TNF-α在吸烟所致肺血管损伤中的作用及Etanercept的干预机制

发布时间：2018-05-09 18:41

本文选题：肺动脉重构 + 香烟烟雾暴露　；参考：《南京医科大学》2013年博士论文

【摘要】：第一部分吸烟者及COPD患者肺小血管TNF-α的表达及肺血管内皮细胞凋亡的研究目的：通过观察吸烟者和慢性阻塞性肺病患者肺组织切片中肺血管形态、肺动脉内皮细胞凋亡、肺组织TNF-α表达变化,探讨香烟暴露所致肺血管损伤可能的机制。方法：2010年6月至2011年8月在南京医科大学第一附属医院胸外科行肺切除术的病例32例,将其分成3组：对照组8例；吸烟非COPD组13例;吸烟并COPD组11例。HE染色和VG染色观察肺血管形态,TUNEL法检测肺血管内皮细胞凋亡指数,免疫组织化学法检测TNF-α蛋白的分布和表达。分析肺小动脉形态学变化特点及吸烟人群中吸烟指数、肺血管内皮细胞凋亡指数、肺血管内皮细胞TNF-α表达三者之间的相关性。结果：1.吸烟组和COPD组肺小动脉血管管壁较正常对照组明显增厚,差异具有统计学意义。2.与正常组相比,吸烟组和COPD组患者肺血管内皮细胞凋亡率明显增加,差异具有统计学显著性。免疫组化结果显示,吸烟组和COPD组患者肺血管TNF-α表达增加。3.经相关性分析,吸烟人群中,肺血管细胞凋亡率与吸烟指数呈正相关(r=0.701,P0.01),肺血管TNF-α平均光密度与吸烟指数呈正相关(r=0.417,P0.05)。结论：与不吸烟对照人群相比,吸烟者和COPD患者都表现出肺小动脉结构改变；其肺血管内皮细胞凋亡率和TNF-α表达量均明显增加,且都与吸烟指数存在相关性。第二部分TNF-a抑制剂Etanercept对香烟烟雾提取物诱导的人肺动脉内皮细胞凋亡中的作用目的：研究肿瘤坏死因子-α抑制剂Etanercept对香烟烟雾提取物(cigarette smoking extract, CSE)诱导的人肺动脉内皮细胞凋亡中的作用。方法：体外培养人肺动脉内皮细胞(HPAEC),应用MTT法检测不同干预对其细胞活力的影响,根据结果分为以下四组,分别给予不同处理：1.对照组(未作特殊处理)；2.10%CSE干预(CSE组)；3. TNF-a抑制剂Etanercept(Entanercept, ECT)预处理后,以10%CSE干预(CSE+ECT组)；4.ECT组。以上各组采用流式细胞术Annexin V/PI双染法检测内皮细胞凋亡率,Hochest染色观察细胞凋亡形态,用Western Blot检测Caspase-3、 Caspase-8、Caspase-9活化情况。结果：1.与对照组相比,CSE组HPAEC凋亡显著增加,ECT+CSE组凋亡较CSE组有所减少,但仍高于对照组,差异具有统计学意义；而ECT组与对照组比较未见显著差异。2.与对照组相比,CSE组HPAEC中Caspase-3活性表达明显增加,ECT+CSE组Caspase-3活性表达较CSE组有所减少,但仍高于对照组,差异具有统计学意义。3.与对照组相比,CSE组HPAEC中Caspase-8、Caspase-9活性表达均明显增加,ECT+CSE组Caspase-8、Caspase-9活性表达较CSE组有所减少,但仍高于对照组,差异具有统计学意义。结论：1.Etanercept干预抑制CSE诱导所致的HPAEC凋亡。2. Etanercept干预通过下调Caspase-3的活化,对CSE刺激诱导的HPAEC凋亡发挥保护作用。3.Caspase-8、Caspase-9的活化水平减低参与了Etanercept抵抗CSE刺激所致细胞凋亡的药理作用机制。第三部分TNF-α抑制剂Etanercept对香烟暴露大鼠肺血管重构的影响及其机制目的：探讨短期香烟烟雾暴露对大鼠肺血管构型重建的机制以及TNF-α抑制剂Etanercept (Entanercept, ECT)的干预作用方法： SD大鼠分为三组,分别予以不同的处理：1.对照组：正常条件饲养;2.吸烟组：置于自制熏箱内进行被动吸烟,每天2次,每次吸烟10支,每周6天,共吸烟2周；3.吸烟同时加用ECT组：每周三次皮下注射Etanercept药液,每次0.4mg/kg。每次给药至少早于香烟烟雾暴露之前4小时。其他饲养和香烟烟雾暴露条件同吸烟组。采用HE染色法和Masson染色法观察各组大鼠肺组织病理变化和胶原沉积,免疫组织化学法和明胶酶谱分析法检测大鼠肺组织MMP-2、MMP-9蛋白表达和活性变化,酶联免疫吸附法检测肺组织匀浆中TNF-α表达水平,以及Western Blot检测NF-κB p65核转位情况。结果：1.与对照组相比,吸烟组大鼠肺小动脉厚度百分比明显增加,肺动脉压力上升,血管外膜胶原组织沉积加重。与吸烟组相比,ECT干预组大鼠肺小动脉厚度减少,肺动脉压降低,血管外膜胶原组织沉积减轻,但上述指标仍高于对照组。2.与对照组相比,吸烟组和Etanercept干预组大鼠肺小动脉MMP-2、 MMP-9蛋白表达水平显著增加。与吸烟组相比,Etanercept干预组大鼠肺小血管MMP-2、MMP-9蛋白表达水平有所下降,差异具有统计学意义。明胶酶谱分析结果示吸烟组大鼠肺组织MMP-2、MMP-9活化程度较对照组明显增加,ECT组大鼠肺组织MMP-2、MMP-9活化程度较吸烟组有所减低,但仍高于对照组。3.吸烟组大鼠肺组织TNF-α浓度较对照组明显升高,ECT干预组大鼠肺组织TNF-α浓度较吸烟组降低,但较正常对照组为高。结论：1.短期香烟烟雾暴露即可导致大鼠肺血管重构和肺动脉压力上升；2.Etanercept可降低香烟烟雾暴露诱导的大鼠肺动脉压升高,抑制肺血管构型重建,减轻肺血管外膜胶原沉积；3.Etanercept可部分逆转香烟烟雾暴露所致的大鼠肺组织MMP-2、MMP-9表达上调和活化；4.抑制香烟烟雾引起的TNF-α表达增加和减低NF-κB p65核转位参与了Etanercept对香烟烟雾暴露的保护性作用过程。
[Abstract]:Part one: the expression of TNF- alpha and apoptosis of pulmonary vascular endothelial cells in smokers and COPD patients. Objective: To investigate the pulmonary vascular morphology, apoptosis of pulmonary artery endothelial cells and the expression of TNF- alpha in lung tissue in smokers and patients with chronic obstructive pulmonary disease, and to explore the possible pulmonary vascular injury caused by cigarette exposure. Methods: 32 cases of pulmonary resection in Department of thoracic surgery, the First Affiliated Hospital of Nanjing Medical University from June 2010 to August 2011 were divided into 3 groups: 8 cases in control group, 13 cases in non COPD smoking group, 11 cases of.HE and VG staining in group COPD, and TUNEL method to detect the apoptosis index of pulmonary vascular endothelial cells and the immune tissue. The distribution and expression of TNF- alpha protein were detected by chemical method. The morphological changes of pulmonary arterioles and the correlation between the smoking index, the apoptosis index of the pulmonary vascular endothelial cells and the expression of TNF- alpha in the pulmonary vascular endothelial cells were