地高辛拮抗RORγt活性减缓实验性腹主动脉瘤进展的研究

发布时间：2018-07-04 19:55

本文选题：IL-17A + ROγt　；参考：《华中科技大学》2015年博士论文

【摘要】：第一部分人腹主动脉瘤病变组织中IL-17A, RORyt蛋白表达的研究目的通过收集腹主动脉瘤患者病变组织标本与正常腹主动脉组织标本,研究IL-17A、RORyt的蛋白表达差异,为实验性腹主动脉瘤模型的研究提供依据。方法收取人腹主动脉瘤病变组织标本以及人正常腹主动脉组织标本各10例,分别分成腹主动脉瘤组(Abdominal Aortic Aneurysm, AAA鍚以及对照组(Control组),对两组标本分别进行组织学以及Western Blot方法研究。通过弹性纤维EVG染色、免疫组化(CD4、Mac2、CD31、α-SM actin)染色研究腹主动脉瘤的病理学改变。通过Western Blot法对两组标本的1L-17A以及RORyt的蛋白表达进行研究。结果组织学研究表明腹主动脉瘤病变组织中发生了明显的弹性纤维断裂、降解,中层平滑肌细胞凋亡,外膜新生毛细血管增生,管壁炎性细胞(CD4+T细胞,Mac2+巨噬细胞)浸润。同时,Western Blot分析表明AAA组中1L-17A、RORyt的蛋白表达较Control组明显增加,两者差异均具有统计学意义(P0.05)。结论目前关于IL-17A在腹主动脉瘤瘤中的作用是不明确的,我们通过研究结果表明IL-17A、RORyt蛋白水平的表达在腹主动脉瘤组织中明显增高,炎性细胞大量浸润,推测IL-17A对腹主动脉瘤的发生发展可能起到促进作用,为后续动物实验的开展提供了理论依据。第二部分地高辛对血管紧张素II诱导的ApoE-/-小鼠实验性腹主动脉瘤模型保护作用的研究目的探讨地高辛药物干预对血管紧张素II诱导的ApoE-/-小鼠实验性腹主动脉瘤模型的作用及其机制。方法常规建立血管紧张素II经Alzet微量渗透泵皮下置入法诱导的ApoE-/-小鼠实验性腹主动脉瘤模型。80只16周龄的雄性ApoE-小鼠随机分为假手术组(Sham组)、对照组(Control组)、低剂量地高辛干预组(Low-dose组)、高剂量地高辛干预组(High-dose组),每组均20只实验鼠。其中Sham组由生理盐水皮下微量泵入并给予同等量的0.5%DMSO溶液腹腔注射治疗；Control组由血管紧张素Ⅱ皮下微量泵入并给予同等量的0.5%DMSO溶液腹腔注射治疗；Low-dose组由血管紧张素Ⅱ皮下微量泵入并给予同等量的0.5%DMSO+Digoxin(20μg/只.天)混合溶液腹腔注射治疗；High-dose组由血管紧张素Ⅱ皮下微量泵入并给予同等量的0.5%DMSO+Digoxin(40μg/只.天)混合溶液腹腔注射治疗。各组小鼠分别于术后第0、7、14、21以及28天对小鼠进行腹主动脉直径超声测量,评估腹主动脉瘤形成情况。术后第0、28天采用尾夹测压法测量小鼠收缩压,评估血管紧张素II对血压的影响。术后第28天收取小鼠腹主动脉,进行HE、EVG、免疫组化(α-SM actin、CD31、CD4以及Mac2)、免疫荧光染色(anti-CD4、IL-17A)等组织学分析、小鼠脾细胞流式细胞仪分析、Western blot蛋白检测炎性相关蛋白(IL-17A,、RORγt、MCP-1、IFN-γ、RANTES以及MMP-2、MMP-9)的表达。结果(1)与对照组相比,高剂量地高辛干预显著的降低了腹主动脉瘤的形成率(35%vs70%,P0.05)并且提高了小鼠存活率。(2)地高辛干预对术后第28天的小鼠血压并无明显影响。(3)与对照组比较,高剂量地高辛干预明显地保护了血管壁结构。(4)与对照组比较,高剂量地高辛干预明显地减轻了腹主动脉组织中炎性细胞(CD4+T细胞,巨噬细胞)的浸润。(5)与对照组比较,高剂量地高辛干预明显地抑制了促炎因子及MMPs的蛋白表达。(6)与对照组比较,高剂量地高辛干预改变了小鼠脾细胞中Th17细胞、调节性T细胞的组成。结论地高辛干预(尤其是高剂量干预)可以有效的抑制炎性因子的表达以及炎性细胞浸润,有效地保护血管正常组织,减缓实验性腹主动脉瘤的发展。第三部分地高辛对猪胰弹性蛋白酶诱导的C57BL/6J小鼠实验性腹主动脉瘤模型保护作用的研究目的探讨地高辛干预对另一种由猪胰弹性蛋白酶诱导的C57BL/6J小鼠实验性腹主动脉瘤模型的作用及其机制。方法建立了改良的猪胰弹性蛋白酶诱导的C57BL/6J小鼠实验性腹主动脉瘤模型。42只10周龄的雄性C57BL/6J小鼠随机分为假手术组(Sham组,生理盐水灌注+0.5%DMSO溶液腹腔注射治疗,100mmHg灌注压,5min, n=12),对照组(Control组)、地高辛干预组(n=15/组)。其中Sham组采用生理盐水5min+0.5%DMSO溶液腹腔注射治疗(n=12); Control组采用4U/ml的PPE溶液lOOmmHg灌注压灌注5min+0.5%DMSO溶液腹腔注射治疗(n=15)；Digoxin组采用4U/ml的PPE溶液100mmHg灌注压灌注5min+Digoxin(40μg/只.天)与0.5%DMSO混合溶液腹腔注射治疗(n=15)。各组小鼠分别于术后第0、7、14天对小鼠进行腹主动脉直径超声测量,评估腹主动脉瘤形成情况。术后第14天收取小鼠腹主动脉,进行EVG、免疫组化(α-SM actin.CD31.CD4以及Mac2)等组织学分析,并进行Western blot蛋白检测炎性相关蛋白(IL-17A,MCP-1,IFN-γ以及MMP-2)的表达。结果(1)与对照组相比,地高辛干预显著的降低了腹主动脉瘤的形成率(33.3%vs71.4%,P0.05)。(2)与对照组比较,地高辛干预明显地保护了血管壁结构。(3)与对照组比较,地高辛干预明显地减轻了腹主动脉组织中炎性细胞(CD4+T细胞,巨噬细胞)的浸润。(4)与对照组比较,高剂量地高辛干预明显地抑制了促炎因子及MMP-2的蛋白表达。结论地高辛干预由猪胰弹性蛋白酶诱导的C57BL/6J小鼠实验性腹主动脉瘤模型可以有效的抑制炎性因子的表达以及炎性细胞浸润,有效地保护血管正常组织,减缓实验性腹主动脉瘤的发展。
