COPD患者外周血Rho激酶水平测定的临床意义

发布时间：2018-06-18 02:48

本文选题：慢性阻塞性肺疾病 + 肺动脉高压　；参考：《广州医学院》2012年硕士论文

【摘要】：研究背景慢性阻塞性肺疾病(COPD)是一种重要的慢性呼吸系统疾病，患病人数多，病死率高。由于其缓慢进行性发展，严重影响患者的劳动能力和生活质量。COPD患者在急性发作期过后，临床症状虽有所缓解，但其肺功能仍在继续恶化，并且由于自身防御和免疫功能的降低以及外界各种有害因素的影响，经常反复发作，而逐渐产生各种心肺并发症。世界卫生组织认为，慢性阻塞性肺疾病可以预防，但目前无法治愈。治疗有助于减缓病情发展，但这一疾病通常在一段时间后逐渐恶化。气道炎症是COPD的起始阶段，气道重塑是COPD气流阻塞的病理基础，是COPD病变持续发展的关键因素。因此，，研究其病理改变及发生机制对改善其防治效果及患者预后具有重要意义。慢性阻塞性肺疾病的一个重要的合并症就是肺动脉高压，慢性阻塞性肺疾病合并肺动脉高压是逐渐发生和缓慢进展的，肺动脉高压是慢性阻塞性肺疾病发展至肺源性心脏病的重要病理生理过程，如果肺动脉压持续升高，可导致右心负荷增加，最终导致右心衰竭。研究表明Rho/Rho激酶信号通路是机体各组织细胞普遍存在的一条信号转导通路，其通路的关键信号分子包括：Rho蛋白、Rho激酶和肌球蛋白磷酸酶。其通过调节细胞肌动蛋白骨架的聚合状态，参与调控了细胞形态维持、细胞粘附与迁移、细胞增殖与凋亡、基因转录、平滑肌收缩等多种生物学行为，介导了多种平滑肌与非平滑肌功能异常相关疾病的发生机制。在多种疾病动物模型平滑肌细胞中均存在Rho激酶的过多表达。Rho/Rho激酶信号通路参与了COPD的发病过程，Rho激酶抑制剂可抑制炎症细胞的迁移和趋化、拮抗炎性因子分泌，阻断炎性因子作用，从而发挥抗炎作用，改善气道重塑。本研究主要研究Rho/Rho激酶信号通路在慢性阻塞性肺疾病(COPD)及COPD伴发肺动脉高压（PAH）患者血清Rho激酶1（ROCK1）及外周血单核细胞ROCK1的表达变化,为临床防治COPD提供依据,为COPD及伴发肺动脉高压提供新的治疗靶点。目的通过酶联免疫吸附法(ELISA)及细胞流式仪技术等检测方法，检测正常人群、慢性阻塞性肺疾病(COPD)伴发或不伴发肺动脉高压（PAH）患者的血清和外周血单核细胞中的Rho关联含卷曲螺旋蛋白1（ROCK1）的表达变化，探讨Rho/Rho激酶信号通路在COPD伴发或不伴发PAH患者发病中的作用。方法 1.研究对象收集2010年1月~2011年1月在广州市第一人民医院鹤洞分院收治的COPD患者40例，其中COPD患者20例，COPD伴发肺动脉高压(PAH)患者20例，其中COPD患者组符合中华医学会呼吸分会2007年制定的慢性阻塞性肺疾病诊治指南的诊断标准；COPD伴发肺动脉高压患者组同时符合中华医学会呼吸分会2007年制定的慢性阻塞性肺疾病诊治指南的诊断标准及ESC2009年制定的肺动脉高压诊断与治疗指南的诊断标准。另取在本院体检中心健康体检者20例作为对照组。三组在年龄、性别、体重上尽量匹配。 2.肺功能测定所有研究对象均需进行肺功能检测，主要的检测指标包括用力肺活量（FVC）、第一秒用力呼气容积（FEV1）、FEV1/FVC及FEV1占预计值的百分比（FEV1％）、吸入支气管扩张剂（沙丁胺醇400ug）后的FEV1/FCV。 3.肺动脉平均压的测定所有研究对象均通过心脏彩色多普勒超声检查，测得安静状态下的肺动脉平均压（mPAP）。 4.血清ROCK1的表达水平检测 4.1标本采集：抽取研究对象空腹静脉血3ml，离心(3000R/15min.20℃)，留取上层血清1ml,于低温－80℃冰箱中保存待测。 4.2测定方法：血清ROCK1的表达水平检测方法为酶联免疫吸附法（ELISA），其中标本处理、测定方法和样本浓度计算均按照相关试剂盒说明书进行。 5.外周血单核细胞ROCK1的表达水平检测 5.1.标本采集：抽取研究对象空腹静脉血20ml，以改进的Ficoll-Hypaque密度分离法和塑料吸附法分离出单核细胞，于低温－80℃冰箱中保存待测。 5.2测定方法：采用流式细胞仪检测研究对象的外周血单核细胞ROCK1的表达水平。其中标本处理、测定方法和样本浓度计算均按照相关试剂盒说明书进行。结果 1.血清ROCK1表达水平在对照组、COPD组、COPD伴发PAH组分别为13.10±2.67、23.55±4.97和36.72±7.18pmol/L，COPD组、COPD伴发PAH组明显高于对照组。三组之间存在显著性差异（P0.01）。 2.外周血单核细胞ROCK1表达水平在对照组、COPD组、COPD伴发PAH组分别为3.61±2.44%、10.56±3.72%和31.21±8.83%，统计学分析显示三组之间存在显著性差异（P0.01）。 3.相关性分析显示受试者血清ROCK1水平与肺动脉平均压呈正相关（r=0.654，P0.05），外周血单核细胞ROCK1表达水平与肺动脉平均压亦呈正相关（r=0.664，P0.05），血清ROCK1水平与外周血单核细胞ROCK1表达水平呈显著正相关（r=0.864，P0.01）。结论 Rho/Rho激酶信号通路的启动参与了COPD疾病肺动脉高压的形成和发展，血清中的ROCK1表达升高可能与ROCK1在单核细胞中的高表达有关。
[Abstract]:Research background