analyzed. The results showed that the vascular wall of the pulmonary arterioles in the 1. smoking group and the COPD group was thicker than the normal control group, and the difference between the three group and the COPD group was significantly different. Compared with the normal group, the apoptosis rate of pulmonary vascular endothelial cells in the smoking group and the COPD group was significantly increased, and the difference was statistically significant. The results of immunohistochemistry showed that the expression of TNF- alpha in pulmonary vessels in smoking and COPD groups increased by.3. correlation analysis, and the apoptosis rate of pulmonary vascular cells and smoking index were in the smoking population. R=0.701 (P0.01), the mean light density of pulmonary vascular TNF- alpha was positively correlated with the smoking index (r=0.417, P0.05). Conclusion: compared with the non smoking control group, both smokers and COPD patients showed structural changes in the pulmonary arteriole, the apoptosis rate and the expression of TNF- a in the pulmonary vascular endothelial cells were significantly increased, and all were related to the smoking index. Second part of the effect of TNF-a inhibitor Etanercept on the apoptosis of human pulmonary artery endothelial cells induced by cigarette smoke extract: the role of tumor necrosis factor - alpha inhibitor Etanercept in the apoptosis of human pulmonary artery endothelial cells induced by cigarette smoke extract (cigarette smoking extract, CSE). Methods: in vitro culture Human pulmonary artery endothelial cell (HPAEC) was used to detect the effect of different intervention on cell viability by MTT method. According to the results, the results were divided into four groups, respectively: 1. control groups (no special treatment); 2.10%CSE intervention (group CSE); 3. TNF-a inhibitor Etanercept (Entanercept, ECT) pretreatment, 10%CSE intervention (CSE+ECT group); 4.EC Group T. The apoptotic rate of endothelial cells was detected by flow cytometry and Annexin V/PI double staining, Hochest staining was used to observe the morphology of apoptosis, and Caspase-3, Caspase-8 and Caspase-9 activation were detected by Western Blot. Results: 1. compared with the control group, HPAEC apoptosis was significantly increased in CSE group, and the apoptosis in ECT+CSE group was less than that in the group, but still higher than that in the control group. There was no significant difference between the control group and the control group, but there was no significant difference between the ECT group and the control group. Compared with the control group, the expression of Caspase-3 activity in the group CSE was significantly increased, and the expression of Caspase-3 activity in the group ECT+CSE was lower than that of the CSE group, but it was still higher than the control group, but it was still higher than the control group. The difference has the significance of.3. in the HPAEC Caspas group and CSE group HPAEC in HPAEC Caspas. The activity expression of E-8, Caspase-9 increased obviously, and the expression of Caspase-8 and Caspase-9 in group ECT+CSE decreased, but it was still higher than that of the control group, but it was still higher than the control group. The difference has statistical significance. Conclusion: 1.Etanercept intervention inhibits HPAEC apoptosis induced by CSE,.2. Etanercept intervention through activation of lower modulation Caspase-3. The protective effect of.3.Caspase-8, the decrease of activation level of Caspase-9 participates in the pharmacological mechanism of Etanercept resistance to CSE induced apoptosis. Third the effect and mechanism of TNF- alpha inhibitor Etanercept