本文選題：系統(tǒng)性硬化癥 + 內(nèi)皮素A型受體抗體��； 參考：《南昌大學(xué)》2017年碩士論文

【摘要】：背景及目的:系統(tǒng)性硬化癥(Systemic sclerosis,SSc)相關(guān)肺動脈高壓(Pulmonary Arterial Hypertension,PAH)是導(dǎo)致SSc患者死亡的重要原因之一。因為其起病隱匿,臨床表現(xiàn)常不典型,早期診斷困難,治療效果不理想。目前對于SSc-PAH早期診斷,治療,預(yù)后評估仍是困擾風(fēng)濕免疫科醫(yī)生的重要難題。肺動脈高壓(PAH)是SSc相關(guān)的肺部疾病中預(yù)后最差的并發(fā)癥之一,其病理變化與內(nèi)皮細(xì)胞功能障礙及局部炎癥有關(guān),在晚期則表現(xiàn)為肺血管平滑肌細(xì)胞過度增殖、凋亡減少。在PAH的病理過程中內(nèi)皮素-1(Endothelin-1,ET-1)起到了非常重要的作用。ET-1是由血管內(nèi)皮細(xì)胞合成的21肽,作為一種強力持久的血管收縮劑,促進血管壁的肥厚和纖維化的發(fā)生。ET1正是通過與其受體(Endothelin receptor,ETR)(A型受體、B型受體)結(jié)合發(fā)揮收縮血管的作用,特別是位于肺動脈平滑肌細(xì)胞和血管內(nèi)皮細(xì)胞的內(nèi)皮素A型受體(Endothelin Receptor Type A,ETRA),是觸發(fā)血管的收縮主要因素,同時在細(xì)胞增殖和肺動脈免疫微環(huán)境的調(diào)控中發(fā)揮了重要的作用。本研究擬通過檢測抗內(nèi)皮素A型受體抗體(Anti-Endothelin Receptor Type A Autoantibodies ETRA-Ab)在SSc-PAH患者血清中的陽性率和特異性,從而探索ETRA-Ab與SSc-PAH發(fā)病及疾病嚴(yán)重程度之間的關(guān)系。進而探討抗內(nèi)皮素A型受體抗體作為協(xié)助早期診斷SSc-PAH生物標(biāo)記物的可能性。并通過動物實驗進一步證實ETRA-Ab作為功能性抗體,在SSc-PAH發(fā)病機制中的作用。方法:1、臨床觀察:共納入24例SSc-PAH樣本。同時選擇24例相匹配SSc非PAH對照,24份正常對照。記錄臨床數(shù)據(jù),心彩超測肺動脈收縮壓(PASP)。其中5例SSc-PAH行右心導(dǎo)管檢測。測定各組6分鐘步行距離(6WMD)。使用酶聯(lián)免疫吸附試驗(ELISA)法檢測以上各組血漿中的抗ETAR抗體,計算陽性率。2、建立MCT肺動脈高壓大鼠模型:共36只雄性SD大鼠,隨機分為正常對照組(n=12)、MCT組(n=12)、MCT+ETRA-Ab組(n=12)。MCT組和MCT+ETRA-Ab組行腹腔注射野百合堿(Monocrotatine,MCT),MCT+ETRA-Ab組在腹腔注射MCT后第1~3周經(jīng)尾靜脈給予注入多克隆IgG ETRA抗體,每周3次。3、平均肺動脈壓力(mPAP)測定:于MCT造模后4周,經(jīng)右頸外靜脈插管,用Medlab-E生物機能信號采集系統(tǒng)測mPAP。并使用SPSS20.0分析各組間的差異。4、右心肥厚指數(shù)(RVHI%)測定:取出心臟,分離左右心室和室間隔,分別稱量右心室壁(RV)的質(zhì)量和左心室和室間隔(LV+S)的質(zhì)量,計算右心肥厚指數(shù)=RV/(LV+S)。分析各組間的差異。5、肺血管形態(tài)學(xué)觀察:切取完整右肺上葉,甲醛固定、脫水、浸蠟、包埋、切片、烘干、HE染色。光鏡下觀察各組大鼠肺小動脈形態(tài)學(xué)變化。比較各組肺血管肌型動脈(MA),部分肌型動脈(PMA),無肌型動脈(NMA)比例的變化。6、檢測各組血漿中炎癥因子白介素(interleukin IL)-6、腫瘤壞死因子(tumor necrosis factor TNF)-α的水平,分析各組間差異。結(jié)果:1、SSc-PAH組與SSc非PAH組ETRA抗體陽性率比較前者ETRA抗體陽性率50.0%。后者ETRA抗體陽性率為12.5%,正常對照組ETRA抗體陽性率為0%。陽性率比較有顯著性差異(P0.01)。2、SSc-PAH患者血清中ETRA抗體滴度水平與肺動脈壓力PASP有相關(guān)性(r=0.3439 P=0.0321)。而ETRA抗體滴度水平與6分鐘步行距離測試結(jié)果之間存在顯著的負(fù)相關(guān)(r=-0.3221,P=0.0391),與NT-proBNP結(jié)果呈現(xiàn)相關(guān)性(r=0.3335,P=0.0318)。與末梢循環(huán)檢測指數(shù)呈正相關(guān)(r=0.3439,P=0.0321)。3、建立MCT模型第4周,注射10%水合氯醛,腹腔麻醉,經(jīng)頸外靜脈插管,行三組間mPAP(mmHg)比較:正常對照組(17.3±1.1)。MCT組(30.8±2.0)、MCT+ETRA-Ab組(34.5±3.1)(P0.05)。4、測壓后處死動物右室分離行右心肥厚指數(shù)比較:MCT組(0.355±0.051)、MCT+ETRA-Ab組(0.402±0.049)(P0.05)。5、肺組織HE染色,比較MA、PMA、NMA在三組中所占比例(PMA、PPMA、PNMA)的變化:MCT+ETRA-Ab組中PMA、PPMA高于MCT對照組(P0.05),兩者均明顯正常對照組。6、血漿炎癥因子測定:MCT組、MCT+ETRA-Ab組中IL-6、TNF-α(pg/ml)水平明顯高于正常對照組。但MCT+ETRA-Ab組較MCT組升高更加顯著(P0.05)。結(jié)論:1、ETRA-Ab對于SSc-PAH的診斷有較好的敏感性和較高的特異性,從而提示ETRA-Ab可以作為SSc-PAH早期診斷的新型生物標(biāo)記物。2、SSc-PAH患者中ETRA-Ab滴度與PASP、NT-proBNP正相關(guān),與6分鐘步行距離測試距離負(fù)相關(guān)。提示ETRA-Ab滴度可以作為評估患者病情,判斷預(yù)后的指標(biāo)。3、ETRA-Ab對MCT大鼠的血流動力學(xué)指標(biāo)、右心肥厚指數(shù)、肺血管形態(tài)變化指數(shù)升高均有上調(diào)作用。并上調(diào)血漿中的炎癥因子IL-6、TNF-α。提示ETRA-Ab作為功能性抗體可能參與到了包括肺血管重塑,右心肥厚等多個方面。