Chronic obstructive pulmonary disease (COPD) is an important chronic respiratory disease, the number of patients and the high mortality. Because of its slow progressive development, the patients' working ability and quality of life seriously affect the patient's working ability and quality of life (.COPD) after the acute attack period, although the clinical symptoms are slowly solved, but the lung function is still deteriorating, and because of its own The decline in defense and immunity, as well as the influence of various harmful factors on the outside world, often recurs and gradually produces various kinds of cardiopulmonary complications. The WHO believes that chronic obstructive pulmonary disease can be prevented but can not be cured at present. Treatment helps to slow the development of the disease, but the disease usually worsens after a period of time. Airway inflammation is the starting stage of COPD. Airway remodeling is the pathological basis of COPD airflow obstruction. It is the key factor for the continuous development of COPD lesions. Therefore, it is of great significance to study its pathological changes and mechanism to improve the effect of prevention and treatment and the prognosis of the patients. Pulmonary hypertension is a gradual and slow progression of pulmonary hypertension. Pulmonary arterial hypertension is an important pathophysiological process for the development of chronic obstructive pulmonary disease to pulmonary heart disease. If the pulmonary arterial pressure continues to rise, it can lead to an increase in the right heart load and eventually lead to right heart failure.
The Rho/Rho kinase signaling pathway is a common signal transduction pathway in the tissues and cells of the body. The key signaling molecules of the pathway include: Rho protein, Rho kinase and myosin phosphatase, which regulate cell morphology maintenance, cell adhesion and migration by regulating the polymerization of actin cytoskeleton. Multiple biological behaviors, such as cell proliferation and apoptosis, gene transcription and smooth muscle contraction, mediate the pathogenesis of a variety of smooth muscle and smooth muscle dysfunction related diseases. There is an overexpression of Rho kinase signaling pathway in the pathogenesis of COPD kinase in the smooth muscle cells of various disease animal models, and the inhibition of Rho kinase inhibition. It can inhibit the migration and chemotaxis of inflammatory cells, inhibit secretion of inflammatory factors and block inflammatory factors, thereby playing an anti-inflammatory role and improving airway remodeling.
