人突变载脂蛋白E(apoE4,apoE7)转基因小鼠模型

为揭示人突变apoE基因表达在整体引起的异常反应并得到人类相关疾病的小鼠模型,用显微注射法制备了人apoE4与apoE7转基因小鼠,用Southern印迹杂交及ELISA证明人apoE基因在首建鼠及其子代体内整合与表达良好,具有遗传稳定性.血清脂质与行为测定表明,人apoE表达使小鼠患高脂血症并出现大脑学习与记忆功能衰退和寿命缩短,高脂食物对正常鼠与转基鼠均可诱发高血脂,但机制不同.同时还证明,氨基端与羧基端突变的apoE具有相同的致病作用.     

The human apoE7 and apoE4 transgenic mice models

To scrutinize the disorders caused by human mutant apoE7/apoE4, human apoE4 and E7 transgenic mice were established with microinjection technique to examine molecular genetic phenomenain vivo. The integration and expression of h-apoE mutant genes in transgenic mice were determined with Southern blot, Northern blot and ELISA. The current studies indicated that the transgenes and the phenotypes regarding expression of transgenes could be transmitted stably in transgenic lines. The levels of serum lipid in transgenic mice showed the characteristics of hyperlipidemia. Besides, behavior tests demonstrated the degeneration of learning and memory in transgenic mice. Short life span was observed in 2 transgenic lines. After fed with high lipid food high serum lipid was found both in normal and transgenic mice, but their mechanism regulating lipid metabolism was different. It was also verified that the human apoE mutants located at either N-terminal or C-terminal had the same pathogenesis regarding disorders of lipid metabolism in murine.     

人ApoE转基因小鼠与TGE_（7－2） TGE_（7－3）转基因鼠系的建立

用含小鼠金属硫蛋白（ＭＴ－１）基因启动子与突变的人ＡｐｏＥ７基因的６．０ＫｂＤＮＡ片段作为目的基因制备转基因小鼠,利用显微注射法将目的基因导入４４１枚受精卵,移植于２０只假孕鼠,其中１５只受孕,共产仔鼠８０只,经ａ－３２Ｐ斑点杂交与Ｓｏｕｔｈｅｒｎ杂交法鉴定出两只整合有人ＡｐｏＥ基因的转基因雌鼠,其拷贝数分别为２和５。将两只首建鼠（雌性Ｆｏ代）与正常雄鼠交配,建立Ｆ１代转基因鼠系ＴＧＥ７－２和ＴＧＥ７－３,为进一步从整体上研究ＡｐｏＥ在脂质代谢中的作用以及ＡｐｏＥ与动脉粥样硬化和Ａｌｚｈｅｉｍｅｒ’ｓ病的关系创造条件。     

人突变appE基因在转基因鼠体内的表达及血清脂质变化

为了研究人突变ａｐｏＥ７基因在血脂代谢中的作用．采用微注射的方法建立了人ａｐｏＥ７转基因鼠,三个首建鼠（ｔｇ１,ｔｇ２,ｔｇ３）整合目的基因的拷贝数相差２倍左右,其血中表达的人ａｐｏＥ７的水平也不相同,低水平表达的ｔｇ１为１．２６ｍｇ／ｄｌ,高水平表达的首建鼠ｔｇ３血清中ａｐｏＥ７浓度可高达２１．１ｍｇ／ｄｌ．异常ａｐｏＥ基因的表达导致了转基因鼠血清甘油三酯和胆固醇明显升高,为对照的１．５～３倍．高密度脂蛋白ＨＤＬ降低,低密度脂蛋白ＬＤＬ和极低密度脂蛋白ＶＬＤＬ升高．经２０ｍｍｏｌ／ＬＺｎＳＯ４诱导后,Ｆ１代Ｔｇ３鼠系血清甘油三酯（ＴＧ）水平高达４４４ｍｇ／ｄｌ,胆固醇（ＴＣ）高达２３４ｍｇ／ｄ１．ＨＤＬ升高和ＬＤＬ／ＶＬＤＬ降低十分明显,表现了高脂血症的指征．     

转基因鼠载脂蛋白E基因异常表达与血脂代谢水平的相关性

探讨载脂蛋白E(apoE)基因在转基因鼠体内的表达及其在血脂代谢中的作用.以单克隆抗体酶联免疫吸附法分别测定apoE4、apoE7转基因鼠(tg4、tg7)的血清apoE含量.用甘油磷酸化酶(GPO)法和胆固醇氧化酶(CHOD-PAP)法对转基因鼠及对照组鼠(control)的血清甘油三酯(TG)和胆固醇(TC)进行测定.tg4血清apoE含量为20.3±7.2ug/dl,tg7血清apoE含量为1.8±5.4ug/dl.血清TG水平转基因鼠[tg4(19.16±0.31)mmol/L,tg7(18.15±0.46)mmol/L]与对照组鼠[(4.95±2.25)mmol/L]的差异均有显著性(p＜0.05).血清TC水平tg4[(4.44±0.04)mmol/L]与对照组鼠[(1.49±0.01)mmol/L]也表现出显著性的差异(p＜0.05).提示apoE基因异常表达影响了转基因鼠的血脂代谢.     

The human apoE7 and apoE4 transgenic mice models

To scrutinize the disorders caused by human mutant apoE7/apoE4, human apoE4 and E7 transgenic mice were established with microinjection technique to examine molecular genetic phenomena in vivo. The integration and expression of h-apoE mutant genes in transgenic mice were determined with Southern blot, Northern blot and ELISA. The current studies indicated that the transgenes and the phenotypes regarding expression of transgenes could be transmitted stably in transgenic lines. The levels of serum lipid in transgenic mice showed the characteristics of hyperlipidemia. Besides, behavior tests demonstrated the degeneration of learning and memory in transgenic mice. Short life span was observed in 2 transgenic lines. After fed with high lipid food high serum lipid was found both in normal and transgenic mice, but their mechanism regulating lipid metabolism was different. It was also verified that the human apoE mutants located at either N-terminal or C-terminal had the same pathogenesis regarding disorders of     

重组人apoE基因表达活性和转基因小鼠研究

人apoE基因组DNA,去除其自身启动子,代之以小鼠金属硫蛋白启动子,重组质粒经脂质体介导转入小鼠NIH／3T3细胞后,以人apoE基因组DNA／EcoRⅠ片段为探针检测mRNA表达,可见apoEmRNA杂交信号很强,经重金属诱导后杂交信号更强,表明MT启动子功能良好,pME表达正常．将人apoE基因组DNA用显微注射法导入小鼠受精卵雄性原核,再将胚胎移植入假孕母鼠输卵管内,仔鼠分娩四周后,自鼠尾提取DNA,鉴定人apoEDNA在小鼠染色体上的整合,最终得到有人apoE基因整合的转基因首建鼠．