本文選題：IL-17A + ROγt��； 參考：《華中科技大學》2015年博士論文

【摘要】：第一部分人腹主動脈瘤病變組織中IL-17A, RORyt蛋白表達的研究 目的通過收集腹主動脈瘤患者病變組織標本與正常腹主動脈組織標本,研究IL-17A、RORyt的蛋白表達差異,為實驗性腹主動脈瘤模型的研究提供依據(jù)。 方法收取人腹主動脈瘤病變組織標本以及人正常腹主動脈組織標本各10例,分別分成腹主動脈瘤組(Abdominal Aortic Aneurysm, AAA鍚以及對照組(Control組),對兩組標本分別進行組織學以及Western Blot方法研究。通過彈性纖維EVG染色、免疫組化(CD4、Mac2、CD31、α-SM actin)染色研究腹主動脈瘤的病理學改變。通過Western Blot法對兩組標本的1L-17A以及RORyt的蛋白表達進行研究。 結果組織學研究表明腹主動脈瘤病變組織中發(fā)生了明顯的彈性纖維斷裂、降解,中層平滑肌細胞凋亡,外膜新生毛細血管增生,管壁炎性細胞(CD4+T細胞,Mac2+巨噬細胞)浸潤。同時,Western Blot分析表明AAA組中1L-17A、RORyt的蛋白表達較Control組明顯增加,兩者差異均具有統(tǒng)計學意義(P0.05)。 結論目前關于IL-17A在腹主動脈瘤瘤中的作用是不明確的,我們通過研究結果表明IL-17A、RORyt蛋白水平的表達在腹主動脈瘤組織中明顯增高,炎性細胞大量浸潤,推測IL-17A對腹主動脈瘤的發(fā)生發(fā)展可能起到促進作用,為后續(xù)動物實驗的開展提供了理論依據(jù)。 第二部分地高辛對血管緊張素II誘導的ApoE-/-小鼠實驗性腹主動脈瘤模型保護作用的研究 目的探討地高辛藥物干預對血管緊張素II誘導的ApoE-/-小鼠實驗性腹主動脈瘤模型的作用及其機制。 方法常規(guī)建立血管緊張素II經Alzet微量滲透泵皮下置入法誘導的ApoE-/-小鼠實驗性腹主動脈瘤模型。80只16周齡的雄性ApoE-小鼠隨機分為假手術組(Sham組)、對照組(Control組)、低劑量地高辛干預組(Low-dose組)、高劑量地高辛干預組(High-dose組),每組均20只實驗鼠。其中Sham組由生理鹽水皮下微量泵入并給予同等量的0.5%DMSO溶液腹腔注射治療；Control組由血管緊張素Ⅱ皮下微量泵入并給予同等量的0.5%DMSO溶液腹腔注射治療；Low-dose組由血管緊張素Ⅱ皮下微量泵入并給予同等量的0.5%DMSO+Digoxin(20μg/只.天)混合溶液腹腔注射治療；High-dose組由血管緊張素Ⅱ皮下微量泵入并給予同等量的0.5%DMSO+Digoxin(40μg/只.天)混合溶液腹腔注射治療。各組小鼠分別于術后第0、7、14、21以及28天對小鼠進行腹主動脈直徑超聲測量,評估腹主動脈瘤形成情況。術后第0、28天采用尾夾測壓法測量小鼠收縮壓,評估血管緊張素II對血壓的影響。術后第28天收取小鼠腹主動脈,進行HE、EVG、免疫組化(α-SM actin、CD31、CD4以及Mac2)、免疫熒光染色(anti-CD4、IL-17A)等組織學分析、小鼠脾細胞流式細胞儀分析、Western blot蛋白檢測炎性相關蛋白(IL-17A,、RORγt、MCP-1、IFN-γ、RANTES以及MMP-2、MMP-9)的表達。 結果(1)與對照組相比,高劑量地高辛干預顯著的降低了腹主動脈瘤的形成率(35%vs70%,P0.05)并且提高了小鼠存活率。(2)地高辛干預對術后第28天的小鼠血壓并無明顯影響。(3)與對照組比較,高劑量地高辛干預明顯地保護了血管壁結構。(4)與對照組比較,高劑量地高辛干預明顯地減輕了腹主動脈組織中炎性細胞(CD4+T細胞,巨噬細胞)的浸潤。(5)與對照組比較,高劑量地高辛干預明顯地抑制了促炎因子及MMPs的蛋白表達。(6)與對照組比較,高劑量地高辛干預改變了小鼠脾細胞中Th17細胞、調節(jié)性T細胞的組成。 結論地高辛干預(尤其是高劑量干預)可以有效的抑制炎性因子的表達以及炎性細胞浸潤,有效地保護血管正常組織,減緩實驗性腹主動脈瘤的發(fā)展。 第三部分地高辛對豬胰彈性蛋白酶誘導的C57BL/6J小鼠實驗性腹主動脈瘤模型保護作用的研究 目的探討地高辛干預對另一種由豬胰彈性蛋白酶誘導的C57BL/6J小鼠實驗性腹主動脈瘤模型的作用及其機制。 方法建立了改良的豬胰彈性蛋白酶誘導的C57BL/6J小鼠實驗性腹主動脈瘤模型。42只10周齡的雄性C57BL/6J小鼠隨機分為假手術組(Sham組,生理鹽水灌注+0.5%DMSO溶液腹腔注射治療,100mmHg灌注壓,5min, n=12),對照組(Control組)、地高辛干預組(n=15/組)。其中Sham組采用生理鹽水5min+0.5%DMSO溶液腹腔注射治療(n=12); Control組采用4U/ml的PPE溶液lOOmmHg灌注壓灌注5min+0.5%DMSO溶液腹腔注射治療(n=15)；Digoxin組采用4U/ml的PPE溶液100mmHg灌注壓灌注5min+Digoxin(40μg/只.天)與0.5%DMSO混合溶液腹腔注射治療(n=15)。各組小鼠分別于術后第0、7、14天對小鼠進行腹主動脈直徑超聲測量,評估腹主動脈瘤形成情況。術后第14天收取小鼠腹主動脈,進行EVG、免疫組化(α-SM actin.CD31.CD4以及Mac2)等組織學分析,并進行Western blot蛋白檢測炎性相關蛋白(IL-17A,MCP-1,IFN-γ以及MMP-2)的表達。 結果(1)與對照組相比,地高辛干預顯著的降低了腹主動脈瘤的形成率(33.3%vs71.4%,P0.05)。(2)與對照組比較,地高辛干預明顯地保護了血管壁結構。(3)與對照組比較,地高辛干預明顯地減輕了腹主動脈組織中炎性細胞(CD4+T細胞,巨噬細胞)的浸潤。(4)與對照組比較,高劑量地高辛干預明顯地抑制了促炎因子及MMP-2的蛋白表達。 結論地高辛干預由豬胰彈性蛋白酶誘導的C57BL/6J小鼠實驗性腹主動脈瘤模型可以有效的抑制炎性因子的表達以及炎性細胞浸潤,有效地保護血管正常組織,減緩實驗性腹主動脈瘤的發(fā)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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R96

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相關碩士學位論文 前10條

1 王建國;影響腹主動脈瘤延展及預后相關因素的臨床研究[D];中國人民解放軍醫(yī)學院;2012年

2 吳s，

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