HCY-2基因和同型半胱氨酸与鸡胚先天性心脏畸形发生的关系

The relationship between HCY 2 gene and congenital heart teratogenesis in early chick embryos

目的 探讨新基因HCY 2和同型半胱氨酸与早期先天性心脏畸形发生的关系。方法 应用鸡胚致畸试验观察同型半胱氨酸 (0～ 1 6 μmol/胚胎 )对鸡胚心血管发育的影响 ;采用脂质体介导基因转移法将已构建的含有全长HCY 2cDNA的真核表达载体转入新生鸡卵 ,培养 4d后再用Western杂交和免疫组织化学等方法观察HCY 2基因对早胚心血管发育的致畸作用及其编码产物的表达和分布。结果 HCY对器官形成期的心脏有明显的致畸作用并呈剂量 反应关系 (P <0 .0 5 )。转1 0 μg和 2 0 μgHCY 2基因能诱导鸡胚心血管系统发育异常 ,心脏畸形发生率分别为 1 6 .7%和 2 4.0 % ,主要表现型是心包积液、异位心、心外露和心脏过小。Western杂交显示转 1 0 μg和 2 0 μg基因组胚胎样品有HCY 2蛋白大量表达 ,其分布以胚胎心脏组织为主。HCY 2基因可引起心脏结构紊乱、心内膜垫缺损和抑制心室心房发育 ,并可损害心肌细胞的超微结构。结论 HCY/HCY 2基因可能是一种新的心脏发育毒性因子 ;HCY 2基因的异常表达在心血管畸形发生过程中起重要作用。

Objective To explore the relationship between novel gene (HCY 2), homocysteine and congenital heart defects, and to study their biological mechanisms. Methods The teratogenic test of chick embryos which were treated with D.L homocysteine (0 16 μmol/embryo) was used to observe the developmental toxicity of homocysteine per se. The eukaryotic expressing vector containing whole length HCY 2 cDNA (2 014 bp) was microinjected into fertile eggs with lipofect AMINE TM reagent; then the effects of HCY 2 gene on cardiovascular damages of embryos, the expression and distribution of HCY 2 gene coding product were investigated with the techniques of Western blot, immunohistochemistry, light and electron microscopy. Results Apparently teratogenic action of homocysteine on developing heart was observed in the critical stage of organogenesis and the damages were concentration dependent ( P <0.05). 10～20 μg HCY 2 gene could cause dysmorphogenesis of the cardiovascular system. The congenital heart defect rates were 16.7% and 24.0% respectively. The abnormal forms were ectopic cardis extrathoracic heart and hypoplastic heart. Western blot and immunohistochemical staining showed that HCY 2 protein was largely expressed in the abnormal embryos, with stronger staining at the sites of embryonic heart and brain as compared with controls. Under light and electron microscope, it was seen that HCY 2 gene resulted in structure disturbance, endocardial cushion defect and hypoplasia of heart. Conclusion Homocysteine/HCY 2 gene may be a new heart developing cytotoxic/genotoxic factor, and HCY 2 gene may play a very important role in the mechanisms of congenital heart defects of chick, and probably humans as well.