两个缺血再灌可诱导新基因的表达与克隆

Expression and cloning of two novel genes induced by ischemia and reperfusion

目的 :发现和克隆短暂缺血再灌注心肌组织内mRNA表达发生改变的基因。方法 :反复短暂结扎猪左冠状动脉前降支引起缺血再灌 ,从受累心肌组织中提取RNA并用于mRNA差异显示。经克隆和测序其cDNA序列后 ,用Northern杂交进一步证实其表达。结果 :克隆并确证两个cDNA(即W12和W2 8)为真正差异表达。它们在缺血再灌心肌中的表达明显高于非缺血的心肌 ,W12 ,W2 8表达水平分别为非缺血心肌的 2 0倍 (P <0 0 5 )和 1 9倍 (P <0 .0 5 )。另外 ,在猪心脏、肝、肺、肾、脾、肠、脑、骨骼肌等组织器官均可检测到它们的mRNA。DNA序列分析显示 ,该两个cDNA与已知基因均无同源性 ,表明它们代表新的基因。结论

Objective To identify and clone the differentially expressed genes in brief ischemia and reperfusion myocadium. Methods Ischemia and reperfusion were induced by repeated brief ligation of the porcine left anterior descending coronary artery. Total RNA which was isolated from myocardium subjected ischemia and reperfusion was used for mRNA differential display. After cloning and sequencing the cDNA fragments which showed change in expression, their expression were further confirmed by Northern Blot analysis. Results Two differentially expressed cDNAs (W12 and W28) were identified and cloned. Their expression were subsequently confirmed to be truly differentially expressed. The expression of both genes in ischemia and reperfusion myocardium was obvious higher than that in nonischemia and reperfusion: W12 expression level was 2 fold (P<0.05), and W28 expression level 1.9 fold (P<0.05). In addition, mRNAs of W12 and W28 were existed in all tested organs including heart, liver, lung, kidney, spleen, intestine, brain and skeletal muscle. DNA sequencing analysis showed that there was no homology between W12, W28 and known genes, implying that they would represent novel gene respectively. Conclusion Two novel genes induced by ischemia and reperfusion are identified, cloned and confirmed.