本文選題：肺動脈重構(gòu) + 香煙煙霧暴露　； 參考：《南京醫(yī)科大學(xué)》2013年博士論文

【摘要】：第一部分吸煙者及COPD患者肺小血管TNF-α的表達及肺血管內(nèi)皮細胞凋亡的研究目的：通過觀察吸煙者和慢性阻塞性肺病患者肺組織切片中肺血管形態(tài)、肺動脈內(nèi)皮細胞凋亡、肺組織TNF-α表達變化,探討香煙暴露所致肺血管損傷可能的機制。方法：2010年6月至2011年8月在南京醫(yī)科大學(xué)第一附屬醫(yī)院胸外科行肺切除術(shù)的病例32例,將其分成3組：對照組8例；吸煙非COPD組13例;吸煙并COPD組11例。HE染色和VG染色觀察肺血管形態(tài),TUNEL法檢測肺血管內(nèi)皮細胞凋亡指數(shù),免疫組織化學(xué)法檢測TNF-α蛋白的分布和表達。分析肺小動脈形態(tài)學(xué)變化特點及吸煙人群中吸煙指數(shù)、肺血管內(nèi)皮細胞凋亡指數(shù)、肺血管內(nèi)皮細胞TNF-α表達三者之間的相關(guān)性。結(jié)果：1.吸煙組和COPD組肺小動脈血管管壁較正常對照組明顯增厚,差異具有統(tǒng)計學(xué)意義。2.與正常組相比,吸煙組和COPD組患者肺血管內(nèi)皮細胞凋亡率明顯增加,差異具有統(tǒng)計學(xué)顯著性。免疫組化結(jié)果顯示,吸煙組和COPD組患者肺血管TNF-α表達增加。3.經(jīng)相關(guān)性分析,吸煙人群中,肺血管細胞凋亡率與吸煙指數(shù)呈正相關(guān)(r=0.701,P0.01),肺血管TNF-α平均光密度與吸煙指數(shù)呈正相關(guān)(r=0.417,P0.05)。結(jié)論：與不吸煙對照人群相比,吸煙者和COPD患者都表現(xiàn)出肺小動脈結(jié)構(gòu)改變；其肺血管內(nèi)皮細胞凋亡率和TNF-α表達量均明顯增加,且都與吸煙指數(shù)存在相關(guān)性。第二部分TNF-a抑制劑Etanercept對香煙煙霧提取物誘導(dǎo)的人肺動脈內(nèi)皮細胞凋亡中的作用目的：研究腫瘤壞死因子-α抑制劑Etanercept對香煙煙霧提取物(cigarette smoking extract, CSE)誘導(dǎo)的人肺動脈內(nèi)皮細胞凋亡中的作用。方法：體外培養(yǎng)人肺動脈內(nèi)皮細胞(HPAEC),應(yīng)用MTT法檢測不同干預(yù)對其細胞活力的影響,根據(jù)結(jié)果分為以下四組,分別給予不同處理：1.對照組(未作特殊處理)；2.10%CSE干預(yù)(CSE組)；3. TNF-a抑制劑Etanercept(Entanercept, ECT)預(yù)處理后,以10%CSE干預(yù)(CSE+ECT組)；4.ECT組。以上各組采用流式細胞術(shù)Annexin V/PI雙染法檢測內(nèi)皮細胞凋亡率,Hochest染色觀察細胞凋亡形態(tài),用Western Blot檢測Caspase-3、 Caspase-8、Caspase-9活化情況。結(jié)果：1.與對照組相比,CSE組HPAEC凋亡顯著增加,ECT+CSE組凋亡較CSE組有所減少,但仍高于對照組,差異具有統(tǒng)計學(xué)意義；而ECT組與對照組比較未見顯著差異。2.與對照組相比,CSE組HPAEC中Caspase-3活性表達明顯增加,ECT+CSE組Caspase-3活性表達較CSE組有所減少,但仍高于對照組,差異具有統(tǒng)計學(xué)意義。3.