本文關(guān)鍵詞：全身炎癥反應(yīng)對(duì)慢性阻塞性肺疾病氣道、肺血管重構(gòu)的影響，，由筆耕文化傳播整理發(fā)布。

        目的：研究慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)大鼠模型發(fā)病過(guò)程中,炎癥因子表達(dá)水平的動(dòng)態(tài)變化以評(píng)估COPD大鼠全身炎癥反應(yīng)的程度及其與氣道及肺血管重塑情況的關(guān)系,同時(shí)探討NF-κ B抑制劑吡咯烷二硫代氨基甲酸鹽(pyrrolidine dithiocarbamate, PDTC))對(duì)慢性阻塞性肺疾病大鼠模型肺組織中炎癥因子表達(dá)的影響及其干預(yù)后COPD大鼠模型氣道及肺血管重塑情況的變化,從而了解全身炎癥反應(yīng)在慢性阻塞性肺疾病發(fā)病過(guò)程中的作用及PDTC對(duì)慢性阻塞性肺疾病全身炎癥反應(yīng)的影響,為慢性阻塞性肺疾病的治療提供新的思路。方法：實(shí)驗(yàn)分為兩部分。第一部分檢測(cè)慢性阻塞性肺疾病(COPD)大鼠模型發(fā)病過(guò)程中炎癥因子：高遷移率族蛋白B1(high mobility group protein B1, HMGB1),核因子-κB (the nuclear factor-κ B, NF-κB), TNF-α、IL-8、血管內(nèi)皮生長(zhǎng)因子(Vascular endothelial growth factor, VEGF)表達(dá)水平的動(dòng)態(tài)變化及其與氣道重構(gòu)和肺血管重塑情況的關(guān)系。實(shí)驗(yàn)采用香煙霧暴露疊加低氧復(fù)制COPD大鼠模型(共分三組,每組隨機(jī)分配SD大鼠8只：正常對(duì)照組(A)：第1d和第14d分別經(jīng)氣道內(nèi)注入生理鹽水,6周后檢測(cè)；COPD組(B)：第1d和第14d分別經(jīng)氣道注入LPS,200μg/次,香煙煙霧暴露1h/d,共6周；COPD并低氧組(C)：第1d和第14d分別經(jīng)氣道注入LPS,200μg/次,香煙煙霧暴露1h/d,,共6周,實(shí)驗(yàn)的最后兩周同時(shí)給予疊加18%低氧(8h/d)。第二部分檢測(cè)PDTC對(duì)慢性阻塞性肺疾病大鼠模型炎癥因子表達(dá)的影響：即干預(yù)前后肺組織中NF-κB蛋白水平,HMGB1mRNA和蛋白表達(dá),血清、BALF中TNF-α、IL-8、VEGF的含量變化；干預(yù)后COPD大鼠模型氣道重構(gòu)和肺血管重塑情況。與第一部分方法相同設(shè)立A、B、C共三個(gè)實(shí)驗(yàn)?zāi)Ｐ徒M,每組隨機(jī)分配SD大鼠8只：PDTC干預(yù)組為：A1(空白對(duì)照組)、B1(COPD干預(yù)組)、C1(COPD并低氧干預(yù)組)。動(dòng)物模型制作同各對(duì)應(yīng)組,從實(shí)驗(yàn)第3周起給各藥物干預(yù)組腹腔注射干預(yù)藥物PDTC,劑量為100mg· kg-1· d-1。Al組自實(shí)驗(yàn)第三周起腹腔注射與PDTC相同劑量的生理鹽水。各組動(dòng)物模型完成造模后均檢測(cè)以下指標(biāo)：①HE染色觀察肺組織病理改變；②VG+維多利亞藍(lán)特殊染色檢測(cè)氣道重構(gòu)及肺血管重塑指標(biāo)：氣道重塑指標(biāo)(細(xì)支氣管管壁厚度與氣管外徑比值(MT%),管壁面積和氣管總面積比值(MA%))；肺血管重塑指標(biāo)(肺小動(dòng)脈血管壁厚度與血管外徑比(WT%),血管壁面積與血管總面積比(WA%))。③采用不同方法檢測(cè)各炎癥因子的表達(dá)情況：Western blot法檢測(cè)各組肺組織NF-κB蛋白表達(dá)情況、RT-PCR及Western blot分別檢測(cè)各組肺組織勻漿中HMGB1mRNA及蛋白表達(dá)情況,ELISA法分別檢測(cè)各組血及清肺泡灌洗液(BALF)中TNF-α、IL-8、VEGF的表達(dá)情況。④分析上述各炎癥因子的表達(dá)情況與氣道重構(gòu)、肺血管重塑指標(biāo)的相關(guān)性,以及PDTC對(duì)各炎癥因子表達(dá)的影響。結(jié)果：1.氣道重塑指標(biāo)(MT%、MA%):與A組比較,B、C組氣道重構(gòu)指標(biāo)細(xì)支氣管管壁厚度與氣管外徑比值(MT%)、管壁面積和氣管總面積比值(MA%)均明顯增高(P<0.05)。B、C組間MT%、MA%無(wú)差異。B1組與B組,C1組與C組比較,細(xì)支氣管MT%、MA%均下降(P<0.05)。2.肺血管重塑指標(biāo)(WT%、WA%):與A組相比,B組肺血管重構(gòu)僅表現(xiàn)為肌化型動(dòng)脈百分比增高,肌化型動(dòng)脈管壁厚度尚未增加,未見(jiàn)肌化型動(dòng)脈WT%、WA%明顯改變(P>0.05),而C組不僅出現(xiàn)肌化動(dòng)脈百分比增高,并且出現(xiàn)肌化型動(dòng)脈WT%、WA%值增高(P<0.05)。使用NF-κB抑制劑吡咯烷二硫代氨基甲酸鹽(PDTC))干預(yù)后,B1組較B組,C1組較C組的WT%、 WA%降低(P<0.05)。而A1組WA%與對(duì)應(yīng)的試驗(yàn)組A組比較無(wú)明顯差異(P>0.05)。3.各炎癥因子的表達(dá)情況3.1NF-κ B的表達(dá)情況：Western blot電泳結(jié)果顯示,B、C組NF-κB蛋白表達(dá)與A組相比明顯增強(qiáng)(P<0.05),藥物PDTC干預(yù)組B1、C1組NF-κB蛋白表達(dá)比較B、C組降低；3.2HMGB1的表達(dá)情況：RT-PCR及Western blot結(jié)果說(shuō)明, B、C組HMGB1mRNA、蛋白表達(dá)與A組相比均明顯增強(qiáng)(P<0.05),且B組、C組表達(dá)有逐漸增加的趨勢(shì),藥物PDTC干預(yù)組B1、CI組與對(duì)應(yīng)的實(shí)驗(yàn)組B、C組比較HMGB1mRNA、蛋白表達(dá)均降低(P<0.05)；3.3TNF-α、IL-8、VEGF的表達(dá)情況：ELISA結(jié)果表明：B組和C組BALF及血清中TNF-α、IL-8、VEGF表達(dá)水平高于A組,藥物PDTC干預(yù)組B1、C1組TNF-α、IL-8、VEGF表達(dá)較對(duì)應(yīng)B、C組下降,(P<0.05)。