[Abstract]:Part one: expression of IL-17A and RORyt protein in human abdominal aortic aneurysm
Objective to study the difference of protein expression between IL-17A and RORyt by collecting the pathological specimens of abdominal aortic aneurysm and normal abdominal aorta, and to provide the basis for the study of experimental abdominal aortic aneurysm model.
Methods 10 specimens of human abdominal aortic aneurysm and 10 specimens of normal abdominal aorta were divided into abdominal aortic aneurysm group (Abdominal Aortic Aneurysm, AAA) and control group (Control group). The two groups were histologically and Western Blot method, respectively, and immunohistochemistry (CD4) (CD4) (CD4). The pathological changes of abdominal aortic aneurysm were studied by Mac2, CD31, and alpha -SM actin. The protein expression of 1L-17A and RORyt in two groups of specimens was studied by Western Blot method.
Results the histological study showed that obvious elastic fiber rupture, degradation, apoptosis of middle smooth muscle cells, neovascularization of outer membrane and infiltration of CD4+T cells (Mac2+ macrophages) were observed in the abdominal aortic aneurysm, and Western Blot analysis showed that the protein expression of 1L-17A and RORyt in AAA group was more than that of Control group. The difference was statistically significant (P0.05).
Conclusion the current role of IL-17A in abdominal aortic aneurysm is not clear. We have shown that the expression of IL-17A, RORyt protein level in abdominal aortic aneurysm is significantly higher, and inflammatory cells infiltrate in large numbers. It is presumed that IL-17A may play a role in the development of abdominal aortic aneurysm, and for subsequent animal experiments. The exhibition provides a theoretical basis.
The second part of digoxin protects against angiotensin II induced experimental abdominal aortic aneurysm in ApoE-/- mice.
Objective to investigate the effect of digoxin drug intervention on angiotensin II induced experimental abdominal aortic aneurysm model in ApoE-/- mice and its mechanism.
