【摘要】：研究目的1. 建立穩(wěn)定的系統(tǒng)性紅斑狼瘡動(dòng)脈粥樣硬化動(dòng)物模型。2. 利用小鼠模型,觀察系統(tǒng)性紅斑狼瘡(Systemic Lupus Erythematosus,SLE)對(duì)動(dòng)脈粥樣硬化嚴(yán)重程度的影響。3. 進(jìn)一步觀察狼瘡動(dòng)脈粥樣硬化病變特點(diǎn),尤其是免疫系統(tǒng)在動(dòng)脈局部以及全身的改變,以及血脂變化特點(diǎn),初步探討狼瘡動(dòng)脈粥樣硬化發(fā)病的潛在機(jī)制。4. 研究常用的抗SLE藥物——羥氯喹(hydroxychloroquine, HCQ)對(duì)動(dòng)脈粥樣硬化的影響,并初步觀察其對(duì)血脂以及炎癥反應(yīng)的影響,為臨床上治療動(dòng)脈粥樣硬化提供新的選擇。研究方法1. 應(yīng)用apoE-/-以及C57BL/6小鼠,8周齡時(shí)通過(guò)腹腔內(nèi)一次性注射0.5ml pristane構(gòu)建SLE模型。部分小鼠通過(guò)飲水給予HCQ。2. 通過(guò)檢測(cè)自身抗體水平檢驗(yàn)狼瘡模型構(gòu)造是否成功,通過(guò)觀察動(dòng)脈粥樣硬化斑塊是否形成明確動(dòng)脈粥樣硬化模型構(gòu)建是否成功。3.6個(gè)月后檢測(cè)尿蛋白,處死小鼠。分離血清,檢測(cè)血脂、白蛋白、總IgG、干擾素a (interferon-α, IFN-α)。4. 分離小鼠主動(dòng)脈、心臟,進(jìn)行主動(dòng)脈大體油紅0染色,主動(dòng)脈流式細(xì)胞術(shù)檢測(cè)CD45+、CD19+、CD68+、CD11c+細(xì)胞,主動(dòng)脈瓣根部組織連續(xù)冰凍切片,進(jìn)行油紅0染色、IgG免疫熒光染色、CD19+、CD68+、CD11c+細(xì)胞免疫熒光染色。5. 制備脾細(xì)胞懸液,流式細(xì)胞術(shù)檢測(cè)CD3+、CD19+、CD11b+、CD11c+細(xì)胞比例。結(jié)果1. 模型構(gòu)建成功：注射了pristane的小鼠dsDNA抗體陽(yáng)性。服用HCQ導(dǎo)致注射了pristane的小鼠dsDNA抗體陽(yáng)性率降低。2. 小鼠外觀變化：部分狼瘡小鼠出現(xiàn)脫毛、皮膚出血,非狼瘡小鼠無(wú)上述現(xiàn)象。3. apoE-/-小鼠體重更重。4. 生化指標(biāo)：apoE-/-小鼠LDL-C、TC高于C57BL/6小鼠。高脂飼料喂養(yǎng)的小鼠LDL-C、TC高于普通飲食小鼠。狼瘡發(fā)病對(duì)LDL-C無(wú)影響,狼瘡小鼠TC低于非狼瘡小鼠。HCQ對(duì)LDL-C、TC水平無(wú)顯著性影響,輕度降低血脂。狼瘡小鼠Alb低于非狼瘡小鼠,羥氯喹對(duì)Alb水平無(wú)影響。5. 動(dòng)脈粥樣硬化斑塊進(jìn)展情況：狼瘡小鼠主動(dòng)脈斑塊面積大于非狼瘡小鼠,高脂飼料喂養(yǎng)小鼠斑塊面積多于普通飲食小鼠。至處死時(shí),只有apoE-/-小鼠主動(dòng)脈出現(xiàn)了斑塊,所有C57BL/6小鼠都沒(méi)有觀察到主動(dòng)脈斑塊。狼瘡小鼠主動(dòng)脈瓣根部斑塊面積大于非狼瘡小鼠,高脂飼料喂養(yǎng)的小鼠主動(dòng)脈瓣根部斑塊面積多于普通飲食小鼠。只有apoE-/-小鼠主動(dòng)脈瓣根部有斑塊,所有C57BL/6小鼠都沒(méi)有觀察到主動(dòng)脈瓣根部斑塊。狼瘡對(duì)主動(dòng)脈瓣根部斑塊的促進(jìn)作用在高脂飼料喂養(yǎng)的apoE-/-小鼠中更明顯。羥氯喹具有抑制主動(dòng)脈斑塊以及主動(dòng)脈瓣根部斑塊形成的作用。6. 斑塊成分特點(diǎn)：狼瘡發(fā)病導(dǎo)致主動(dòng)脈細(xì)胞增加,apoE-/-小鼠主動(dòng)脈細(xì)胞數(shù)目多于C57BL/6小鼠。使用羥氯喹減少主動(dòng)脈細(xì)胞數(shù)目。狼瘡小鼠主動(dòng)脈白細(xì)胞總數(shù)、樹(shù)突狀細(xì)胞、巨噬細(xì)胞數(shù)量增加,B細(xì)胞減少。羥氯喹降低狼瘡小鼠主動(dòng)脈白細(xì)胞、樹(shù)突狀細(xì)胞、巨噬細(xì)胞數(shù)目,增加狼瘡小鼠B細(xì)胞數(shù)目,對(duì)非狼瘡小鼠主動(dòng)脈細(xì)胞數(shù)目無(wú)影響。狼瘡小鼠CD68+巨噬細(xì)胞和CD11c+樹(shù)突狀細(xì)胞定位基本一致。大量IgG沉積在主動(dòng)脈瓣根部,B細(xì)胞也聚集在IgG沉積部位。狼瘡小鼠斑塊中IgG沉積增多,且更加質(zhì)密,高脂飼料喂養(yǎng)導(dǎo)致IgG沉積增多。羥氯喹顯著減少apoE-/-小鼠主動(dòng)脈斑塊IgG沉積。狼瘡小鼠血清IgG高于非狼瘡小鼠,高脂飼料喂養(yǎng)的小鼠血清IgG低于普通飲食小鼠,apoE-/-小鼠IgG低于C57BL/6小鼠。HCQ對(duì)血清IgG水平無(wú)影響。主動(dòng)脈斑塊IgG沉積與血清IgG含量呈低度負(fù)相關(guān)。7. 狼瘡發(fā)病、小鼠動(dòng)脈硬化易感的基因型導(dǎo)致脾臟指數(shù)增加,高脂飼料、羥氯喹對(duì)脾臟指數(shù)無(wú)影響。狼瘡小鼠脾臟淋巴細(xì)胞減少,高脂飼料也導(dǎo)致脾臟淋巴細(xì)胞減少。羥氯喹對(duì)小鼠脾臟B細(xì)胞比例無(wú)影響。羥氯喹增加狼瘡小鼠脾臟T細(xì)胞比例,對(duì)非狼瘡小鼠脾臟T細(xì)胞比例無(wú)影響。狼瘡小鼠脾臟CD11c+細(xì)胞增多,高脂飼料導(dǎo)致脾臟CD11c+細(xì)胞減少。羥氯喹對(duì)小鼠脾臟CD11c+細(xì)胞比例無(wú)顯著影響。狼瘡小鼠脾臟CD11b+細(xì)胞無(wú)顯著變化,羥氯喹對(duì)脾臟CD11b+細(xì)胞比例無(wú)影響。8. 狼瘡小鼠尿蛋白高于非狼瘡小鼠。HCQ不影響apoE-/-小鼠尿蛋白水平,但是降低C57BL/6小鼠尿蛋白水平。