4.各炎癥因子的表達(dá)情況與氣道重構(gòu)、肺血管重塑指標(biāo)的相關(guān)性分析：炎癥因子NF-κB、HMGB1的表達(dá)量與氣道重構(gòu)指標(biāo)(MT%、MA%)及肺血管重塑指標(biāo)(WT%、WA%)成正相關(guān)；大鼠肺組織內(nèi)NF-κB蛋白的表達(dá)量與大鼠肺組織內(nèi)HMGB1的表達(dá)量及肺泡灌洗液(BALF)中IL-8、TNF-α、VEGF的表達(dá)成正相關(guān)；大鼠肺組織內(nèi)HMGB1的表達(dá)量與肺泡灌洗液(BALF)中IL-8、TNF-α、VEGF的表達(dá)成正相關(guān)。使用NF-κB抑制劑吡咯烷二硫代氨基甲酸鹽(PDTC))干預(yù)后,各炎癥因子的表達(dá)量下降,氣道重構(gòu)、肺血管重塑部分緩解。結(jié)論：①COPD的炎癥不僅局限于肺部,同時(shí)存在全身炎癥反應(yīng)；全身炎癥反應(yīng)參與了COPD病情發(fā)生發(fā)展的全過(guò)程,并隨著COPD病情的加重,炎癥因子的表達(dá)逐漸增高。全身炎癥反應(yīng)可能通過(guò)影響肺血管、氣道重構(gòu)參與COPD病情的進(jìn)展。②NF-κB可通過(guò)調(diào)節(jié)HMGB1、TNF-α、IL-8、VEGF的表達(dá)影響氣道、肺血管的重構(gòu),NF-κB在細(xì)胞因子網(wǎng)絡(luò)的調(diào)節(jié)中的起核心作用。

    Objective:To detect the changes of inflammatory cytokines expression in the development of COPD and To assess the degree of systemic inflammatory response in COPD,Find out the relationship between systemic inflammatory response and airway remodeling, also the relationship between systemic inflammatory response and the pulmonary vascular remodeling. To understand the effect of PDTC to inflammatory cytokines expression in animal mode lung tissue of COPD. To research the changes of airway remodeling and pulmonary vascular remodeling after use PDTC.Find out the effect of systemic inflammatory response in the development of COPD and the influence of the PDTC to airway remodeling and pulmonary vascular remodeling in COPD. Giving a new way to the treatment of COPD.Methods:There are two part of this research. Part1:To detect the changes of inflammatory cytokines(high mobility group protein B1,the nuclear factor-Kb, Tumor necrosis facto-a,Interleukin-8, Vascular endothelial growth factor)expression in the development of COPD. Find out the relationship between systemic inflammatory response and the airway and pulmonary vascular remodeling. The experiment uses the smog exposition with the low oxygen to make the animal mode of COPD.(There are three in this part of the research,8SD rat in each group randomly):Healthy control group (group A):0.2ml saline water was installed intratracheally once at first and forteenth day for all rats, testing after6weeks. COPD group (group B):200μg LPS was installed intratracheally once at first and forteenth day and the rats were exposed to cigarette smoke lh/d for6weeks. low oxygen with COPD group (group C):200μg LPS was installed intratracheally once at first and forteenth day and the rats were exposed to cigarette smoke1h/d for6weeks. In last2weeks chronic hypoxia (FiO2=0.18)8h/d were performed simultaneously.Part2:To understand the effect of NF-κB inhibitor pyrrolidine two dithiocarbamate (PDTC)), to inflammatory cytokines (HMGB1, IL-8, TNF-a, VEGF, NF-κB) expression in animal mode of COPD. To research the changes of airway and pulmonary vascular remodeling after use PDTC.