎心脏近端流出道组织中转录因子的改变,从分子水平探讨 Cx43基因敲除小鼠胚胎心脏近端流出道发育异常的原因。方法以胎龄13.5 d 和14.5 d 的 Cx43基因敲除、Cx43杂合子和 Cx43野生型鼠胚心脏近端流出道部分为研究对象。分别提取总 RNA,反转录成 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小鼠全基因组芯片进行杂交。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。用实时定量逆转录(RT)PCR 的方法对基因芯片筛选出的与心脏发育相关的部分转录因子进行验证。结果与 Cx43野生型组相比,Cx43基因敲除组表达差异的基因中,有6个是与心脏发育相关的转录因子。实时定量 RT-PCR 验证了其中3个基因:Sox11、Foxp1和 Tbx20。在胎龄13.5 d,Cx43基因敲除鼠胚 Sox11、Foxp1的表达量均明显低于 Cx43野生型组(4.76±0.19 vs 5.34±0.25,5.08±0.28 vs 5.64±0.15,均 P<0.01);Tbx20在各组间差异不明显。胎龄14.5 d,各基因Sox11、Foxp1以及 Tbx20的表达量在基因敲除组均明显低于 Cx43野生型组(4.71±0.27 vs 5.00±0.19,5.25±0.31 vs 5.77±0.16,7.05±0.17 vs 7.43±0.25,均 P<0.05)。其变化趋势与基因芯片结果基本一致。结论心脏特异性的转录因子 Foxp1、Sox11和Tbx20等的表达异常可能与 Cx43基因敲除小鼠流出道的异常发育有关。
Objective To investigate the changes in the expression of cardiac transcription factors in the cardiac outflow tract （OlaF） tissues in the connexin43 knockout homozygotes （Cx43 KO） , connexin43 heterozygotes, and connexin43 wild-type mice （Cx43 WT）. Methods The cDNA was retrotranscribed from the RNA extracted from the Olaf 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 ： Soxl 1, Foxpl, and Tbx20 were measured by real time quantitative RT-PCR. Results 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 Soxll and Foxpl 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 Soxll, Foxpl, 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. Conclusion The cardiac transcription factors such as Sox11, Foxp1, and Thx20 that are differently expressed in the Cx43 KO OFF tissue may be involved in the pathogenesis of the OFF defects.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2007年第24期1709-1712,共4页
National Medical Journal of China
基金
国家自然科学基金(30471823)
