【摘要】 为阐明人清道夫受体AI (hSR -AI)的功能及在动脉粥样硬化发生中的作用 ,本研究首先构建了含鼠tie- 1启动子和人清道夫受体AIcDNA的表达载体 ,经酶切及测序鉴定后 ,用显微注射的方法将制备好的tie - 1-hSR-A -BGHpolyA片段注入受精卵 ,将注射后存活的受精卵移入ICR假孕母鼠 ,产下的仔鼠经聚合酶链反应和Southernblot分析 ,筛选出整合有外源目的基因的阳性转基因鼠 ;对小鼠组织RNA行逆转录聚合酶链反应及组织切片免疫组织化学染色 ,检测人清道夫受体AI在小鼠体内的表达水平及表达部位 ;光镜及电镜观察转基因鼠血管及其它组织的病理变化。结果发现 ,重组Tie - 1/hSR -AI质粒中鼠tie - 1启动子和人清道夫受体AIcDNA序列正确 ,显微注射后存活的 5 6 1枚受精卵分别移入 19只ICR假孕母鼠 ,有 13只受孕 ,共产下 5 6只仔鼠 ,存活 5 4只 ,经过整合检测 ,检出 7只阳性鼠 ,整合效率为 13%。在G1、G2、G3代及纯合子转基因鼠中PCR阳性率分别为 47.8%、71.3%、75 .0 %和 10 0 %。 5只雄性转基因鼠的主动脉、肾、肝等组织中均有人清道夫受体AI表达 ,且主要集中在血管的内皮细胞上 ;转基因鼠主动脉内皮细胞明显水肿 ,表面呈多囊状和虫蚀样改变 ,胞质中有较多水泡 ,中膜有糖原沉积样灶性病变 ,平滑肌细胞中亦有水肿 ;血浆?更多还原

【Abstract】 Aim To establish a new transgenic mice model for determining the function and roles of human scavenger receptor A in atherosclerosis in vivo. Methods Human scavenger receptor A-I minigene-driven tie-1 promoter was constructed by endonuclease digestion and confirmed by sequence analysis. Transgenic mice were produced via microinjection method. PCR, Southern blot were used to screen the positive transgenic mice. Transgenic mice line established by mating with C57BL/6 mice and offspring were identified by PCR so as to research transgenic mice transmission. Northern blot, RT-PCR, immunohistochemical analysis, light and transmission electron microscopy were used to investigate the expression location of human SR-A and lesions of arteries and vascular endothelial cells in transgenic mice. Results The fragment sequence of mouse tie-1 promoter and human SR-AI cDNA are similar to their sequences in Genebank and no ATG before the translation initiation sites of human SR-A by sequence analysis. About 3.4 kb tie-1-promoter-hSR-AI-cDNA-BGH polyA fragment were obtained by AatⅡand XhoⅠdigests. 561 survival embryos injected with purified human SR-A minigene were implanted into the oviducts of 19 ICR pseudopregnant mice.Among the 54 survived pups from 13 foster mothers, 7 founders were identified with PCR and Southern blot analysis. Integration rate of exogenous was 13%. PCR positive rates were 47.8%, 71.3%, 75.0% and 100% in G1, G2, G3 and homozygous transgenic mice, respectively. The results of RT-PCR and immunohistochemical analysis showed human SR-A I specifically expressed on endothelial cells of aorta, liver and renal artery of transgenic mice. Plasma triglyceride level of transgenic mice was significantly higher than the level of non-transgenic mice (P<0.05 vs control). There was no difference in total plasma cholesterol level between transgenic and non-transgenic mice. But in transgenic mice, total plasma cholesterol level was significantly higher of males than of females (P<0.01). SEM of the luminal surface of aorta revealed an irregular, elevated bumpy and swelling surface. There was disruption of the endothelial layer and some blood cell was observed adhering to the surface of them in transgenic mice. TEM of aorta of transgenic mice showed that vesicles, multivesicle bodies and swelling mitochondria filled in plasma of endothelial cells. Vacuolar and mucoid degeneration were observed in the media of aortic ardh sections in transgenic mice with HE staining. Conclusions A transgenic mice model with overexpressed human SR-A on ECs were successfully established in this study. The transgene stable inherited across generation after generation. The high level of plasma lipid, hydropic and mucoid degeneration of arterial wall in transgenic mice may accelerate the development of atherosclerosis. Our studies provide a new transgenic model for investigation of the function and roles of SR-A in atherosclerosis.更多还原