Methods the experimental abdominal aortic aneurysm model of ApoE-/- mice induced by Alzet microosmotic pump subcutaneous implantation was routinely established. The male ApoE- mice of 16 weeks old were randomly divided into the sham operation group (group Sham), the control group (Control group), the low dose digoxin intervention group (Low-dose group), the high dose digoxin intervention group (High-dose group), and the 16 weeks old male ApoE- mice were randomly divided into the control group (group Control), the low dose digoxin intervention group (Low-dose group) and the high dose digoxin intervention group (High-dose group). Groups of 20 rats in each group were treated with the subcutaneous micropump of the physiological saline and the same amount of 0.5%DMSO solution for intraperitoneal injection; the Control group was injected with the angiotensin II subcutaneous micropump and given the same amount of 0.5%DMSO solution intraperitoneally, and the Low-dose group was given the same amount of blood vessel tight Zhang Su subcutaneous micropump and given the same amount. The 0.5%DMSO+Digoxin (20 g/ only. Day) mixed solution was injected into the abdominal cavity for injection, and the High-dose group was injected with angiotensin II subcutaneous micropump and given the same amount of 0.5%DMSO+Digoxin (40 g/ only day) mixed solution. The formation of abdominal aortic aneurysm was evaluated. The systolic blood pressure of mice was measured by tail clamp pressure measurement on day 0,28 after operation, and the effect of angiotensin II on blood pressure was assessed. The abdominal aorta of mice was collected twenty-eighth days after operation, HE, EVG, immunohistochemical staining (alpha -SM actin, CD31, CD4 and Mac2), immunofluorescence staining (anti-CD4, IL-17A), etc. Cell flow cytometry analysis, Western blot protein detection of inflammatory related protein (IL-17A, ROR gamma T, MCP-1, IFN- gamma, RANTES and MMP-2, MMP-9) expression.
Results (1) compared with the control group, high dose digoxin significantly reduced the formation rate of abdominal aortic aneurysm (35%vs70%, P0.05) and increased the survival rate of mice. (2) digoxin intervention did not significantly affect the blood pressure of mice at twenty-eighth days after the operation. (3) high dose digoxin intervention significantly protected the vascular wall structure. (4) Compared with the control group, high dose digoxin intervention significantly alleviated the infiltration of inflammatory cells (CD4+T cells, macrophages) in the abdominal aorta. (5) compared with the control group, high dose digoxin intervention significantly inhibited the protein expression of pro-inflammatory factors and MMPs. (6) the intervention of high dose of digoxin changed the spleen cells of mice compared with those in the control group. Th17 cells, the composition of regulatory T cells.
Conclusion digoxin intervention (especially high dose intervention) can effectively inhibit the expression of inflammatory factors and inflammatory cell infiltration, effectively protect the normal vascular tissue and slow the development of experimental abdominal aortic aneurysm.
The protective effect of digoxin on experimental abdominal aortic aneurysm model induced by porcine pancreatic elastase in third C57BL/6J mice
Objective to investigate the effect and mechanism of digoxin intervention on another experimental abdominal aortic aneurysm model induced by porcine pancreatic elastase in C57BL/6J mice.
Methods a modified C57BL/6J mouse model of abdominal aortic aneurysm induced by modified pig pancreatic elastase was established. The male C57BL/6J mice of 10 weeks old were randomly divided into sham group (group Sham, intraperitoneal injection of +0.5%DMSO solution with saline, 100mmHg perfusion pressure, 5min, n= 12), control group (Control group), and digoxin intervention group (n=15/ group). Among them, group Sham was treated by intraperitoneal injection of saline 5min+0.5%DMSO solution (n=12), and group Control was injected with 4U/ml PPE solution lOOmmHg perfusion pressure and intraperitoneal injection of 5min+0.5%DMSO solution (n=15), and Digoxin group was treated with 4U/ml PPE solution perfusion pressure perfusion (40 mu only. N=15. The mice were measured by the abdominal aorta diameter at the 0,7,14 day after the operation to assess the formation of abdominal aortic aneurysm. The abdominal aorta of mice was collected fourteenth days after operation, EVG, immunohistochemistry (alpha -SM actin.CD31.CD4 and Mac2) were collected, and Western blot protein was used to detect inflammatory related protein (IL-17A). The expression of MCP-1, IFN- gamma and MMP-2).
Results (1) compared with the control group, digoxin intervention significantly reduced the formation rate of abdominal aortic aneurysm (33.3%vs71.4%, P0.05). (2) compared with the control group, digoxin intervention obviously protected the vascular wall structure. (3) compared with the control group, digoxin intervention significantly reduced the inflammatory cells (CD4+T cells, macrophages) in the abdominal aorta. Infiltration (4) compared with the control group, high dose digoxin intervention significantly inhibited proinflammatory cytokines and MMP-2 protein expression.
Conclusion the intervention of digoxin induced experimental abdominal aortic aneurysm model in C57BL/6J mice induced by porcine pancreatic elastase can effectively inhibit the expression of inflammatory factors and infiltration of inflammatory cells, effectively protect the normal vascular tissue and slow the development of experimental abdominal aortic aneurysm.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R96

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