红斑狼疮动脉粥样硬化小鼠模型建立、发病机制及羟氯喹干预研究
发布时间：2018-07-17 16:41
【摘要】：研究目的1. 建立稳定的系统性红斑狼疮动脉粥样硬化动物模型。2. 利用小鼠模型,观察系统性红斑狼疮(Systemic Lupus Erythematosus,SLE)对动脉粥样硬化严重程度的影响。3. 进一步观察狼疮动脉粥样硬化病变特点,尤其是免疫系统在动脉局部以及全身的改变,以及血脂变化特点,初步探讨狼疮动脉粥样硬化发病的潜在机制。4. 研究常用的抗SLE药物——羟氯喹(hydroxychloroquine, HCQ)对动脉粥样硬化的影响,并初步观察其对血脂以及炎症反应的影响,为临床上治疗动脉粥样硬化提供新的选择。研究方法1. 应用apoE-/-以及C57BL/6小鼠,8周龄时通过腹腔内一次性注射0.5ml pristane构建SLE模型。部分小鼠通过饮水给予HCQ。2. 通过检测自身抗体水平检验狼疮模型构造是否成功,通过观察动脉粥样硬化斑块是否形成明确动脉粥样硬化模型构建是否成功。3.6个月后检测尿蛋白,处死小鼠。分离血清,检测血脂、白蛋白、总IgG、干扰素a (interferon-α, IFN-α)。4. 分离小鼠主动脉、心脏,进行主动脉大体油红0染色,主动脉流式细胞术检测CD45+、CD19+、CD68+、CD11c+细胞,主动脉瓣根部组织连续冰冻切片,进行油红0染色、IgG免疫荧光染色、CD19+、CD68+、CD11c+细胞免疫荧光染色。5. 制备脾细胞悬液,流式细胞术检测CD3+、CD19+、CD11b+、CD11c+细胞比例。结果1. 模型构建成功：注射了pristane的小鼠dsDNA抗体阳性。服用HCQ导致注射了pristane的小鼠dsDNA抗体阳性率降低。2. 小鼠外观变化：部分狼疮小鼠出现脱毛、皮肤出血,非狼疮小鼠无上述现象。3. apoE-/-小鼠体重更重。4. 生化指标：apoE-/-小鼠LDL-C、TC高于C57BL/6小鼠。高脂饲料喂养的小鼠LDL-C、TC高于普通饮食小鼠。狼疮发病对LDL-C无影响,狼疮小鼠TC低于非狼疮小鼠。HCQ对LDL-C、TC水平无显著性影响,轻度降低血脂。狼疮小鼠Alb低于非狼疮小鼠,羟氯喹对Alb水平无影响。5. 动脉粥样硬化斑块进展情况：狼疮小鼠主动脉斑块面积大于非狼疮小鼠,高脂饲料喂养小鼠斑块面积多于普通饮食小鼠。至处死时,只有apoE-/-小鼠主动脉出现了斑块,所有C57BL/6小鼠都没有观察到主动脉斑块。狼疮小鼠主动脉瓣根部斑块面积大于非狼疮小鼠,高脂饲料喂养的小鼠主动脉瓣根部斑块面积多于普通饮食小鼠。只有apoE-/-小鼠主动脉瓣根部有斑块,所有C57BL/6小鼠都没有观察到主动脉瓣根部斑块。狼疮对主动脉瓣根部斑块的促进作用在高脂饲料喂养的apoE-/-小鼠中更明显。羟氯喹具有抑制主动脉斑块以及主动脉瓣根部斑块形成的作用。6. 斑块成分特点：狼疮发病导致主动脉细胞增加,apoE-/-小鼠主动脉细胞数目多于C57BL/6小鼠。使用羟氯喹减少主动脉细胞数目。狼疮小鼠主动脉白细胞总数、树突状细胞、巨噬细胞数量增加,B细胞减少。羟氯喹降低狼疮小鼠主动脉白细胞、树突状细胞、巨噬细胞数目,增加狼疮小鼠B细胞数目,对非狼疮小鼠主动脉细胞数目无影响。狼疮小鼠CD68+巨噬细胞和CD11c+树突状细胞定位基本一致。大量IgG沉积在主动脉瓣根部,B细胞也聚集在IgG沉积部位。狼疮小鼠斑块中IgG沉积增多,且更加质密,高脂饲料喂养导致IgG沉积增多。羟氯喹显著减少apoE-/-小鼠主动脉斑块IgG沉积。狼疮小鼠血清IgG高于非狼疮小鼠,高脂饲料喂养的小鼠血清IgG低于普通饮食小鼠,apoE-/-小鼠IgG低于C57BL/6小鼠。HCQ对血清IgG水平无影响。主动脉斑块IgG沉积与血清IgG含量呈低度负相关。7. 狼疮发病、小鼠动脉硬化易感的基因型导致脾脏指数增加,高脂饲料、羟氯喹对脾脏指数无影响。狼疮小鼠脾脏淋巴细胞减少,高脂饲料也导致脾脏淋巴细胞减少。羟氯喹对小鼠脾脏B细胞比例无影响。羟氯喹增加狼疮小鼠脾脏T细胞比例,对非狼疮小鼠脾脏T细胞比例无影响。狼疮小鼠脾脏CD11c+细胞增多,高脂饲料导致脾脏CD11c+细胞减少。羟氯喹对小鼠脾脏CD11c+细胞比例无显著影响。狼疮小鼠脾脏CD11b+细胞无显著变化,羟氯喹对脾脏CD11b+细胞比例无影响。8. 狼疮小鼠尿蛋白高于非狼疮小鼠。HCQ不影响apoE-/-小鼠尿蛋白水平,但是降低C57BL/6小鼠尿蛋白水平。9. 个别狼疮小鼠血清IFN-α升高,无统计学显著性。结论1. apoE-/-、C57BL/6小鼠通过腹腔注射pristane可以成功诱导狼疮疾病模型,2. apoE-/-小鼠容易出现主动脉粥样硬化斑块,C57BL/6小鼠不出现明显斑块。3. 狼疮小鼠动脉粥样硬化加重。4. 狼疮小鼠的心血管传统危险因素(总胆固醇)降低,但是狼疮小鼠主动脉内炎性反应异常活跃(树突状细胞、巨噬细胞增多,B细胞减少,IgG沉积增多)；同时出现全身免疫状态异常(脾脏淋巴细胞减少,树突状细胞增多,血清白蛋白降低,IgG升高)。5. 羟氯喹可以降低dsDNA抗体,可以减轻动脉粥样硬化。6. 羟氯喹对血脂无显著性影响,可以部分逆转狼疮所致的动脉粥样硬化的斑块成分异常以及脾脏细胞比例异常。7. 高脂饲料也可以通过升高血脂以及加重免疫系统异常从而加重动脉粥样硬化。8. 狼疮小鼠血清IFN-α无显著变化。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R593.241;R543.5;R-332
本文编号：2130271
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R593.241;R543.5;R-332
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