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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?
In order to elucidate the function of human scavenger receptor AI (hSR-AI) and its role in the development of atherosclerosis, we first constructed the expression vector containing mouse tie-1 promoter and human scavenger receptor AIcDNA. After digestion and sequencing, the prepared tie - 1 - hSR - A - BGHpolyA fragment was injected into the fertilized egg by microinjection, and the fertilized eggs after the injection were transferred into the ICR fake pregnant rats and the offspring The positive transgenic mice were screened by polymerase chain reaction and Southern blot analysis. The RNA of the scavenger receptor was detected by reverse transcriptase-polymerase chain reaction and immunohistochemical staining. The expression level and expression site of the mice in vivo were observed. The pathological changes in the blood vessels and other tissues of the transgenic mice were observed under light microscope and electron microscope. The results showed that the sequence of AIcDNA of tie - 1 promoter and human scavenger receptor in the recombinant Tie - 1 / hSR - AI plasmid was correct. After the microinjection, 561 fertilized eggs were transferred into 19 ICR females , There are 13 conception, a total of 5,6 off-generation offspring, survival 54, after integration testing, detection of seven positive mice, the integration efficiency of 13%. The positive rates of PCR in G1, G2, G3 generation and homozygous transgenic mice were 47.8%, 71.3%, 75.0% and 100% respectively. Five male transgenic mice showed scavenger receptor AI expression in the aorta, kidney, liver and other tissues, and mainly concentrated on the vascular endothelial cells. The endothelial cells of transplanted aorta showed obvious edema, Erosion samples change, there are more blisters in the cytoplasm, the membrane has focal lesions of glycogen deposition, smooth muscle cells also have edema;