Analysis of the induction mechanisms of atherosclerosis using C-reactive protein transgenic rabbits

在本研究中，我们将人C反应蛋白(h-CRP)基因插入plivHEG/LE6载体中，构建了pLIVLE6-hCRP载体，并将其注射到兔受精卵原核中，在肝脏中特异性表达高水平的h-CRP。经显微注射后，将453枚胚胎移植到hCG诱导假孕的寄养兔输卵管内，共产仔47只。结果表明，47只仔兔中有5只在血浆中表达了h-CRP，其中2只(品系C205和C215)存活至断奶。对C205和C215分别进行菌落扩增后，检测h-C反应蛋白在各转基因株系中的表达水平，证实C205和C215分别为7.30±1.0和122.0±13.4 mg/dL。比较转基因兔和非转基因兔的血脂谱如总胆固醇、高密度脂蛋白胆固醇和甘油三酯，转基因和非转基因兔之间没有差异。为了分析动脉粥样硬化的诱导机制，转基因和非转基因兔饲喂高胆固醇饲料16周。每周测量一次血浆胆固醇浓度，并通过改变饲料中胆固醇的含量(0.075-0.3%)调整到两组之间相同的水平。胆固醇负荷已经完成，我们收集了所有兔的主动脉和心脏进行病理分析。目前，正在为手稿的准备进行更详细的组织病理学分析。

In this study, we made plivLE6-hCRP construct which inserted human C-reactive protein (h-CRP) cDNA in plivHEG/LE6 vector to express high h-CRP in liver specifically, and it was injected into rabbit fertilized egg pronuclei by microinjection method. After microinjection, 453 embryos were transplanted in fallopian tubes of the foster rabbits induced pseudopregnancy by hCG injection and they gave birth to 47 pups in all. PCR analysis revealed transgene was introduced in 5 of 47 pups, and we confirmed h-CRP expression in plasma of 2 rabbits (Line C205 and C215) survived until weaning. After expand of the each colony of C205 and C215, we examined the expression level of h-CRP in each transgenic line, and we confirmed that C205 and C215 showed 7.3±1.0 and 122.0±13.4 mg/dL, respectively. In comparison of plasma lipid profile such as total cholesterol, HDL-cholesterol and triglyceride, there were no differences between transgenic and non-transgenic rabbits.For analysis of the induction mechanisms of atherosclerosis, transgenic and non-transgenic rabbits were fed cholesterol-rich diet for 16 weeks. The concentration of plasma cholesterol was measured weekly and adjusted to same level between 2 groups by changing the amount of cholesterol in the diet (0.075-0.3%). Cholesterol loading was finished already, and we have collected aortas and hearts from all rabbits for pathological analysis. At present, more detailed histopathological analysis is doing for preparation of manuscript.