吸烟对气道平滑肌细胞增殖和凋亡的影响及HSP70在凋亡中保护作用的研究
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摘要
第一部分:吸烟对气道平滑肌细胞增殖中可能信号通路的影响的研究
气道平滑肌细胞(airway smooth muscle cells,ASMCs)不仅在气道重建中起着决定性的作用,而且在气道慢性炎症的形成和调节方面也发挥重要的作用。吸烟可以导致小气道的炎症、纤维化、平滑肌增生,使气道壁增厚、气道狭窄。在气道平滑肌细胞增殖的过程中有许多的信号通路被涉及到。在气道平滑肌细胞有丝分裂的信号通路中,细胞外信号调节蛋白激酶(extracellular signal regulated kinase,ERK)和磷酰肌醇3激酶(phosphatidylinositol 3-kinase, PI-3K)通路是气道平滑肌有丝分裂的主要的正向调节者。然而,细胞外信号调节蛋白激酶和磷酰肌醇3激酶通路是否参与了吸烟引起的气道平滑肌细胞增殖的过程中尚不清楚。因而,本课题部分主要探讨在体内外引起细胞增殖的可能的信号通路及其它们之间的联系。
论文一:香烟提取物对气道平滑肌细胞增殖中ERKs及NF-κB的影响
在吸烟的慢性阻塞性肺疾病患者以及熏香烟复制的大鼠慢性阻塞性肺疾病模型中,气道平滑肌都显著增生。而且香烟提取物(cigarette smoke extract,CSE)能刺激体外气道平滑肌细胞(airway smooth muscle cells,ASMCs)增殖。在影响细胞增殖的因素中,细胞外信号调节蛋白激酶(extracellular regulated protein,ERK)为丝裂原激活蛋白激酶(mitogen activated protein kinase,MAPK)家族重要成员,ERK1/2信号通路是多数生长因子、细胞因子调控细胞增殖的重要途径。研究显示ERK1/2通路是调节有丝分裂原诱导气道平滑肌增殖事件中的一个关键信号。另外,核因子-κB(nuclearfactor-kappaB,NF-κB)能被多种因素激活,激活后的NF-кB不仅参与机体的免疫应答和炎症反应,而且还可调控细胞增殖和凋亡相关基因,在熏香烟复制的大鼠COPD模型中肺泡巨噬细胞及气道上皮细胞中NF-кB表达增加,而且NF-кB在正常平滑肌细胞增殖中也发挥重要作用。本研究探讨香烟提取物对气道平滑肌细胞细胞增殖中ERKs及NF-κB的影响。
方法:
体外培养Wistar大鼠的气道平滑肌细胞,给予不同浓度的香烟提取物刺激24小时后,MTT法检测增殖情况,采用Western blot的方法检测细胞外信号调节蛋白激酶以及磷酸化的细胞外信号调节蛋白激酶和核因子-κB的表达。
结果:
1.在不同浓度香烟提取物对气道平滑肌细胞刺激后,气道平滑肌细胞的A值分别为0.247±0.027(对照组)、0.298±0.038(1/32CSE组)、0.353±0.017(1/16CSE组)和0.376±0.024(1/8CSE组)。随CSE浓度的增高细胞增殖程度逐渐增加,与对照组比较均有显著性增高(P<0.05,n=4)。
2.在不同浓度香烟提取物对气道平滑肌细胞刺激后:ERK1的表达分别为0.586±0.032(对照组)、0.801±0.067(1/32CSE组)、0.842±0.067(1/16CSE组)及0.913±0.101(1/8CSE组);ERK2的表达分别为0.589±0.061(对照组)、0.765±0.046(1/32CSECSE组)、0.836±0.054(1/16组)及0.919±0.086(1/8CSE组);p-ERK的表达分别为0.579±0.058(对照组)、0.773±0.051(1/32CSE组)、0.848±0.075(1/16CSE组)及0.941±0.067(1/8CSE组);NF-κB的表达分别为0.629±0.069(对照组)、0.879±0.058(1/32CSE组)、0.901±0.066(1/16CSE组)及0.936±0.087(1/8CSE组)。经过对数据统计分析后显示,ERKs、p-ERK、NF-κB的表达均随CSE浓度的增高而逐渐增高,且与对照组相比有显著性差异(P<0.05,n=4)。
3.将对照组及不同浓度的CSE组中气道平滑肌细胞p-ERK和NF-κB免疫印迹的表达的A值与相应β- actin的A值比较后所得的数据进行相关分析,发现NF-κB的表达水平随p-ERK的表达的增加呈增加趋势,其r=0.858(P<0.05, n=4)。
结论:
一定浓度的CSE作用气道平滑肌细胞后可引起其增殖,而且随CSE浓度的增高细胞增殖程度逐渐增加;同时ERKs、p-ERK及NF-κB的表达增加,磷酸化激活后的ERKs可能通过NF-κB的活化参与气道平滑肌细胞的增殖。
论文二: PI-3K在吸烟大鼠COPD模型气道平滑肌增生中的表达的作用