on pulmonary vascular remodeling in rats exposed to cigarette smoke and its mechanism: To explore the reconstruction of pulmonary vascular configuration in rats by short term smoke smoke exposure The mechanism and the intervention method of TNF- alpha inhibitor Etanercept (Entanercept, ECT): SD rats were divided into three groups, which were treated in different ways: 1. control groups: normal conditions; 2. smoking groups: smoking in a self-made smoked box, 2 times a day, smoking 10 cigarettes each time, 6 days a week for 2 weeks; 3. smoking and E at the same time. Group CT: three times a week, Etanercept solution was injected subcutaneously. Each time 0.4mg/kg. was given at least 4 hours before cigarette smoke exposure. Other feeding and cigarette smoke exposure were the same as smoking group. HE staining and Masson staining were used to observe the pathological changes of lung tissue and collagen deposition in each group, immunohistochemistry and gelatinase spectrum. The expression and activity of MMP-2 and MMP-9 protein in lung tissue of rats were detected by analysis. The expression of TNF- alpha in lung tissue homogenate was detected by enzyme linked immunosorbent assay, and NF- kappa B p65 nuclear transposition was detected by Western Blot. Results: 1. compared with the control group, the percentage of pulmonary arteriole thickness increased significantly in the smoking group, the pulmonary artery pressure increased, and the pulmonary artery pressure increased, and the extravascular blood vessels were increased. Compared with the smoking group, the thickness of the pulmonary arteriole decreased, the pulmonary artery pressure decreased, and the collagen tissue deposited in the epicardial membrane in the ECT intervention group, but the above index was still higher than that of the control group.2., the small pulmonary artery of the smoking group and the Etanercept intervention group was MMP-2, and the expression of MMP-9 protein was significantly increased. Compared with the smoke group, the lung small blood vessels of the Etanercept intervention group were MMP-2, the expression level of MMP-9 protein decreased and the difference was statistically significant. The results of gelatinase analysis showed that the lung tissue of the smoking group rats was MMP-2, the activation degree of MMP-9 was significantly higher than that of the control group. The activation degree of the lung tissue in the ECT group was lower than that in the smoking group, but still higher than that in the smoking group. The concentration of TNF- alpha in lung tissue in the control group of.3. smoking group was significantly higher than that in the control group. The concentration of TNF- alpha in lung tissue in the ECT intervention group was lower than that in the smoking group, but it was higher than that in the normal control group. Conclusion: 1. short term cigarette smoke exposure can lead to pulmonary vascular remodeling and pulmonary arterial pressure rise in rats; 2.Etanercept can reduce the exposure of cigarette smoke. The pulmonary arterial pressure in rats was increased, the pulmonary vascular configuration was inhibited and the collagen deposition was reduced in the pulmonary vascular outer membrane. 3.Etanercept could partly reverse the MMP-2 and MMP-9 expression of lung tissue in rats induced by cigarette smoke exposure. 4. inhibition of the expression of TNF- alpha caused by cigarette smoke increased and reduced NF- kappa B p65 nuclear transposition to participate in Etanercept against incense. The protective action of smoke exposure.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R563.9

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