[Abstract]:Background and purpose: Systemic sclerosis (SSc) associated pulmonary arterial hypertension (Pulmonary Arterial Hypertension, PAH) is one of the important causes of death in SSc patients. Because of its insidious onset, the clinical manifestations are often untypical, early diagnosis is difficult, and the treatment effect is not ideal. At present, the early diagnosis, treatment, and prognosis evaluation of SSc-PAH is not satisfactory. The evaluation of pulmonary hypertension (PAH) is one of the worst complications in SSc related pulmonary diseases. The pathological changes are related to endothelial dysfunction and local inflammation. In the late stage, the proliferation of pulmonary vascular smooth muscle cells and the decrease of apoptosis. The endothelium in the pathological process of PAH -1 (Endothelin-1, ET-1) plays a very important role in.ET-1, a 21 peptide synthesized from vascular endothelial cells, as a powerful and persistent vasoconstrictor, promoting the genesis of the hypertrophy and fibrosis of the vascular wall, and.ET1 is the combination of its receptor (Endothelin receptor, ETR) (A receptor, B receptor) to play the role of vasoconstrictor. The endothelin A receptor (Endothelin Receptor Type A, ETRA), which is located in the pulmonary artery smooth muscle cells and vascular endothelial cells, is the main factor that triggers the contraction of the blood vessels, and plays an important role in the regulation of cell proliferation and the regulation of the pulmonary artery immune microenvironment. This study is intended to detect the anti endothelin A receptor antibody (Anti-Endoth). The positive rate and specificity of Elin Receptor Type A Autoantibodies ETRA-Ab in the serum of SSc-PAH patients, thus exploring the relationship between ETRA-Ab and SSc-PAH pathogenesis and the severity of disease, and further exploring the possibility of anti endothelin A receptor antibody as an early diagnosis of SSc-PAH biomarkers, and further evidence by animal experiments. The role of real ETRA-Ab as a functional antibody in the pathogenesis of SSc-PAH. Methods: 1, clinical observation: a total of 24 SSc-PAH samples were included. 24 cases of matched SSc non PAH controls and 24 normal controls were selected. The clinical data were recorded and the systolic pressure of pulmonary artery (PASP) was measured by cardiac color Doppler echocardiography. Among them, the right cardiac catheterization was detected in 5 cases of SSc-PAH. The 6 minute walk was measured in each group. Distance (6WMD). Using enzyme linked immunosorbent assay (ELISA) to detect the anti ETAR antibody in the plasma of the above groups, the positive rate of.2 was calculated, and the MCT pulmonary hypertension rat model was established: 36 male SD rats were randomly divided into normal control group (n=12), MCT group (n=12), MCT+ ETRA-Ab group (n=12) group and abdominal injection of monocrotaline. ROTATINE, MCT), group MCT+ETRA-Ab was injected with the polyclonal IgG ETRA antibody through the caudal vein after 1~3 intraperitoneal injection of MCT, 3 times a week, and the average pulmonary artery pressure (mPAP) was measured: 4 weeks after the MCT model, the right cervical vein was intubated by the right external jugular vein, and the Medlab-E biological function signal acquisition system was used to measure the mPAP. and the differences between the groups were analyzed and the right heart was analyzed. Right heart RVHI% determination: take out the heart, separate the left and right ventricles and the ventricular septum, weigh the mass of the right ventricular wall (RV) and the mass of the left ventricle and interventricular septum (LV+S), calculate the right heart hypertrophy index =RV/ (LV+S). Analysis the difference between each group.5, the