【摘要】：類風(fēng)濕關(guān)節(jié)炎(rheumatoid arthritis,RA)被認(rèn)為是增加心血管疾病(cardiovascular disease,CVD)發(fā)生率與死亡率的非傳統(tǒng)風(fēng)險(xiǎn)因素。臨床研究表明,與健康受試者相比,RA患者內(nèi)皮功能異常,頸動(dòng)脈粥樣硬化程度和平均頸動(dòng)脈中膜厚度增加,提示RA患者血管結(jié)構(gòu)與功能異常可能是引起CVD發(fā)生率與死亡率增加的重要原因,探索RA炎癥與CVD相互影響及其可能機(jī)制具有重要意義。血管緊張素Ⅱ(angiotensinⅡ,AngⅡ)是血管緊張素Ⅰ(angiotensin I,Ang I)在血管緊張素轉(zhuǎn)化酶(angiotensin converting enzyme,ACE)的作用下水解產(chǎn)生的八肽物質(zhì),主要作用于細(xì)胞膜上的兩個(gè)受體(angiotensinⅡreceptors,ATR)即AT1R、AT2R發(fā)揮生物學(xué)效應(yīng)。AngⅡ?qū)κ湛s血管、調(diào)節(jié)血壓、維持電解質(zhì)平衡起到重要作用。有研究表明,佐劑性關(guān)節(jié)炎(adjuvant-induced arthritis,AA)大鼠體內(nèi)灌注AngⅡ可加重高血壓反應(yīng)、內(nèi)皮功能紊亂及血管肥厚,但其對(duì)AA炎癥嚴(yán)重程度及血管結(jié)構(gòu)與功能產(chǎn)生怎樣的影響及作用機(jī)制仍有待深入研究。RA患者CVD死亡率是正常人群的1.5~2倍,因此RA疾病的正確治療對(duì)減少CVD的發(fā)生有重要意義。RA是一種慢性、系統(tǒng)性的自身免疫病,以受累關(guān)節(jié)的滑膜炎癥和血管翳為基本病變。成纖維樣滑膜細(xì)胞(fibroblast-like synovial cell,FLS)增生是RA疾病的主要病理特征之一。研究表明,RA患者血清、滑膜液中ACE水平升高,ACE能將Ang I轉(zhuǎn)化為AngⅡ,間接提示AngⅡ參與了RA疾病病程。AngⅡ作用于AT1R發(fā)揮促炎、促增殖作用,作用于AT2R發(fā)揮抗炎、抗增殖作用。課題組前期研究表明,AT1R阻斷劑氯沙坦(Losartan)、AT2R激動(dòng)劑(CGP42112)均能改善AA大鼠的炎癥反應(yīng)。AngⅡ作用于ATR如何影響AA炎癥及血管損傷的相關(guān)機(jī)制尚不清楚。目的:建立大鼠AHD模型,觀察2K1C-HT與AA疾病之間的相互影響,探索高水平AngⅡ?qū)A大鼠炎癥及血管損傷的影響及部分機(jī)制,為臨床認(rèn)識(shí)RA患者罹患CVD風(fēng)險(xiǎn)的機(jī)制提供實(shí)驗(yàn)依據(jù)。方法:分別在大鼠左后足跖皮內(nèi)注射0.1ml完全弗氏佐劑(complete freund's adjuvant,CFA)建立AA模型,實(shí)施外科手術(shù)用0.25mm銀夾縮窄大鼠左腎動(dòng)脈建立兩腎一夾高血壓(two-kidney-one-clip hypertension,2K1C-HT)大鼠模型,以及同時(shí)施加上述兩種方法建立類風(fēng)濕關(guān)節(jié)炎復(fù)合高血壓疾病(adjuvant-induced arthritis combined with two-kidney-one-clip hypertension disease,AHD)模型。免疫11天后,每三天測(cè)量大鼠的體重和足爪腫脹度,并進(jìn)行炎癥評(píng)分;用尾動(dòng)脈測(cè)壓儀每周檢測(cè)大鼠的尾動(dòng)脈收縮壓;第35天處死大鼠,取出左腎、右腎和心臟并稱重,同時(shí)收集踝關(guān)節(jié)、脾臟、胸主動(dòng)脈用于病理檢查;Western blot、激光共聚焦法檢測(cè)AA、2K1C-HT、AHD關(guān)節(jié)FLS上AT1R、AT2R的表達(dá)與定位;血管環(huán)實(shí)驗(yàn)檢測(cè)胸主動(dòng)脈內(nèi)皮依賴的血管舒張功能;酶聯(lián)免疫吸附實(shí)驗(yàn)(enzyme-linked immunosorbent assay,ELISA)檢測(cè)血清中AngⅡ、TNF-α的變化;免疫組化(Immunohistochemistry,IHC)法檢測(cè)AngⅡ及其受體的表達(dá)及定位;Western blot法檢測(cè)胸主動(dòng)脈組織AngⅡ及下游信號(hào)分子如p-ERK1/2、p-NF-κB的表達(dá)變化;分別以CCK-8和Western blot法檢測(cè)Losartan、CGP42112、PD123319對(duì)FLS增殖及AT1R、AT2R表達(dá)的影響。結(jié)果:1.采用CFA足跖免疫法聯(lián)合2K1C法可成功建立AHD大鼠模型與正常組相比,AA大鼠體重明顯降低,關(guān)節(jié)明顯腫脹、炎癥指標(biāo)評(píng)分明顯升高,HT大鼠左腎指數(shù)明顯降低,血壓值明顯升高;AHD大鼠體重明顯降低、關(guān)節(jié)炎指標(biāo)評(píng)分明顯升高;病理學(xué)檢查可見(jiàn)關(guān)節(jié)腔有大量炎性細(xì)胞浸潤(rùn)、滑膜增生和軟骨破壞,脾臟生發(fā)中心數(shù)量增多、淋巴細(xì)胞聚集成團(tuán)、紅髓彌漫性浸潤(rùn),血清AngⅡ、TNF-α水平升高,同時(shí),AHD大鼠第2周開(kāi)始血壓值升高,第5周顯著升高,左腎指數(shù)明顯降低,心臟指數(shù)明顯升高,胸主動(dòng)脈血管橫截面積(cross-sectional area,CSA)、管壁中膜厚度(media thickness,MT)增大、管腔直徑(lumen diameter,LD)降低,心肌細(xì)胞排列紊亂、斷裂、腫大,CSA與軸長(zhǎng)增加。提示,AHD大鼠同時(shí)具有RA與2K1C-HT模型的特征,表明AHD模型成功建立。2.2K1C-HT對(duì)AA炎癥的影響及部分機(jī)制與AA大鼠比較,AHD大鼠發(fā)病時(shí)程提前,發(fā)病率有升高趨勢(shì),足爪腫脹度、關(guān)節(jié)炎指數(shù)、關(guān)節(jié)及脾臟病理評(píng)分明顯升高,血清中AngⅡ水平明顯升高,提示2K1C-HT加重了AA炎癥病理情況。進(jìn)一步研究發(fā)現(xiàn),AA大鼠關(guān)節(jié)滑膜細(xì)胞上AT1R表達(dá)明顯增加而AT2R無(wú)明顯變化,AHD大鼠滑膜細(xì)胞的AT1R、AT2R表達(dá)均明顯升高且AT1R與AT2R的比值明顯升高,表明在炎癥條件下,兩腎一夾誘導(dǎo)的高水平AngⅡ主要作用于AT1R發(fā)揮促炎作用。