Apoe和Ldlr基因敲除对大鼠巨噬细胞炎症，免疫功能及脂质溶出功能的影响

发布时间：2018-05-20 15:53

本文选题：Apoe + Ldlr　；参考：《华东师范大学》2016年硕士论文

【摘要】：Apo和Lddlr基因敲除小鼠均是研究动脉粥样硬化症常用模型,任一基因的敲除均能使小鼠表现出血管斑块形成,高胆固醇血症以及严重的慢性炎症反应。大鼠相比小鼠具有脂代谢和基因组同源性更接近人类,取样简便,易长期实时监控等优势,并且有关Apoe,Ldlr基因敲除大鼠模型中免疫调控功能的系统评价并未报道。本实验室前期已经利用CRISPR技术成功建立了Apoe,Ldlr单基因敲除以及双基因敲除大鼠模型。由于在人类动脉粥样硬化炎症反应中内皮下巨噬细胞在起着关键作用,且在小鼠研究中发现Apoe,Ldlr影响巨噬细胞及前体单核细胞不同方面的功能,因此本课题旨在不同的喂养条件下,研究基因敲除对大鼠巨噬细胞功能造成的影响,进一步理清这两个基因对巨噬细胞的调节作用及其在动脉粥样硬化发生发展的功能,为针对性研究参与动脉粥样硬化疾病复杂炎症反应中选择最佳动脉粥样硬化症大鼠模型提供理论依据。实验结果显示无论是普通饲料还是高胆固醇饲料喂养条件下,相比于野生型大鼠,Apoe~(-/-),L lr~(-/-)和Apoe~(-/-)Ldlr~(-/-)大鼠血液中总胆固醇及低密度脂蛋白(LDL)明显上调,而氧化低密度脂蛋白(ox-LDL)则并无差异。脾脏脏器系数则显示只有在高胆固醇饲料喂养组中Ldlr~(-/-)大鼠相比野生型明显上调,但此时Apoe~(-/-)和LdLdr/-/脾脏中巨噬细胞比例均有下调,外周血中的巨噬细胞比例则没有明显变化。随后我们进一步检测了动脉粥样硬化病程密切相关的巨噬细胞功能,发现Apoe~(-/-)大鼠单核细胞迁移能力明显强于野生型和Ldlr~(-/-)及Apoe~(-/-)Ldlr~(-/-)。Ldlr~(-/-)在高胆固醇喂养组中凋亡细胞数量上远远超过其余三组,并且表现出胆固醇溶出障碍。巨噬细胞的吞噬能力相比于野生型也表现为Ldlr~(-/-)组最强。另外我们发现影响吞噬的基因CD36和SR-A1的表达并未表现出与小鼠上的报道一致的相关性。Apoe,Lddlr双敲除大鼠总体在以上结果中表现为倾向介于单敲除大鼠之间的水平,并没有显示叠加效应。综上所述,我们首次在相同遗传背景的Apoe,Ldlr基因敲除大鼠中系统分析比较了巨噬细胞数量和功能的变化,发现Apoe,Ldlr基因敲除引起大鼠体内炎症程度上升但具体影响巨噬细胞的不同功能,如Apoe~(-/-)大鼠主要影响单核细胞迁移,Ldlr~(-/-)则表现出细胞凋亡增强及胆固醇溶出障碍等变化。巨噬细胞这些功能的变化与慢性炎症的恶化息息相关,在大鼠动脉粥样硬化的发生发展中起着促进作用。本文结果也表明,利用AS模型进行不同慢性炎症或免疫调节机制的研究应根据具体需要选取不同的模型。
[Abstract]:Both Apo and Lddlr knockout mice are commonly used to study atherosclerosis. Knockout of either gene can induce plaque formation hypercholesterolemia and severe chronic inflammation in mice. Compared with mice, the lipid metabolism and genomic homology of rats are more similar to humans, the sampling is simple and easy to monitor in real time for a long time, and the systematic evaluation of immune regulation in the rat model of Ldlr gene knockout (Ldlr gene knockout) has not been reported. The rat models of single gene knockout and double gene knockout have been successfully established by CRISPR in our laboratory. As endodermic macrophages play a key role in the inflammatory response to human atherosclerosis, and in mouse studies, it has been found that Apoetin Ldlr affects the functions of macrophages and precursor monocytes in different ways. The purpose of this study was to investigate the effects of gene knockout on macrophage function in rats under different feeding conditions, and to further clarify the regulatory effects of these two genes on macrophages and their role in the development of atherosclerosis. To provide theoretical basis for selecting the best rat model of atherosclerosis in the complex inflammatory reaction of atherosclerosis. The results showed that the total cholesterol and low density lipoprotein (LDL) in the blood of rats fed with normal diet or high cholesterol diet were significantly up-regulated than those of wild type rats (Apoeophane) and Apoestrin / r-Ldlr-r-r / -, but no difference was found in oxidized low density lipoprotein ox-LDL (ox-LDL) in the blood compared with those in the wild type rats and the rats fed with Apoetin or high cholesterol diet significantly increased the total cholesterol and low density lipoprotein (LDL) in the blood compared with those in the wild type rats. The spleen organ coefficient showed that the ratio of macrophages in the spleen of Ldlrr-r / r-/ -) rats was significantly up-regulated than that of the wild type rats, but the ratio of macrophages in the spleen of LdLdr/-/ and Apoeto-r-r -) was down-regulated, while the percentage of macrophages in peripheral blood had no significant change. Then we further examined the macrophage function, which is closely related to the course of atherosclerosis. It was found that the migration ability of monocytes in Apoetin / r /-() rats was significantly stronger than that in wild type and Ldlrr-r-r / -) and Apoestrin -r / -r-P. Ldlrrrr-r-% -r-lr-% -r-% -r--% -r-P -P -P -P The phagocytic ability of macrophages was also stronger than that of wild type Ldlrr-r-r group. In addition, we found that the expression of CD36 and SR-A1, the genes affecting phagocytosis, did not show the same correlation with the reports in mice. There is no superposition effect. In conclusion, for the first time, we systematically compared the number and function of macrophages in the Ldlr knockout rats with the same genetic background. It was found that Ldlr gene knockout induced the increase of inflammatory degree but affected the different functions of macrophages in rats. For example, the Ldlrr-r-% of monocytes mainly affected the migration of monocytes in rats, but the apoptosis increased and cholesterol dissolving disorder was observed in the Ldlr gene knockout rats. These changes of macrophages are closely related to the deterioration of chronic inflammation and play an important role in the development of atherosclerosis in rats. The results also show that different models should be selected according to the specific needs for the study of different chronic inflammation or immunomodulation mechanisms using as model.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R543.5

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