论文部分内容阅读
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 1启动
Objective To study the function of human scavenger receptor A (hSR A) and its role in the development of atherosclerosis by establishing a transgenic mouse model. Methods The expression vector (pTie / hSR A) containing mouse tie 1 promoter and hSR AIcDNA was constructed. After digestion and sequencing, the transgenic mice were established by microinjection. PCR and Southern blot were used to screen transgenic mice. RT-PCR and immunohistochemistry were used to detect the expression and expression of hSR A in mice. The pathological changes of blood vessels and other tissues in transgenic mice were observed by electron microscopy. Results The results of electrophoresis showed that the pTie 1 / hSR A plasmids were digested with SmaI to obtain four bands of 0 9,1 1,1 2 and 4 3 kb. The two bands of 0 8 and 67 kb were digested with Bgl Ⅱ, which was consistent with the expected results. The sequence The analysis further confirmed that the Tie 1 / hSR A plasmid in mouse Tie 1 promoter and hSR AI cDNA sequence and insertion direction correct. After the microinjection, 5 61 fertilized eggs were transferred into 19 ICR fake pregnant rats, 13 of which were conceived. A total of 56 offspring were co-produced and 54 survived. After the integration test, 7 positive mice were detected. The integration efficiency is 13%. The expression of hSR A in the aorta, kidney, liver and other tissues of 5 male transgenic mice was mainly concentrated in the vascular endothelial cells and sinusoidal endothelial cells. Under the transmission electron microscope, a large number of phagocytic vesicles, phagosomes and swollen mitochondria were found in the aorta endothelial cells of transgenic mice. Conclusion This study has successfully established a mouse transgenic mice that mouse tie-1 promoter drives hSR A-specific expression in vascular endothelial cells: the transgene has been integrated into the transgenic mouse chromosome, tie 1 start