［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AI cDNA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-BGH poly A片段注入受精卵，将注射后存活的受精卵移入IC R假孕母鼠，产下的仔鼠经聚合酶链反应和Southern blot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AI cDNA序列正确，显微注射后存活的 561枚受 精卵分别移入19只ICR假孕母鼠，有13只受孕，共产下56只仔鼠，存活54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 100%。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 ；血浆甘油三酯水平明显高于非C57BL/6鼠（P＜0.05），总胆固醇水平虽 不比C57BL/6高，但雄 性鼠总胆固醇水平明显高于雌性（P＜0.01）。这些结果提示，已成功建立 了鼠tie-1启动子 驱动人清道夫受体AI在血管内皮细胞特异性表达的转基因鼠。外源基因能在子代鼠中稳定遗 传。转 基因鼠血管出现内膜水样变性及中膜粘液样变性等动脉粥样硬化的早期病理改变，表明人 清道夫受体AI转基因鼠可能易感动脉粥样硬化，成为新的动脉粥样硬化模型。

［文章编号］1007-3949(2000)-01-0005-08

Genetics Stability and Expression Specificity of Human Scavenger Receptor -A I o n Endothelial Cells in Transgenic Mice

WU Meng-JinWAN Zai-YangWAN La-XiangYANG Yong-Zong

（Researching Center of Molecular Biology, Hengyang Medical College, Hengyang 421001, China）

Sookja Kim CHUNGStephen S.M.CHUNG

（Institute Of Molecular Biology, University of Hong Kong, Hon g Kong, China）

CAO De-Liang

（Department of Pharmacology, School of Medicine, New Haven, Con necticut 06510,USA）

CHEN Xiu

（Department of Pharmacology, Hunan Medical University, Changsha 410078, China)

ABSRACTAimTo establish a new transgenic mice model for determi ning the function and roles of human scavenger receptor A in atherosclerosis in vivo.Methods Human scavenger receptor A-I minige ne-driven tie-1 promoter was constr ucted by endonuclease digestion and confirmed by sequence analysis. Transgenic m ice were produced via microinjection method. PCR, Southern blot were used to s creen the positive transgenic mice. Transgenic mice line established by mating with C57BL/6 mice and offspring were identified by P CR so as to research transgenic mice transmission.Northern blot, RT-PCR, immun oh istochemical analysis, light and transmission electron microscopy were used to i nvestigate the expression location of human SR-A and lesions of arteries and vas cular endothelial cells in transgenic mice.Results The fragment sequence of mousetie-1 promoter and human SR-AI cDNA are similar to their sequences in Genebank and no ATG before the translation initia tion sites of human SR-A by sequence analysis. About 3.4 kb tie-1-promoter-hS R- AI-cDNA-BGH polyA fragment were obtained by AatⅡand XhoⅠdigests. 561 survival embryos injected with purified human SR-A minigene were implanted into the ovidu cts of 19 ICR pseudopregnant mice.Among the 54 survived pups from 13 foster m ot hers, 7 founders were identified with PCR and Southern blot analysis. Integrat io n rate of exogenous was 13%.PCR positive rates were 47.8%, 71.3%, 75.0% and 10 0% 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 tr iglyceride level of transgenic mice was significantly higher than the level of n on-transgenic mice (P＜0.05 vs control). There was no differenc e in total plasm a cholesterol level between transgenic and non-transgenic mice. But in transge ni c mice, total plasma cholesterol level was significantly higher of males than of females (P＜0.01). SEM of the luminal surface of aorta reveal ed an irregular, elevated bumpy and swelling surface. There was disruption of the endothelial la yer and some blood cell was observed adhering to the surface of them in transgen ic mice. TEM of aorta of transgenic mice showed that vesicles, multivesicle bo di es and swelling mitochondria filled in plasma of endothelial cells. Vacuolar a nd mucoid degeneration were observed in the media of aortic ardh sections in trans genic mice with HE staining.Conclusions A transgenic mice model with overexpressed human SR-A on ECs were s uccessfully established in this study. The transgene stable inherited across g en eration after generation.The high level of plasma lipid, hydropic and mucoid d eg eneration 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.

MeSHReceptor， Scavenger； Mice, Transgenic；Genetics； Ge ne Expression;Endothelium,Vascular；Atherosclerosis

清道夫受体A（scavenger receptor A,SR-A）是一类三聚体糖蛋白，其配体非常广泛，能结合和内化多种多聚阴离子化合物如修饰脂蛋白、马来酰牛血清白蛋白、多聚次黄苷酸、多糖、某些磷脂等生物大分子，可能参与机体的防御、细胞粘附和信号转导等多种生理及病理过程^［1-3］。由于清道夫受体不受细胞内游离胆固醇的反馈调节，巨噬细胞能不断摄取修饰脂蛋白，致使胆固醇酯在细胞内聚集，形成泡沫细胞。研究发现，在人类动脉粥样硬化不同阶段的斑块中，尤其是早期斑块的巨噬细胞上SR-A活性增强^［4］，提示SR-A在动脉粥样硬化（ath erosclerosis, As）斑块中泡沫细胞形成过程中可能起着重要作用。

清道夫受体(SR－A)有Ⅰ和Ⅱ型两种异构体，二者由同一基因编码，但剪切方式不同。牛、鼠、兔、人的Ⅰ、Ⅱ型SR的cDNA序列已被克隆，两型SR-A的氨基酸序列及蛋白质结构均已基本查清^［5］，两型之间的主要区别在于Ⅱ型缺乏富含半胱氨酸的Ⅵ区，而被6～17个C－末端氨基酸残基所取代，但二者有相似的配体结合活性。目前，对SR-A功能的了解多为体外研究结果，或源于对小鼠和牛SR-A的研究^［6，7］，为了进一步探讨人SR-A在体内的功能及在AS斑块形成中的作用，本研究利用tie-1基因启动子的血管内皮细胞组织特异性，成功地建立了能在血管内皮细胞特异表达人SR-AI的转基因鼠，高表达人SR-AI的血管内皮细胞吞饮作用增强，形态结构也发生明显改变。

1材料和方法

1.1材料

1.1.1试剂AatⅡ、XhoⅠ、NruⅠ、ApaⅠ、AflⅡ、BglⅡ、SmaⅠ等限制性内切酶及1kb和100bpDNA标准分子质量等购自New England Biolabs Inc.; SDS 和Tri s碱购自Serva公司；蛋白酶K、琼脂糖、dNTP、随机引物标记试剂盒、逆转录试剂盒等购自Promega公司；RNA抽提试剂盒购自Qiagene公司；测序试剂盒购自PE公司；α-³²P-CTP购自北京亚辉生物医学工程公司。引物均由上海生工合成。抗人SR-A的单克隆抗体由日本T.Kodama教授制备并赠送.

1.1.2质粒含有人SR-AcDNA全长的pXhSR1质粒由日本T.Kodama教授制备并赠送。含小鼠tie-1启动子DNA的pTie/Sma质粒由荷兰K.Alitalo教授构建、香港大学分子生物学研究所保存。

1.1.3实验动物F1(来源于C57BL/6×DBA)、ICR假孕母鼠由香港大学实验动物部提供，C57BL/6小鼠由香港大学实验动物部和协和医科大学实验动物繁育场提供。所有动物均喂饲常规颗粒饲料，自由饮水。超排卵F1与C57BL/6小鼠交配提供显微注射用受精卵。

1.2Tie/hSR-AI表达载体的构建及鉴定

<