同時(shí),我們進(jìn)一步觀察了AngⅡ受體靶點(diǎn)藥物對(duì)AA關(guān)節(jié)局部效應(yīng)細(xì)胞—滑膜細(xì)胞增殖的影響,結(jié)果顯示AT1R受體阻斷劑Losartan(10-6mol/L)、AT2R受體激動(dòng)劑CGP42112(10-6mol/L)均能抑制AA-FLS的增殖能力,而AT2R受體拮抗劑PD123319(10-6mol/L)對(duì)AA-FLS增殖無(wú)明顯影響。Losartan聯(lián)合PD123319與單獨(dú)使用藥物相比無(wú)明顯差異,Losartan聯(lián)合CGP42112抑制FLS增殖能力比單獨(dú)使用藥物強(qiáng)但無(wú)顯著性差異。Losartan、CGP42112、PD123319均能降低AT1R的表達(dá),Losartan聯(lián)合CGP42112比單獨(dú)使用CGP42112明顯降低AT1R的表達(dá)。CGP42112能顯著提高AT2R的表達(dá),PD123319明顯抑制AT2R的表達(dá),Losartan聯(lián)合CGP42112比單獨(dú)使用Losartan、CGP42112明顯提高AT2R的表達(dá)。3.AA炎癥加重了2K1C-HT大鼠血管及心肌損傷與2K1C-HT大鼠相比,AHD大鼠血壓值、心臟指數(shù)明顯升高,血清中AngⅡ水平顯著升高,胸主動(dòng)脈的MT和CSA明顯增加、LD明顯降低。心臟組織心肌細(xì)胞排列紊亂、出現(xiàn)斷裂、心肌細(xì)胞CSA和軸長(zhǎng)明顯增加,胸主動(dòng)脈內(nèi)皮依賴的血管舒張功能明顯降低。提示,AA炎癥加重了2K1C-HT大鼠的血管及心肌損傷。4.AngⅡ可能通過(guò)AT1R/ERK1/2信號(hào)通路參與了AHD大鼠的血管損傷AHD大鼠血清中AngⅡ水平顯著升高,大鼠胸主動(dòng)脈組織AngⅡ、AT1R、AT2R、GRK2的表達(dá)均明顯增加,且主要在血管壁內(nèi)側(cè)表達(dá)。與2K1C-HT大鼠相比,AHD大鼠胸主動(dòng)脈組織AngⅡ及下游信號(hào)分子(p-ERK1/2,p-NF-κB)的表達(dá)水平也明顯增加,且AT1R/AT2R比值明顯較高,提示AngⅡ可能主要作用于AT1R激活ERK1/2/NF-κB信號(hào)通路而加重了血管損傷。結(jié)論:1.采用CFA足跖免疫聯(lián)合2K1C法可成功建立大鼠AHD模型;2.2K1C-HT可使AA發(fā)病率有增高趨勢(shì),并且加重AA大鼠關(guān)節(jié)炎病理情況;3.AngⅡ及其受體在2K1C-HT血管損傷中發(fā)揮了重要作用,AA炎癥加劇了AngⅡ引起的血管損傷;4.AngⅡ激活A(yù)T1R/ERK1/2信號(hào)通路可能是其加重AA大鼠血管損傷的重要機(jī)制。
[Abstract]:Rheumatoid arthritis (RA) is considered to be an unconventional risk factor for increased incidence and mortality of cardiovascular disease (CVD). Clinical studies have shown that RA patients have endothelial dysfunction, increased carotid atherosclerosis and increased mean carotid media thickness compared with healthy subjects, suggesting that RA patients have increased risk of cardiovascular disease (CVD) and mortality. Abnormal vascular structure and function may be an important cause of increased CVD incidence and mortality. It is important to explore the interaction between RA inflammation and CVD and its possible mechanism. The octapeptide produced by hydrolysis of CE mainly acts on two receptors (ATR) on the cell membrane, namely AT1R and AT2R. Ang II plays an important role in contracting blood vessels, regulating blood pressure and maintaining electrolyte balance. Endothelial infusion of Ang II can aggravate hypertensive response, endothelial dysfunction and vascular hypertrophy, but its effect on the severity of AA inflammation, vascular structure and function remains to be further studied. Fibroblast-like synovial cell (FLS) hyperplasia is one of the main pathological features of RA. Studies have shown that serum and synovial fluid ACE levels in patients with RA increase, ACE can convert Ang I into Ang I I, indirectly. It is suggested that Ang II plays an important role in the pathogenesis of RA. Ang II acts on AT1R to promote inflammation, proliferation and anti-inflammatory and anti-proliferative effects on AT2R. Previous studies showed that AT1R blockers Losartan and AT2R agonists (CGP42112) can improve the inflammatory response of AA