(There are three group in this part of the research,8SD rat in each group randomly):PDTC intervention group include:A1(Healthy control group),B1(COPD intervented group), C1(low oxygen with COPD intervented group). Animal model were made the same as the corresponding group, PDTC100mg-kg-1·d-1were injected into the rats in each drug group from the third week. The saline water which was the same dose of PDTC was injected into the rats in A1group from the third week.We examine the following target of rat in each group after animal model made completely:HE staining of lung tissue and VG+Victoria blue triple staining was used to observe the airway and pulmonary vascular remodeling in pathological changes, includeing MT%and MA%,WT%and WA%. Using different methods to detect the expression of various inflammatory factors: Western blot detect the NF-κB protein expression. RT-PCR and Western blot were used to detect HMGB1mRNA and protein expression in each group in lung tissue, ELISA was used to detect TNF-a, IL-8, VEGF expression in lavage fluid (BALF) and serum in each group. Analysis of the expression of various inflammatory factors and airway remodeling, pulmonary vascular remodeling related indicators, as well as expression of PDTC on various inflammatory factors.Results:1.The target of airway remodeling(MT%and MA%):Compare to group A, the target of airway remodeling of group B and C were higher than that of group A(P<0.05). After using PDTC, the target of airway remodeling of group B1and C1were significantly decreased than that of the corresponding group B and C(P<0.05). The above target of group Al which were used saline were no different to that of the corresponding group A(P>0.05).2. The target of pulmonary vascular remodeling(WT%and WA%): Compare to group A, Pulmonary vascular remodeling expressed as the percentage of muscular arteries were increase in group B, But muscle type of artery wall thickness has not been increased.The target of pulmonary vascular remodeling of group C were higher than that of group A(P<0.05). After using PDTC, the target of pulmonary vascular remodeling of group B1and C1were significantly decreased than that of the corresponding group B and C(P<0.05). The above target of group A1which were used saline were no different to that of the corresponding group A(P>0.05).3. The expression of inflammatory factors:3.1The expression of NF-κB Western blot showed that NF-κB