岩藻糖基转移酶Ⅳ在缺血后心肌细胞凋亡和心脏成纤维细胞增生中的作用

Ischemic heart disease is increasing worldwide. The transfected embryonic stem cells with FUT4 cDNA differentiate into myocardial cells more efficiently than the cell clones without fucosyltransferase Ⅳ (FUT4) cDNA. Furthermore, the differentiated myocardial cells from the cell clones with FUT4 cDNA survive longer than those from the cell clones without FUT4 cDNA. However, the roles of FUT4 in pathologic remodeling after myocardial ischemia (MI) have not been clarified. Our previous studies have found that downregulation of FUT4 suppresses activation of integrin αvβ3/FAK signaling in epithelial cells( Fertil Steril 2011;95:1446-51 ). We also found that peroxisome proliferator-activated receptor γ（PPARγ）and nuclear factor κB (NF-κB), both of which belong to nuclear transcription factors, are involved in modulation of FUT4 expression in epithelium. It has been reported that integrin αvβ3 and PPARγ as well as NF-κB play critical roles in myocardial remodeling due to loss of cardiomyocytes and proliferation of cardiac fibroblast after MI. This study is objective to apply a model of MI rat, combing the system of three dimensional coculture of cardiomyocyte-fibroblast and the protocols in molecular biology to investigate the followings in depth: 1. the roles of FUT4 in cardiomyocyte apoptosis and cardiac fibroblast proliferation induced by ischemia; 2. the influence of changes in FUT4 expression on activations of integrin αvβ3/FAK and MAPK, as well as PI3K/AKT signal pathways; 3. the effects of activation of PPARγ or NF-κB on FUT4 expression. Our study may provide a new target for the therapy of heart injury after MI.

心肌缺血（MI）的发病率逐年升高。岩藻糖基转移酶Ⅳ(FUT4)促进胚胎干细胞分化成心肌细胞并延长分化的心肌细胞的存活时间。但是，FUT4在MI后心肌重构中的作用不清楚。我们的前期研究揭示下调上皮细胞的FUT4基因抑制整合素αvβ3的激活，过氧化物酶体增殖物激活受体（PPARγ）及核因子κB（NF-κB）参与调节FUT4的表达。MI时，整合素αvβ3,PPARγ及NF-κB被激活引起心肌细胞凋亡和心脏成纤维细胞增生导致心肌重构。本研究拟用MI大鼠模型，结合心肌细胞-心脏成纤维细胞立体共培养，运用分子生物学技术，深入研究：①FUT4在心肌细胞凋亡及心脏成纤维细胞增生中的作用；②FUT4对整合素αvβ3/FAK、MAPK及PI3K/AKT信号通路激活的影响；③激活的PPARγ及NF-κB对FUT4的调节。该研究将为临床减轻MI后心肌损伤提供新的治疗靶点。

心肌梗死时心肌细胞发生凋亡或坏死，是诱发心衰发生的最主要的病理变化。然而，心肌缺血后心肌细胞凋亡发生的机制仍未被完全阐明。糖基化修饰的变化是否参与缺血诱发的心肌细胞凋亡仍未被研究。我们在该实验的研究中发现，唾液酸转移酶7A（Siat7A），即唾液酸糖基转移酶家族中的一员，在缺血的心肌组织中表达明显增加；同时体外细胞培养时发现，在缺氧的状态下，人源的心肌细胞系AC16中的Siat7A蛋白表达也明显增加，并呈现时间及缺氧程度依赖性。Siat7A的合成底物，Sialyl-Tn抗原（Neu5Acα2–6GalNAc-O-Ser/Thr）随之显著增加，并且促进心肌细胞凋亡。无论是体内还是体外，只要敲低Siat7A基因即会明显抑制缺血或缺氧导致的心肌细胞凋亡。细胞外信号调节激酶1和2（ERK1/2）参与缺血后Siat7A诱导的心肌细胞凋亡。通过运用染色质免疫共沉淀技术检测发现，转录因子Klf4能够特异性地与Siat7A启动子区(nt -655 to -636 bp)结合，并且转录激活Siat7A基因的表达。通过检测心肌梗死组织发现，Klf4蛋白与Siat7A蛋白表达平行增加。我们的实验研究结果为Siat7A转化为临床治疗心衰发生的一个靶点提供了基础理论依据。