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

本研究制作了plivLE6-hCRP构建体，将人c反应蛋白（h-CRP） cDNA插入plivHEG/LE6载体中，特异表达肝脏中高含量的h-CRP，并采用微量注射法将其注射到兔受精卵原核中。经显微注射后，将453个胚胎移植到经hCG诱导假妊娠的养兔输卵管内，共产仔兔47只。PCR分析显示，47只家兔中有5只引入了转基因，2只存活至断奶的家兔（C205系和C215系）血浆中表达了h-CRP。对C205和C215的每个菌落进行扩增后，我们检测了h-CRP在每个转基因系中的表达水平，我们证实C205和C215分别为7.3±1.0和122.0±13.4 mg/dL。在血浆总胆固醇、高密度脂蛋白胆固醇和甘油三酯等血脂指标的比较中，转基因家兔与非转基因家兔无显著差异。为了分析动脉粥样硬化的诱导机制，分别饲喂转基因和非转基因家兔高胆固醇饲料16周。每周测定血浆胆固醇浓度，通过改变饮食中胆固醇含量（0.075 ~ 0.3%）调整至两组相同水平。胆固醇负荷已完成，我们采集了所有家兔的主动脉和心脏进行病理分析。目前正在进行更详细的组织病理分析，准备稿件。

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.