protein expression of group B and C were higher than that of group A(P<0.05); After using PDTC NF-κB protein expression of group B1and C1were significantly decreased than that of the corresponding group B and C(P<0.05).3.2The expression of HMGB1RT-PCR and Western blot shows that in group B, C, of HMGB1mRNA, protein expression compared with group A were significantly increased(P<0.05), The expression of NF-κB protein in group C is higher than group B(P<0.05). After using PDTC HMGB1mRNA and protein expression of group B1and C1were significantly decreased than that of the corresponding group B and C (P<0.05).3.3The expression of TNF-a, IL-8, VEGF ELISA results showed that These above target include concentration of TNF-a,IL-8, VEGF in BALF and in serum of group B and C were all higher than that of group A ((P<0.05). After using PDTC, Compared to the group B and C,the expression of TNF-a, IL-8, VEGF in BALF and in serum of group B1and C1, were decreased(P<0.05).4. The releationship between the expression of inflammatory cytokines and the airway and pulmonary vascular remodeling:There was positive correlation between the expressions of inflammatory cytokines (NF-κB,HMGB1) and airway remodeling index (MT%,MA%) and pulmonary vascular remodeling index (WT%,WA%) and inflammatory cytokines(IL-8,TNF-a,VEGF). There was positive correlation between the expressions of NF-κB and HMGB1. After using PDTC, the expression of inflammatory cytokines (NF-κB,HMGB1,IL-8, TNF-a,VEGF) were decreased and the airway remodeling and pulmonary vascular remodeling were improve partly.Conclusions:①With the development of the desease, the expression of TNF-α、 IL-8、VEGF in Serum and bronchoalveolar lavage fluid were gradually increased, there was positive correlation between the expressions of TNF-a, IL-8, VEGF and NF-κB,HMGB1. Prompted the inflammation of COPD is not only confined to the lungs, while there is a systemic inflammatory response. Systemic inflammatory response participated in all process of the development of COPD, Systemic inflammatory response expression advances gradually with the aggravation of COPD. It was possibly promotes the COPD course progress through the airway and pulmonary vascular remodeling.②NF-κB influence pulmonary vascular and airway reconstruction by regulating the expression of HMGB1, TNF-alpha, IL-8, VEGF. NF-κB plays an important role in COPD inflammation factor network.

        全身炎癥反應(yīng)對(duì)慢性阻塞性肺疾病氣道、肺血管重構(gòu)的影響

中文摘要4-8Abstract8-12英漢縮略詞對(duì)照表13-14前言14-171 材料與方法17-242 結(jié)果24-473 討論47-514 結(jié)論51-52參考文獻(xiàn)52-56綜述56-71    參考文獻(xiàn)68-71攻讀學(xué)位期間發(fā)表的學(xué)術(shù)論文目錄71-72致謝72-73

  本文關(guān)鍵詞：全身炎癥反應(yīng)對(duì)慢性阻塞性肺疾病氣道、肺血管重構(gòu)的影響，由筆耕文化傳播整理發(fā)布。