吸烟是引起慢性阻塞性肺疾病(COPD)的最重要的环境危险因素。在吸烟的慢性阻塞性肺疾病患者以及熏香烟复制的大鼠慢性阻塞性肺疾病模型中,气道平滑肌都显著增生。而且香烟提取物(cigarette smoke extract,CSE)能刺激体外气道平滑肌细胞(airway smooth muscle cells , ASMCs)增殖。磷脂酰肌醇3激酶( phosphoinositide3-kinases, PI-3K)是多磷脂酰肌醇通路中一个重要的效应器和涉及信号转导、细胞转化的关键酶。PI-3K产生的特异肌醇脂质3,4,5-三磷酸肌醇和3,4-二磷酸肌醇是调节Akt活性的重要作用分子,与多种细胞功能有关。研究发现PI-3K通过效应分子Akt能促进细胞增殖、抑制细胞凋亡,而且与癌症的发展有关。目前研究表明人的气道平滑肌细胞中,PI-3K能够诱导其DNA的合成,从而诱导气道平滑肌细胞的加速生长。本研究通过建立吸烟大鼠COPD模型来探讨PI -3K在COPD气道平滑肌增生中的作用机制。
方法:
制作大鼠吸入香烟烟雾后的COPD模型,测定大鼠气道阻力及呼吸系统总顺应性,对气道平滑肌进行HE染色及PCNA免疫组化观察,并且逆转录聚合酶链反应(RT-PCR)检测气道平滑肌中PI-3KmRNA表达。
结果:
1.呼吸功能检测两组的呼吸功能检测显示:在气道阻力(cmH2O/L/s)方面CODP组和对照组分别为2.898±0.856和1.686±0.142,COPD组比对照组有显著性增高(P<0.05,n=8);而在肺顺应性(L/cmH2O)方面COPD组和对照组分别为0.386±0.046和0.426±0.042,两者相比无统计学差异(P>0.05,n=8)。
2.肺组织的HE染色在吸烟4月组,见大鼠支气管气道平滑肌增厚,部分肺泡壁变薄、断裂、肺泡融合、肺泡腔扩大以及炎症细胞侵润;而对照组的支气管及肺组织结构正常。
3.气道平滑肌PCNA的表达平滑肌细胞核棕黄色为提示PCNA阳性。采用HPIAS - 100高清晰度彩色病理图文分析系统测定PCNA的表达。结果表明:COPD组和对照组大鼠气道平滑肌PCNA表达分别为0.773±0.076和0.573±0.062,两者相比有显著性差异(P<0.05,n=8)。
4. COPD大鼠组和对照组PI-3KmRNA的表达将PI-3KmRNA的A值与β-actinA值比较后,结果发现:COPD后大鼠和对照组气道平滑肌PI-3KmRNA的表达分别为0.872±0.068和0.648±0.066,两者相比有显著性差异(P<0.05,n=8)。
5.将对照组及COPD大鼠组中气道平滑肌中PI-3KmRNA表达的A值与相应β- actin的A值比较后所得的值和PCNA表达的值进行相关分析后发现,PI-3KmRNA的表达水平与PCNA的表达水平的呈明显正相关(r=0.816,P<0.05, n=8)。
结论:
香烟烟雾可能通过PI-3K信号途径在COPD大鼠气道平滑肌增生中发挥作用。
第二部分:吸烟对气道平滑肌细胞凋亡中p-JNK的影响及HSP70在凋亡中保护作用的研究
吸烟是引起慢性阻塞性肺疾病发生的主要危险因素。较高浓度的香烟提取物(cigarette smoke extract ,CSE)刺激气道平滑肌细胞(airway smooth muscle cells,ASMCs)后可导致其凋亡。凋亡程序牵涉到几个主要的执行者,应激活化蛋白激酶(C-J unN-terminal kinase,JNKs)是其中之一。JNK是丝裂原激活蛋白激酶(mitogen activated protein kinase,MAPK)家族重要成员之一,它在气道平滑肌细胞中也有表达。JNK信号途径的活化可激活下游的细胞凋亡信号通路。较低浓度的香烟提取物刺激气道平滑肌细胞后,可以引起气道平滑肌细胞HSP70表达的增加,而增加表达的HSP70能对细胞起保护作用。施维舒(Geranylgeranylacetone,GGA)是一种树木香气和树汁中含有的萜类物质,具有组织修复作用,特别能强化抗溃疡作用。作为一种非毒性的诱导剂,无论是在体内还是在体外都能诱导胃肠道组织和细胞HSP70的表达。因而我们通过GGA增加气道平滑肌细胞中HSP70的表达,探讨HSP70表达的增高对较高浓度CSE所致气道平滑肌细胞凋亡中p-JNK的影响。
论文一:施维舒对气道平滑肌细胞HSP70表达的影响
施维舒(Geranylgeranylacetone,GGA)是一种树木香气和树汁中含有的萜类物质,具有组织修复作用,特别能强化抗溃疡作用。作为一种非毒性的诱导剂,无论是在体内还是在体外都能诱导胃肠道组织和细胞HSP70的表达。本研究探讨施维舒对气道平滑肌细胞(airway smooth muscle cells,ASMCs)中的热休克蛋白70(heat shock protein 70, HSP70)表达的影响。
方法:
将体外培养Wistar大鼠的气道平滑肌细胞进行分组:(1)A组,空白对照组:为未加任何处理的气道平滑肌细胞;(2)B组,含2μg/mlα-tocopherol的乙醇处理的气道平滑肌细胞组;(3)C组,10-6MGGA处理组;(4)D组,10-5MGGA处理组。用Western blot的方法检测气道平滑肌细胞中HSP70的表达情况。
结果:
各组气道平滑肌细胞HSP70的表达免疫印迹的表达:A组,0.252±0.018;B组,0.264±0.082;C组,0.601±0.092;D组,0.842±0.062。将所得数据进行统计学分析发现:与A组比较,C组和D组HSP70的表达均增加,而且随GGA浓度增高而增加(P<0.05,n=4);A组与B组之间HSP70的表达无显著差异(P>0.05, n=4)。结论:
一定浓度的GGA能诱导气道平滑肌细胞HSP70表达的增加,而且随GGA浓度增高而增加。
论文二:香烟提取物对气道平滑肌细胞凋亡中p-JNK的影响
吸烟是引起慢性阻塞性肺疾病发生的主要危险因素。较高浓度香烟提取物(cigarette smoke extract ,CSE)的刺激可导致气道平滑肌细胞的凋亡。凋亡程序牵涉到几个主要的执行者,应激活化蛋白激酶(C-J unN-terminal kinase,JNKs)是其中之一。JNK是丝裂原激活蛋白激酶(mitogen activated protein kinase,MAPK)家族重要成员之
一,它在气道平滑肌细胞中也有表达。目前,JNK是否参与了香烟提取物引起的气道平滑肌细胞的凋亡过程尚不清楚。因此,我们通过给予不同浓度的香烟提取物刺激气道平滑肌细胞后,观察香烟提取物对气道平滑肌细胞凋亡中p-JNK的表达的影响。
方法:
将体外培养Wistar大鼠的气道平滑肌细胞分组:(1)对照组;(2)15%香烟提取物处理组;(3)30%香烟提取物处理组;(4)45%香烟提取物处理组。给予不同浓度的香烟提取物刺激3小时后,流式细胞仪检测凋亡细胞的发生率法;用Western blot的方法检测气道平滑肌细胞中p-JNK的表达。
结果:
1.给予不同浓度的香烟提取物刺激后,各组的平滑肌细胞凋亡情况如下:对照组为5.33±0.27;15%香烟提取物处理组为17.67±1.24; 30%香烟提取物处理组为26.38±3.08; 45%香烟提取物处理组30.42±4.28。将所得的数据经统计学分析后发现:与对照组相比,不同浓度的香烟提取物组的凋亡均显著的升高,而且随香烟提取物浓度的增加升高越明显(P<0.05,n=4)。
2.加入不同浓度的香烟提取物处理的气道平滑肌细胞后p-JNK的表达结果:对照组为0.451±0.035;15%香烟提取物处理组为0.624±0.059; 30%香烟提取物处理组为0.736±0.075; 45%香烟提取物处理组0.886±0.079。所得数据经统计学分析后显示与对照组相比,不同浓度的香烟提取物处理组的p-JNK的表达均显著的升高,而且随香烟提取物浓度的增加升高越明显(P<0.05,n=4)。
结论:
通过我们的研究表明较高浓度的香烟提取物作用气道平滑肌细胞后引起气道平滑肌细胞的的凋亡增加,而且香烟提取物浓度越高气道平滑肌细胞的凋亡率越高;同时,气道平滑肌细胞中p-JNK表达的增加,而且其表达也随着香烟提取物浓度的增高而增加。较高浓度的香烟提取物作用气道平滑肌细胞后可能通过JNK的活化启动细胞凋亡程序导致细胞凋亡。
论文三: HSP70对CSE引起的气道平滑肌细胞凋亡中p-JNK的影响
较低浓度的香烟提取物(cigarette smoke extract ,CSE)刺激气道平滑肌细胞(airway smooth muscle cells,ASMCs)后,可以引起气道平滑肌细胞HSP70表达的增加,而增加表达的HSP70能对细胞起保护作用。较高浓度的香烟提取物刺激气道平滑肌细胞后可导致其凋亡。JNK是丝裂原激活蛋白激酶(mitogen activated protein kinase,MAPK)家族重要成员之一,它在气道平滑肌细胞中也有表达。JNK信号途径的活化可激活下游的细胞凋亡信号通路。因而我们通过GGA增加气道平滑肌细胞中HSP70的表达,探讨HSP70表达的增高对较高浓度CSE所致气道平滑肌细胞凋亡中p-JNK的影响。
方法:
将体外培养Wistar大鼠的气道平滑肌细胞分组:(1)A组,空白对照组;(2)B组,10-5MGGA处理组;(3)C组,30%CSE处理组,CSE的终浓度为30%;(4)D组,预处理10-5MGGA后加30%CSE处理组,CSE的终浓度为30%。给予不同刺激3小时后,流式细胞仪检测凋亡细胞的发生率法;用Western blot的方法检测气道平滑肌细胞中HSP70及p-JNK的表达。
结果:
1.各组中气道平滑肌细胞的凋亡情况气道平滑肌细胞的凋亡率:在A组中为5.33±0.27;在B组中为4.15±0.23;在C组中为26.38±3.08;在D中为14.49±1.48。将各组的数据做统计学分析后显示:与A组相比较,C组气道平滑肌细胞的凋亡率明显升高(P<0.05,n=4);B组和D组中气道平滑肌细胞的凋亡率明显降低(P<0.05,n=4);B组气道平滑肌细胞的凋亡率比D组中的低(P<0.05,n=4)。
