定位转HCY-2基因诱发鸡胚神经管畸形的时间-效应关系研究  

Time effect relationship between the positional microinjection of HCY 2 gene and the neural tube teratogenesis of chick embryos

作　　者：李勇[1] 李竹[1] 张晨晖[1] 陈星[1] 孟昭亨[1] 刘虹[1] 赵智榕[1] 李松[1] 

机构地区：[1]北京医科大学中国妇婴保健中心

出　　处：《卫生研究》1999年第4期221-224,共4页Journal of Hygiene Research

基　　金：国家自然科学基金;卫生部基金

摘　　要：本文旨在研究定位转新基因（ＨＣＹ－２）与神经管畸形发生的时间－效应关系及可能的作用机理。采用脂质体介导基因转移技术将已构建的含有全长ＨＣＹ－２ｃＤＮＡ的真核表达载体定位转入不同发育时期的鸡胚（Ｈａｍｂｕｒｇｅｒ－Ｈａｍｉｌｔｏｎ１、６和１２期），培养后再用ＲＴ－ＰＣＲ、免疫组化、扫描和透射电镜及光镜等技术探讨ＨＣＹ－２基因在４天龄鸡胚中的表达、分布和致畸作用。结果在３个不同时期定位导入ＨＣＹ－２基因后，均可诱导胚胎发生神经管畸形，以１天龄头侧微注射的胚胎最为敏感，神经管畸形发生率高达３５．３％，呈明显时间－效应关系，其表现型是：脑膨出、无脑、脊柱裂和小头。转基因组胚胎能够表达ＨＣＹ－２ｍＲＮＡ和相应编码蛋白，主要分布于胚胎脑组织中。扫描和透射电镜下发现ＨＣＹ－２基因能损伤细胞表面和内部的超微结构；在畸形部位还观察到大量的凋亡细胞。结论是，ＨＹＣ－２基因可能是一种新的基因毒性因子，它在胚胎早期神经管畸形发生机制中起重要作用。The time effect relationship between positional transfered novel gene(HCY 2)and neural tube teratogenesis,and their possible mechanisms were studied.An eukaryotic expressing vector which containing whole length HCY 2 cDNA was microinjected into chick embryos in culture at days 0,1 and 2(≈Hamburger Hamilton stages 1,6 and 12)mediated by lipofect AMINE TM reagent.The techniques of RT PCR,immunohistochemical staining,scanning electron microscope(SEM)and transmission electron microscope (TEM)were used to investigate the expression and distribution of HCY 2 mRNA with its coding product and dysmorphogenesis of the neural tube at 96h (≈stage 22). Neural tube defects (NTDs) were discovered in every transferred gene group, only the day 1 embryos which positional site injected was the area pellucida of head,however,the rate of NTDs was the highest (35 3%).There was an obvious time effect relationship.The phenotypes of NTD were encephalocele,anencephaly,spina bifida and microcephaly.The embryos with transferred gene could express HCY 2 mRNA and its coding product,and the HCY 2 protein mainly distributed in embryonic brain cells as compared to controls.It was found that HCY 2 gene could result in abnormal ultrastructure at the surface and inside of cells under SEM and TEM. It has been observed apoptosis at the sites with NTDs. It is concluded that HCY 2 gene may be a new genotoxic factor,which plays an improtant role in the mechanisms of neural tube teratogenesis during the early developing statge of embryos.

关 键 词：HYC-2基因 神经管畸形 胚胎 时间-效应关系 

分 类 号：R321.6[医药卫生—人体解剖和组织胚胎学] Q593.4[医药卫生—基础医学]