9. 個(gè)別狼瘡小鼠血清IFN-α升高,無(wú)統(tǒng)計(jì)學(xué)顯著性。結(jié)論1. apoE-/-、C57BL/6小鼠通過(guò)腹腔注射pristane可以成功誘導(dǎo)狼瘡疾病模型,2. apoE-/-小鼠容易出現(xiàn)主動(dòng)脈粥樣硬化斑塊,C57BL/6小鼠不出現(xiàn)明顯斑塊。3. 狼瘡小鼠動(dòng)脈粥樣硬化加重。4. 狼瘡小鼠的心血管傳統(tǒng)危險(xiǎn)因素(總膽固醇)降低,但是狼瘡小鼠主動(dòng)脈內(nèi)炎性反應(yīng)異常活躍(樹(shù)突狀細(xì)胞、巨噬細(xì)胞增多,B細(xì)胞減少,IgG沉積增多)；同時(shí)出現(xiàn)全身免疫狀態(tài)異常(脾臟淋巴細(xì)胞減少,樹(shù)突狀細(xì)胞增多,血清白蛋白降低,IgG升高)。5. 羥氯喹可以降低dsDNA抗體,可以減輕動(dòng)脈粥樣硬化。6. 羥氯喹對(duì)血脂無(wú)顯著性影響,可以部分逆轉(zhuǎn)狼瘡所致的動(dòng)脈粥樣硬化的斑塊成分異常以及脾臟細(xì)胞比例異常。7. 高脂飼料也可以通過(guò)升高血脂以及加重免疫系統(tǒng)異常從而加重動(dòng)脈粥樣硬化。8. 狼瘡小鼠血清IFN-α無(wú)顯著變化。
[Abstract]:Objective 1. to establish a stable atherosclerotic animal model of systemic lupus erythematosus.2. model, and to observe the effect of systemic lupus erythematosus (Systemic Lupus Erythematosus, SLE) on the severity of atherosclerosis..3. further observed the characteristics of atherosclerotic lesions in lupus, especially the immune system in the arterial part. As well as the changes in the body and the characteristics of blood lipid changes, the potential mechanism of atherosclerosis in lupus is preliminarily discussed. The effect of the common anti SLE drug hydroxychloroquine (hydroxychloroquine, HCQ) on atherosclerosis and its effect on blood lipid and inflammatory reaction are preliminarily observed in order to treat atherosclerosis in clinical. Provide a new choice. Study method 1. apoE-/- and C57BL/6 mice were used to construct a SLE model by intraperitoneal injection of 0.5ml pristane at 8 weeks of age. Some mice were given HCQ.2. by drinking water to test the success of the lupus model by testing the level of autoantibodies, by observing whether atherosclerotic plaques formed a definite movement. Whether the arteriosclerosis model was successfully constructed.3.6 months later, the urine protein was detected, the mice were executed, the mice were killed, the serum was separated, the blood lipid, albumin, total IgG, a (interferon- alpha, IFN- alpha).4. were isolated from the aorta, the heart, the aorta gross oil red 0, and the active pulse flow cytometry for the detection of CD45+, CD19+, CD68+, CD11c+ cells, and aortic valve The root tissue was frozen in continuous frozen section to perform oil red 0 staining, IgG immunofluorescence staining, CD19+, CD68+, CD11c+ cell immunofluorescence staining.5. to prepare splenocytes suspension. Flow cytometry was used to detect CD3+, CD19+, CD11b+, and CD11c+ cell ratio. Results the 1. model was constructed successfully: the mice injected with pristane were positive. The positive rate of dsDNA antibody in istane mice decreased the appearance of.2. mice: some lupus mice were depilatory, skin bleeding, non lupus