rats. How Ang II acts on ATR affects AA inflammation and vascular injury AIM: To establish a rat model of AHD, observe the interaction between 2K1C-HT and AA disease, explore the effects of high-level Ang II on inflammation and vascular injury in AA rats, and provide experimental evidence for clinical understanding of the risk of CVD in RA patients. METHODS: Intradermal injection of 0.1ml of Ang II into the left hind plantar of rats respectively. Complete Freund's adjuvant (CFA) was used to establish AA model, left renal artery was constricted with 0.25 mm silver clip for surgical operation to establish two-kidney-one-clip hypertension (2K1C-HT) rat model, and rheumatoid arthritis combined with hypertension (adjuvant-indu) was established by both methods. After 11 days of immunization, body weight and paw swelling were measured every three days, and inflammation scores were made; tail artery systolic blood pressure was measured weekly with tail artery manometer; rats were sacrificed on the 35th day, left kidney, right kidney and heart were taken out and weighed. Ankle, spleen and thoracic aorta were used for pathological examination; Western blot and laser confocal focusing were used to detect the expression and localization of AT1R and AT2R on FLS of AA, 2K1C-HT and AHD joints; vascular ring test was used to detect endothelium-dependent vasodilation of thoracic aorta; enzyme-linked immunosorbent assay (ELISA) was used to detect Ang II and TNF-alpha in serum. Immunohistochemistry (IHC) was used to detect the expression and localization of Ang II and its receptors; Western blot was used to detect the expression of Ang II and downstream signal molecules such as p-ERK1/2, p-NF-kappa B in thoracic aorta; CCK-8 and Western blot were used to detect the effects of Losartan, CGP42112, PD123319 on the proliferation of FLS and the expression of AT1R and AT2R, respectively. Results: 1. Compared with the normal group, the weight of AA rats was significantly reduced, the joint swelled, the inflammatory index score was significantly increased, the left kidney index of HT rats was significantly decreased, the blood pressure value was significantly increased, the weight of AHD rats was significantly reduced, and the arthritis index score was significantly increased. There were a lot of inflammatory cells infiltration in the joint cavity, synovial hyperplasia and cartilage destruction, the number of splenic germinal center increased, lymphocyte aggregation, diffuse infiltration of red pulp, serum Ang II, TNF-a levels increased. At the same time, AHD rats began to increase blood pressure in the second week, increased significantly in the fifth week, left renal index decreased significantly, heart index increased significantly. High, cross-sectional area (CSA), media thickness (MT) increased, lumen diameter (LD) decreased, myocardial cells arranged disorderly, ruptured, enlarged, CSA and axial length increased. These results suggest that AHD rats have the characteristics of