This study mainly studies the changes of the expression of Rho/Rho kinase signaling pathway in serum Rho kinase 1 (ROCK1) and peripheral blood mononuclear cells (ROCK1) in patients with chronic obstructive pulmonary disease (COPD) and COPD associated pulmonary hypertension (PAH), providing a basis for clinical prevention and treatment of COPD and providing new therapeutic targets for COPD and associated pulmonary hypertension.
objective
The expression of Rho in serum and peripheral blood mononuclear cells in patients with chronic obstructive pulmonary disease (COPD) with or without pulmonary hypertension (PAH) was detected by enzyme linked immunosorbent assay (ELISA) and cell flow cytometry, and the Rho/Rho kinase signaling pathway was discussed in COPD The role of the patients with or without PAH.
Method
1. research objects
40 patients with COPD were admitted to Guangzhou No.1 People's Hospital in January 2010 ~2011, including 20 cases of COPD patients and 20 cases of COPD associated with pulmonary hypertension (PAH), of which the COPD patients were in accordance with the diagnostic criteria for the diagnosis and treatment of chronic obstructive pulmonary disease in 2007; COPD accompanied by lung movement. Patients with pulse hypertension conformed to the diagnostic criteria of the guidelines for the diagnosis and treatment of chronic obstructive pulmonary disease in 2007, and the diagnostic criteria for the diagnosis and treatment of pulmonary hypertension in ESC2009, which were formulated in the year of the Chinese Medical Association in 2007. In addition, 20 patients in the physical examination center of the medical center of our hospital were taken as the group. The three groups were in the age, sex, and weight as much as possible. Match.
2. pulmonary function measurement
All the subjects were required to perform lung function tests, including the forced expiratory volume (FVC), the first second forced expiratory volume (FEV1), the percentage of FEV1/FVC and FEV1 (FEV1%), and the FEV1/ FCV. after inhalation of bronchodilator (salbutamol 400ug).
3. pulmonary artery mean pressure measurement
The mean pulmonary arterial pressure (mPAP) was measured by color Doppler echocardiography in all subjects.
Detection of expression level of 4. serum ROCK1
4.1 specimen collection: extract the fasting venous blood 3ml of the subjects, centrifugation (3000R/15min.20 C), leave the upper serum 1ml, and store it in the low temperature 80 degree refrigerator.
4.2 determination method: the expression level of serum ROCK1 was detected by enzyme linked immunosorbent assay (ELISA), in which the sample treatment, determination method and sample concentration were calculated according to the instructions of the related kit.
Detection of expression level of ROCK1 in 5. peripheral blood mononuclear cells
Sample collection of 5.1.: extracting 20ml from fasting venous blood, and separating mononuclear cells by improved Ficoll-Hypaque density separation and plastic adsorption, and preserved in the refrigerator at low temperature to 80 degrees C.
5.2 determination method: flow cytometry was used to detect the expression level of ROCK1 in peripheral blood mononuclear cells of the study subjects. The sample treatment, determination method and sample concentration were calculated according to the instructions of the related kit.
Result
1. the level of serum ROCK1 expression in the control group, the COPD group and the group of COPD with PAH were 13.10 + 2.67,23.55 + 4.97 and 36.72 + 7.18pmol/L respectively. The COPD group and the COPD accompanying PAH group were significantly higher than those in the control group. There was a significant difference between the three groups (P0.01).
2. the expression level of ROCK1 in peripheral blood mononuclear cells was 3.61 + 2.44%, 10.56 + 3.72% and 31.21 + 8.83% in the control group, COPD group and COPD group with PAH, respectively. Statistical analysis showed significant difference between the three groups (P0.01).
3. correlation analysis showed that the serum ROCK1 level was positively correlated with the average pressure of pulmonary artery (r=0.654, P0.05), and the level of ROCK1 expression in peripheral blood mononuclear cells was also positively correlated with the average pressure of pulmonary artery (r=0.664, P0.05). The serum ROCK1 level was significantly positively correlated with the level of ROCK1 table of peripheral blood mononuclear cells (r=0.864, P0.01).
conclusion
The initiation of Rho/Rho kinase signaling pathway is involved in the formation and development of pulmonary hypertension in COPD disease. The increase of ROCK1 expression in serum may be related to the high expression of ROCK1 in mononuclear cells.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.9

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