mice had no above phenomena, and.3. apoE-/- mice weighed more.4. biochemical indexes: apoE-/- mice LDL-C, TC higher than C57BL/6 mice. The disease had no effect on LDL-C. The TC of lupus mice was lower than that of non lupus mice,.HCQ had no significant effect on LDL-C, TC level had no significant effect on the level of blood lipid. The Alb of lupus mice was lower than non lupus mice. The level of hydroxychloroquine on Alb level had no effect on the progression of atherosclerotic plaque in.5.: the area of atherosclerotic plaque in lupus mice was greater than that of non lupus mice and high fat feed feeding. The patch area of the mice was more than that of the normal diet mice. Only the aorta appeared in the apoE-/- mice at the time of death. All the C57BL/6 mice did not observe the aortic plaque. The area of the aortic valve root plaque in the lupus mice was greater than that of the non lupus mice, and the area of the root plaque of the main artery valve in the high fat diet mice was more than that of the normal diet mice. Only apoE-/- mice had plaque at the root of the aortic valve, and no plaque in the aortic valve root was observed in all C57BL/6 mice. The promoting effect of lupus on the aortic valve root plaque was more obvious in the apoE-/- mice fed by high fat diet. Hydroxychloroquine has the effect of inhibiting the atherosclerotic plaque of the aorta and the plaque formation of the main artery valve at the root of the aorta. Composition characteristics: the incidence of lupus caused the increase of aortic cells, and the number of aorta cells in apoE-/- mice was more than that of C57BL/6 mice. The number of aortic cells was reduced by hydroxychloroquine. The total number of leukocytes, dendritic cells, macrophages and B cells decreased in lupus mice. The hydroxychloroquine decreased the white blood cells and dendritic cells in lupus mice The number of macrophages and macrophages increased the number of B cells in lupus mice. There was no effect on the number of aortic cells in non lupus mice. The localization of CD68+ macrophages and CD11c+ dendritic cells in lupus mice was basically the same. A large number of IgG were deposited at the root of the aortic valve, and the B cells were also gathered at the IgG deposition position. The IgG deposition in lupus mice increased and increased. Dense, high fat feed led to the increase of IgG deposition. Hydroxychloroquine significantly reduced the IgG deposition in aortic plaque in apoE-/- mice. The serum IgG of lupus mice was higher than non lupus mice, the serum IgG of mice fed with high fat diet was lower than that of normal diet mice, and IgG in apoE-/- mice was less than.HCQ in C57BL/6 