both RA and 2K1C-HT models, indicating that the AHD model was successfully established.2.2K1C-HT on A. Compared with AA rats, the effect of A-inflammation and some mechanisms in AHD rats showed that the course of disease was earlier, the incidence of AHD rats increased, the degree of paw swelling, arthritis index, pathological scores of joints and spleens were significantly increased, and the level of Ang II in serum was significantly increased, suggesting that 2K1C-HT aggravated the inflammatory pathology of AA rats. The expression of AT1R and AT2R in synovial cells of AHD rats were significantly increased and the ratio of AT1R to AT2R was significantly increased. It indicated that the high level of Ang II induced by two kidneys one clip mainly acted on AT1R to promote inflammation under inflammatory conditions. The effect of AT1R receptor antagonist Losartan (10-6mol/L) and AT2R receptor agonist CGP42112 (10-6mol/L) on the proliferation of AA-FLS was observed, while AT2R receptor antagonist PD123319 (10-6mol/L) had no significant effect on the proliferation of AA-FLS. Losartan, CGP42112, PD123319 all decreased the expression of AT1R, Losartan combined with CGP42112 significantly decreased the expression of AT1R compared with CGP42112 alone. CGP42112 significantly increased the expression of AT2R, PD123319 significantly inhibited the expression of AT2R. Losartan combined with CGP42112 significantly increased the expression of AT2R compared with Losartan alone. 3. AA inflammation aggravated vascular and myocardial injury in 2K1C-HT rats. Compared with 2K1C-HT rats, the blood pressure and cardiac index of AHD rats were significantly increased, the levels of Ang II in serum were significantly increased, the levels of CT and CSA in thoracic aorta were significantly increased, and LD was significantly decreased. These results suggest that AA inflammation aggravates vascular and myocardial injury in 2K1C-HT rats. 4. Ang II may participate in the serum Ang II water in AHD rats through AT1R/ERK1/2 signaling pathway. The expression of Ang II, AT1R, AT2R and GRK 2 in the thoracic aorta of AHD rats was significantly higher than that of 2K1C-HT rats. The expression of Ang II and downstream signal molecule (p-ERK1/2, p-NF-kappa B) in the thoracic aorta of AHD rats was also significantly higher than that of 2K1C-HT rats, and the AT1R/AT2R ratio was significantly higher, suggesting that Ang II might be the main factor. Conclusion: 1. The rat model of AHD can be successfully established by CFA plantar immunization combined with 2K1C method; 2.2K1C-HT can increase the incidence of AA and aggravate the pathological condition of AA rat arthritis; 3. Ang II and its receptor play an important role in the vascular injury of 2K1C-HT, AA. Ang II activates AT1R/ERK1/2 signaling pathway, which may play an important role in aggravating vascular injury in AA rats.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R593.22;R54

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