摘要
Objectives To establish a new transgenic mouse model for determining the function and role of human scavenger receptor A (SR-A) in atherosclerosis in vivo.Methods Human scavenger receptor minigene-driven mouse tie-1 promoter was constructed and confirmed by endonuclease digestion and sequence analysis. Transgenic mice were generated via the microinjection method. PCR and Southern blot were used to screen the positive transgenic mice. RT-PCR and immunohistochemical analysis were used to detect the level and location of human SR-A Ⅰ expression in transgenic mice. The activity of human SR-A Ⅰ was determined by morphologic observation of aortic endothelial cells of transgenic mice under transmission electron microscopy.Results The electrophoresis assay showed the expected 4 fragments of 0.9 kb, 1.1 kb, 1.2 kb and 4.2 kb in the Sma Ⅰ digest and 2 fragments of 0.8 kb and 6.7 kb in Bgl Ⅱ digest of plasmids pTie-1/hSR-A. The fragment sequence of tie-1 promoter and human SR-A cDNA in plasmids pTie-1/hSR-A was correct and no ATG before the translation initiation sites of human SR-A was found by sequence analysis. 561 injected and surviving embryos with the purified human SR-A minigene were implanted into the oviducts of 19 ICR pseudopregnant mice. Among the 54 surviving pups from 13 foster mothers, 7 were identified by PCR and Southern blot analysis. The results of RT-PCR and imrnunohistochemical analysis showed human SR-A was specifically expressed on vascular endothelial cells of the aorta and renal artery, as well as hepatic sinusoidal endothelial cells in transgenic mice. Transmmion electron microscope (TEM) of aorta of transgenic mice showed that a large number of vesicles, multivesicle bodies and swollen mitochondria filled the plasma of endothelial cells.Conclusions A transgenic mouse model with overexpression of human SR-A in endothelial cells was successfully established. The transgene was integrated and transmitted into the chromosome of transgenic mice. Tie-1 promoter controlled the transgene to express in endothelial cells in mice. Pinocytic activity of aortic endothelial cells in transgenic mice was higher than that of C57BL/6J mice. Our studies will provide a new transcgenic model for investigation of atherosclerosis and functions of human SR-A.
目的 通过建立转基因鼠模型研究人A类清道夫受体 (hSR A)的功能及在动脉粥样硬化发生中的作用。方法 首先构建含鼠tie 1启动子和hSR AIcDNA的表达载体 (pTie/hSR A) ,通过酶切及测序鉴定后 ,利用显微注射的方法建立转基因鼠 ,PCR和Southernblot分析用于转基因鼠筛选 ,RT PCR和免疫组化方法用于检测hSR A在小鼠体内的表达水平及表达部位 ,电镜观察转基因鼠血管及其它组织的病理变化。结果 电泳结果显示pTie 1/hSR A质粒用SmaⅠ酶切得到 0 9、1 1、1 2和 4 3kb四条带 ,用BglⅡ酶切得到 0 8和6 7kb两条带 ,与预期的结果一致 ;序列分析进一步证明重组Tie 1/hSR A质粒中鼠tie 1启动子和hSR AIcDNA序列及插入方向正确。显微注射后存活的 5 61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%。 5只雄性转基因鼠的主动脉、肾、肝等组织中均有hSR A表达 ,且主要集中在血管的内皮细胞和肝窦内皮细胞上。透射电镜下 ,转基因鼠主动脉内皮细胞内含有大量的吞噬小泡和吞噬小体及肿胀的线粒体。结论 本研究已成功建立了鼠tie 1启动子驱动hSR A在血管内皮细胞特异性表达的转基因鼠 :转基因已完整整合到转基因鼠染色体上 ,tie
基金
ThisprojectwassupportedbytheNationalNaturalScienceFoundationofChina (No 3990 0 0 6 1)
