抗内皮素A型受体抗体与系统性硬化症相关肺动脉高压的相关性研究

发布时间：2018-04-30 03:35

本文选题：系统性硬化症 + 内皮素A型受体抗体　；参考：《南昌大学》2017年硕士论文

【摘要】：背景及目的:系统性硬化症(Systemic sclerosis,SSc)相关肺动脉高压(Pulmonary Arterial Hypertension,PAH)是导致SSc患者死亡的重要原因之一。因为其起病隐匿,临床表现常不典型,早期诊断困难,治疗效果不理想。目前对于SSc-PAH早期诊断,治疗,预后评估仍是困扰风湿免疫科医生的重要难题。肺动脉高压(PAH)是SSc相关的肺部疾病中预后最差的并发症之一,其病理变化与内皮细胞功能障碍及局部炎症有关,在晚期则表现为肺血管平滑肌细胞过度增殖、凋亡减少。在PAH的病理过程中内皮素-1(Endothelin-1,ET-1)起到了非常重要的作用。ET-1是由血管内皮细胞合成的21肽,作为一种强力持久的血管收缩剂,促进血管壁的肥厚和纤维化的发生。ET1正是通过与其受体(Endothelin receptor,ETR)(A型受体、B型受体)结合发挥收缩血管的作用,特别是位于肺动脉平滑肌细胞和血管内皮细胞的内皮素A型受体(Endothelin Receptor Type A,ETRA),是触发血管的收缩主要因素,同时在细胞增殖和肺动脉免疫微环境的调控中发挥了重要的作用。本研究拟通过检测抗内皮素A型受体抗体(Anti-Endothelin Receptor Type A Autoantibodies ETRA-Ab)在SSc-PAH患者血清中的阳性率和特异性,从而探索ETRA-Ab与SSc-PAH发病及疾病严重程度之间的关系。进而探讨抗内皮素A型受体抗体作为协助早期诊断SSc-PAH生物标记物的可能性。并通过动物实验进一步证实ETRA-Ab作为功能性抗体,在SSc-PAH发病机制中的作用。方法:1、临床观察:共纳入24例SSc-PAH样本。同时选择24例相匹配SSc非PAH对照,24份正常对照。记录临床数据,心彩超测肺动脉收缩压(PASP)。其中5例SSc-PAH行右心导管检测。测定各组6分钟步行距离(6WMD)。使用酶联免疫吸附试验(ELISA)法检测以上各组血浆中的抗ETAR抗体,计算阳性率。2、建立MCT肺动脉高压大鼠模型:共36只雄性SD大鼠,随机分为正常对照组(n=12)、MCT组(n=12)、MCT+ETRA-Ab组(n=12)。MCT组和MCT+ETRA-Ab组行腹腔注射野百合碱(Monocrotatine,MCT),MCT+ETRA-Ab组在腹腔注射MCT后第1~3周经尾静脉给予注入多克隆IgG ETRA抗体,每周3次。3、平均肺动脉压力(mPAP)测定:于MCT造模后4周,经右颈外静脉插管,用Medlab-E生物机能信号采集系统测mPAP。并使用SPSS20.0分析各组间的差异。4、右心肥厚指数(RVHI%)测定:取出心脏,分离左右心室和室间隔,分别称量右心室壁(RV)的质量和左心室和室间隔(LV+S)的质量,计算右心肥厚指数=RV/(LV+S)。分析各组间的差异。5、肺血管形态学观察:切取完整右肺上叶,甲醛固定、脱水、浸蜡、包埋、切片、烘干、HE染色。光镜下观察各组大鼠肺小动脉形态学变化。比较各组肺血管肌型动脉(MA),部分肌型动脉(PMA),无肌型动脉(NMA)比例的变化。6、检测各组血浆中炎症因子白介素(interleukin IL)-6、肿瘤坏死因子(tumor necrosis factor TNF)-α的水平,分析各组间差异。结果:1、SSc-PAH组与SSc非PAH组ETRA抗体阳性率比较前者ETRA抗体阳性率50.0%。后者ETRA抗体阳性率为12.5%,正常对照组ETRA抗体阳性率为0%。阳性率比较有显著性差异(P0.01)。2、SSc-PAH患者血清中ETRA抗体滴度水平与肺动脉压力PASP有相关性(r=0.3439 P=0.0321)。而ETRA抗体滴度水平与6分钟步行距离测试结果之间存在显著的负相关(r=-0.3221,P=0.0391),与NT-proBNP结果呈现相关性(r=0.3335,P=0.0318)。与末梢循环检测指数呈正相关(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,经颈外静脉插管,行三组间mPAP(mmHg)比较:正常对照组(17.3±1.1)。MCT组(30.8±2.0)、MCT+ETRA-Ab组(34.5±3.1)(P0.05)。4、测压后处死动物右室分离行右心肥厚指数比较:MCT组(0.355±0.051)、MCT+ETRA-Ab组(0.402±0.049)(P0.05)。5、肺组织HE染色,比较MA、PMA、NMA在三组中所占比例(PMA、PPMA、PNMA)的变化:MCT+ETRA-Ab组中PMA、PPMA高于MCT对照组(P0.05),两者均明显正常对照组。6、血浆炎症因子测定:MCT组、MCT+ETRA-Ab组中IL-6、TNF-α(pg/ml)水平明显高于正常对照组。但MCT+ETRA-Ab组较MCT组升高更加显著(P0.05)。结论:1、ETRA-Ab对于SSc-PAH的诊断有较好的敏感性和较高的特异性,从而提示ETRA-Ab可以作为SSc-PAH早期诊断的新型生物标记物。2、SSc-PAH患者中ETRA-Ab滴度与PASP、NT-proBNP正相关,与6分钟步行距离测试距离负相关。提示ETRA-Ab滴度可以作为评估患者病情,判断预后的指标。3、ETRA-Ab对MCT大鼠的血流动力学指标、右心肥厚指数、肺血管形态变化指数升高均有上调作用。并上调血浆中的炎症因子IL-6、TNF-α。提示ETRA-Ab作为功能性抗体可能参与到了包括肺血管重塑,右心肥厚等多个方面。
[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.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.25;R544.1

【参考文献】