2.各组中气道平滑肌细胞HSP70的表达气道平滑肌细胞的HSP70的表达免疫印迹的表达,经与β- actin的A值比较后结果分别为:在A组中为0.601±0.092;在B组中为0.873±0.078;在C组中为0.434±0.082;在D组中为0.784±0.072。将各组的数据做统计学分析后显示:与A组相比较,C组气道平滑肌细胞的HSP70的表达明显降低(P<0.05,n=4);B组和D组中气道平滑肌细胞的HSP70的表达明显增多(P<0.05,n=4);B组中的气道平滑肌细胞HSP70的表达较D组中的高(P<0.05,n=4)。
3.各组中气道平滑肌细胞p-JNK的表达气道平滑肌细胞的p-JNK的表达免疫印迹的表达,经与β- actin的A值比较后结果分别为:在A组中为0.216±0.026;在B组中为0.157±0.018;在C组中为0.736±0.075;在D组中为0.468±0.058。将各组的数据做统计学分析后显示:与A组相比较,C组气道平滑肌细胞p-JNK的表达明显增加(P<0.05,n=4);B组和D组中气道平滑肌细胞p-JNK的表达明显降低(P<0.05,n=4);B组中的气道平滑肌细胞p-JNK的表达较D组中的少(P<0.05,n=4)。
结论:
我们的研究表明,在气道平滑肌细胞中HSP70表达增加时,一方面可以降低较高浓度的CSE诱导的凋亡,另一方面可下调气道平滑肌细胞中p-JNK的表达。我们推测增加表达的HSP70可能通过下调细胞中p-JNK的表达而降低细胞的凋亡。
附属部分:吸烟对肺功能及外周血T淋巴细胞IL-2mRNA的影响
吸烟是慢性阻塞性肺疾病(COPD)发生的危险因素[1]。大量的研究表明,在COPD患者的气道、肺实质和肺血管中均存在着慢性炎症,并且影响肺功能。T淋巴细胞及其分泌的细胞因子等参与吸烟引起的炎症过程。白介素-2(IL-2)是一种有效的T淋巴因子,IL-2在淋巴细胞发育、淋巴细胞激活和维持免疫反应中都发挥着重要的作用。目前,吸烟对肺功能及外周血T淋巴细胞中IL-2mRNA表达的影响尚不清楚。本研究通过检测吸烟者外周血T淋巴细胞IL-2mRNA表达的变化,以及吸烟对肺功能的影响,探讨吸烟在COPD发病中的作用。
方法:
随机选择健康吸烟者和非吸烟者各24名,检测肺功能指标、检测外周血T淋巴细胞中IL-2mRNA表达。
结果:
吸烟组中FEV1、FEV1/ FVC %、MMEF75/25较对照组有明显下降(P<0.05,n=24);吸烟组中IL-2mRNA较对照组有显著性增高(P<0.05,n=24)。肺功能中FEV1随IL-2mRNA的表达水平增加呈降低趋势,其r=-0.758(P<0.05,n=24)。
结论:
吸烟者在尚无临床症状时,肺功能已经受损。吸烟可能通过增加T淋巴细胞IL-2mRNA的表达促进炎症反应导致肺功能损害。
PartⅠ: Studies on the effect of possible signal pathways in the proliferation of airway smooth muscle cells by smoking
Airway smooth muscle cells (ASMCs) have an important role not only on airway remodeling but also on chronic airway inflammation. Smoking can cause the inflammation, fibrosi, the hyperplasis of airway smooth muscle, the thicknesses of airway wall and the stenosis of airway in the small airway There are lots of signal pathways involved in the course of proliferation of airway smooth muscle cells. In the mitogen-activated signal pathways of airway smooth muscle cells, the extracellular signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI-3K) pathways appear to be key positive regulators of airway smooth muscle mitogenesis.But it remains unknown if ERK and PI-3K pathways participate in the course of proliferation of airway smooth muscle cells by smoking. So we study the effect of possible signal pathways in the proliferation of airway smooth muscle cells by smoking and their relations in vitro and vivo in this part.