與對照組相比,CSE組HPAEC中Caspase-8、Caspase-9活性表達均明顯增加,ECT+CSE組Caspase-8、Caspase-9活性表達較CSE組有所減少,但仍高于對照組,差異具有統(tǒng)計學(xué)意義。結(jié)論：1.Etanercept干預(yù)抑制CSE誘導(dǎo)所致的HPAEC凋亡。2. Etanercept干預(yù)通過下調(diào)Caspase-3的活化,對CSE刺激誘導(dǎo)的HPAEC凋亡發(fā)揮保護作用。3.Caspase-8、Caspase-9的活化水平減低參與了Etanercept抵抗CSE刺激所致細胞凋亡的藥理作用機制。第三部分TNF-α抑制劑Etanercept對香煙暴露大鼠肺血管重構(gòu)的影響及其機制目的：探討短期香煙煙霧暴露對大鼠肺血管構(gòu)型重建的機制以及TNF-α抑制劑Etanercept (Entanercept, ECT)的干預(yù)作用方法： SD大鼠分為三組,分別予以不同的處理：1.對照組：正常條件飼養(yǎng);2.吸煙組：置于自制熏箱內(nèi)進行被動吸煙,每天2次,每次吸煙10支,每周6天,共吸煙2周；3.吸煙同時加用ECT組：每周三次皮下注射Etanercept藥液,每次0.4mg/kg。每次給藥至少早于香煙煙霧暴露之前4小時。其他飼養(yǎng)和香煙煙霧暴露條件同吸煙組。采用HE染色法和Masson染色法觀察各組大鼠肺組織病理變化和膠原沉積,免疫組織化學(xué)法和明膠酶譜分析法檢測大鼠肺組織MMP-2、MMP-9蛋白表達和活性變化,酶聯(lián)免疫吸附法檢測肺組織勻漿中TNF-α表達水平,以及Western Blot檢測NF-κB p65核轉(zhuǎn)位情況。結(jié)果：1.與對照組相比,吸煙組大鼠肺小動脈厚度百分比明顯增加,肺動脈壓力上升,血管外膜膠原組織沉積加重。與吸煙組相比,ECT干預(yù)組大鼠肺小動脈厚度減少,肺動脈壓降低,血管外膜膠原組織沉積減輕,但上述指標(biāo)仍高于對照組。2.與對照組相比,吸煙組和Etanercept干預(yù)組大鼠肺小動脈MMP-2、 MMP-9蛋白表達水平顯著增加。與吸煙組相比,Etanercept干預(yù)組大鼠肺小血管MMP-2、MMP-9蛋白表達水平有所下降,差異具有統(tǒng)計學(xué)意義。明膠酶譜分析結(jié)果示吸煙組大鼠肺組織MMP-2、MMP-9活化程度較對照組明顯增加,ECT組大鼠肺組織MMP-2、MMP-9活化程度較吸煙組有所減低,但仍高于對照組。3.吸煙組大鼠肺組織TNF-α濃度較對照組明顯升高,ECT干預(yù)組大鼠肺組織TNF-α濃度較吸煙組降低,但較正常對照組為高。結(jié)論：1.短期香煙煙霧暴露即可導(dǎo)致大鼠肺血管重構(gòu)和肺動脈壓力上升；2.Etanercept可降低香煙煙霧暴露誘導(dǎo)的大鼠肺動脈壓升高,抑制肺血管構(gòu)型重建,減輕肺血管外膜膠原沉積；3.Etanercept可部分逆轉(zhuǎn)香煙煙霧暴露所致的大鼠肺組織MMP-2、MMP-9表達上調(diào)和活化；4.抑制香煙煙霧引起的TNF-α表達增加和減低NF-κB p65核轉(zhuǎn)位參與了Etanercept對香煙煙霧暴露的保護性作用過程。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R563.9

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7 史記;Vam3對香煙煙霧誘導(dǎo)肺上皮細胞自噬的影響及作用機理研究[D];北京協(xié)和醫(yī)學(xué)院;2012年

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