本文選題：Apoe + Ldlr��； 參考：《華東師范大學(xué)》2016年碩士論文

【摘要】：Apo和Lddlr基因敲除小鼠均是研究動脈粥樣硬化癥常用模型,任一基因的敲除均能使小鼠表現(xiàn)出血管斑塊形成,高膽固醇血癥以及嚴重的慢性炎癥反應(yīng)。大鼠相比小鼠具有脂代謝和基因組同源性更接近人類,取樣簡便,易長期實時監(jiān)控等優(yōu)勢,并且有關(guān)Apoe,Ldlr基因敲除大鼠模型中免疫調(diào)控功能的系統(tǒng)評價并未報道。本實驗室前期已經(jīng)利用CRISPR技術(shù)成功建立了Apoe,Ldlr單基因敲除以及雙基因敲除大鼠模型。由于在人類動脈粥樣硬化炎癥反應(yīng)中內(nèi)皮下巨噬細胞在起著關(guān)鍵作用,且在小鼠研究中發(fā)現(xiàn)Apoe,Ldlr影響巨噬細胞及前體單核細胞不同方面的功能,因此本課題旨在不同的喂養(yǎng)條件下,研究基因敲除對大鼠巨噬細胞功能造成的影響,進一步理清這兩個基因?qū)奘杉毎恼{(diào)節(jié)作用及其在動脈粥樣硬化發(fā)生發(fā)展的功能,為針對性研究參與動脈粥樣硬化疾病復(fù)雜炎癥反應(yīng)中選擇最佳動脈粥樣硬化癥大鼠模型提供理論依據(jù)。實驗結(jié)果顯示無論是普通飼料還是高膽固醇飼料喂養(yǎng)條件下,相比于野生型大鼠,Apoe~(-/-),L lr~(-/-)和Apoe~(-/-)Ldlr~(-/-)大鼠血液中總膽固醇及低密度脂蛋白(LDL)明顯上調(diào),而氧化低密度脂蛋白(ox-LDL)則并無差異。脾臟臟器系數(shù)則顯示只有在高膽固醇飼料喂養(yǎng)組中Ldlr~(-/-)大鼠相比野生型明顯上調(diào),但此時Apoe~(-/-)和LdLdr/-/脾臟中巨噬細胞比例均有下調(diào),外周血中的巨噬細胞比例則沒有明顯變化。隨后我們進一步檢測了動脈粥樣硬化病程密切相關(guān)的巨噬細胞功能,發(fā)現(xiàn)Apoe~(-/-)大鼠單核細胞遷移能力明顯強于野生型和Ldlr~(-/-)及Apoe~(-/-)Ldlr~(-/-)。Ldlr~(-/-)在高膽固醇喂養(yǎng)組中凋亡細胞數(shù)量上遠遠超過其余三組,并且表現(xiàn)出膽固醇溶出障礙。巨噬細胞的吞噬能力相比于野生型也表現(xiàn)為Ldlr~(-/-)組最強。另外我們發(fā)現(xiàn)影響吞噬的基因CD36和SR-A1的表達并未表現(xiàn)出與小鼠上的報道一致的相關(guān)性。Apoe,Lddlr雙敲除大鼠總體在以上結(jié)果中表現(xiàn)為傾向介于單敲除大鼠之間的水平,并沒有顯示疊加效應(yīng)。綜上所述,我們首次在相同遺傳背景的Apoe,Ldlr基因敲除大鼠中系統(tǒng)分析比較了巨噬細胞數(shù)量和功能的變化,發(fā)現(xiàn)Apoe,Ldlr基因敲除引起大鼠體內(nèi)炎癥程度上升但具體影響巨噬細胞的不同功能,如Apoe~(-/-)大鼠主要影響單核細胞遷移,Ldlr~(-/-)則表現(xiàn)出細胞凋亡增強及膽固醇溶出障礙等變化。巨噬細胞這些功能的變化與慢性炎癥的惡化息息相關(guān),在大鼠動脈粥樣硬化的發(fā)生發(fā)展中起著促進作用。本文結(jié)果也表明,利用AS模型進行不同慢性炎癥或免疫調(diào)節(jié)機制的研究應(yīng)根據(jù)具體需要選取不同的模型。
[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.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R543.5

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