mice. The product and serum IgG content showed low negative correlation with.7. lupus, and the susceptible genotype of arteriosclerosis in mice resulted in the increase of spleen index. High fat diet, hydroxychloroquine had no effect on spleen index. The spleen lymphocyte of lupus mice decreased, and high fat diet also resulted in the decrease of spleen lymphocyte. Hydroxychloroquine had no effect on the proportion of spleen B cells in mice. The proportion of T cells in the spleen of lupus mice was increased by chloroquine. The proportion of spleen T cells in non lupus mice was not affected. The spleen CD11c+ cells in lupus mice increased and the high fat feed led to the decrease of spleen CD11c+ cells. The proportion of CD11c+ cells in spleen of mice was not significantly affected by hydroxychloroquine. The spleen CD11b+ cells in lupus mice were not significantly changed, and hydroxychloroquine was used to the spleen CD11b+. No effect of cell ratio on urinary protein of.8. lupus mice was higher than that of non lupus mice,.HCQ did not affect the level of urine protein in apoE-/- mice, but the level of IFN- alpha in serum of C57BL/6 mice was lower than that of apoE-/- mice, and there was no statistical significance. Conclusion 1. apoE-/-, C57BL/6 mice can successfully induce lupus disease through abdominal cavity injection of pristane. Models, 2. apoE-/- mice were prone to atherosclerotic atherosclerotic plaques, C57BL/6 mice did not have obvious plaque in.3. lupus mice and the cardiovascular traditional risk factors (total cholesterol) decreased, but the inflammatory reaction in the aorta of lupus mice was abnormally active (dendritic cells, macrophages increased, B thin). At the same time, the immune state of the spleen was decreased, the immune state of the spleen was decreased, the increase of dendritic cells, the decrease of serum albumin, the increase of serum albumin, and the increase of IgG..5. hydroxychloroquine could reduce the dsDNA antibody, which could reduce the unmarked effect of.6. hydroxychloroquine on the blood lipid, and could partly reverse the atherosclerotic artery atherosclerosis caused by lupus. Abnormal atherosclerotic plaque composition and abnormal proportion of spleen cells.7. high fat diet can also increase serum IFN- alpha in atherosclerotic.8. lupus mice by increasing blood lipid and aggravating immune system abnormalities.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R593.241;R543.5;R-332

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10 簡(jiǎn)丹;謝紅付;李吉;;羥氯喹治療輕中度酒糟鼻臨床療效觀察30例[A];中華醫(yī)學(xué)會(huì)第十八次全國(guó)皮膚性病學(xué)術(shù)年會(huì)論文匯編[C];2012年

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