pulmonary vascular morphological observation: the complete right upper lobe, formaldehyde fixation, dehydration, paraffin impregnation, embedding and slicing The morphological changes of the pulmonary arterioles in each group were observed by light microscopy. The changes of pulmonary arterioles (MA), partial muscle artery (PMA) and no muscle artery (NMA) in each group were compared with.6. The levels of serum inflammatory factor IL (interleukin IL) -6 and tumor necrosis factor (tumor necrosis factor TNF) - alpha were detected in each group, and the level of the serum level of the tumor necrosis factor (tumor necrosis factor TNF) - alpha was analyzed. Results: 1, the positive rate of ETRA antibody in group SSc-PAH and SSc non PAH group was compared with the former ETRA antibody positive rate 50.0%. positive rate of ETRA antibody was 12.5%, and the positive rate of ETRA antibody in normal control group was significant difference (P0.01).2. The level of antibody titer in the sera of the SSc-PAH patients was related to the pulmonary artery pressure. Sex (r=0.3439 P=0.0321). There was a significant negative correlation between the ETRA antibody titer level and the 6 minute walking distance test (r=-0.3221, P=0.0391), and the correlation with the results of NT-proBNP (r=0.3335, P=0.0318). There was a positive correlation between the end circulation detection index (r=0.3439, P=0.0321).3, the establishment of a MCT model for fourth weeks, the injection of 10% chloral chloral, the abdominal cavity. The three groups of mPAP (mmHg) were compared: the normal control group (17.3 + 1.1).MCT group (30.8 + 2) and MCT+ETRA-Ab group (34.5 + 3.1) (P0.05).4. The right ventricular hypertrophy index was compared in the right ventricle after the pressure measurement: MCT group (0.355 + 0.051), MCT+ETRA-Ab group (0.402 + 0.049) (P0.05).5, HE staining of lung tissue, MA, three, three The changes in the proportion (PMA, PPMA, PNMA) in the group: PMA and PPMA in the group MCT+ETRA-Ab were higher than that of the MCT control group (P0.05). Both were obviously in the normal control group, and the plasma inflammatory factors were significantly higher than those in the normal control group. However, the level of the IL-6 in the MCT+ETRA-Ab group was significantly higher than that in the normal control group. The diagnosis of SSc-PAH has good sensitivity and high specificity, suggesting that ETRA-Ab can be used as a new biomarker for early diagnosis of SSc-PAH. The ETRA-Ab titer in SSc-PAH patients is positively related to PASP, NT-proBNP, and is negatively correlated with the distance test distance of 6 minutes. It suggests that the ETRA-Ab titer can be used as a assessment of the patient's condition and to judge the precondition of the patient's condition. The later index.3, ETRA-Ab has an up - regulation effect on the hemodynamic index, right heart hypertrophy index and pulmonary vascular morphologic change index in MCT rats. It also up-regulates the inflammatory factor IL-6, TNF- alpha in plasma, suggesting that ETRA-Ab as a functional antibody may be involved in many aspects, including pulmonary vascular reshaping, right heart hypertrophy and so on.

【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R593.25;R544.1

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