Article one: Effect of cigarette smoke extract on the ERKs and NF-κB in the proliferation of airway smooth muscle cells
The airway smooth muscles were hyperplastic in the airway of COPD patients and COPD model of rats, and cigarette smoke extract can induced the proliferation of airway smooth muscle cells. In the effect factors of the cell proliferation, extracellular regulated protein(ERK) is the important member of mitogen activated protein kinase(MAPK). ERK pathway is the important approach in regulating the cell proliferation by grow factors and cytokines. The study indicated ERK pathway was the key signal in regulating the cell proliferation by mitogen. The transcription factor nuclear factor- kappaB(NF-κB) regulates various genes involved in immunoresponses, cell proliferation and apoptosis. The expression levels of NF-κB were increased in the alveolar macrophages and airway primary epithelial cells of COPD model of rats induced by smoking. NF-κB play the important role in the proliferation of airway smooth muscle cells. We study the effect of cigarette smoke extract on the ERKs and NF-κB in the proliferation of airway smooth muscle cells.
Methods:
The airway smooth muscle cells in Wistar rats were cultured in vitro and stimulated with CSE for 24h. Cell proliferation was determined with mitochondrial dehydrogenase activty (MTT assay). Western bloting was used to detect the expression level of ERKs, p-ERK and NF-κB.
Results:
1. After airway smooth muscle cells stimulating by different concentration CSE, the absorbency results in different groups: 0.247±0.027(control group), 0.298±0.038(1:32CSE), 0.353±0.017(1:16CSE) and 0.376±0.024(1:8CSE).Cell proliferation was gradually increased with 1:32, 1:16 and 1:8 of CSE compared by control group, and it was associated with the rising CSE concentration(P<0.05, n=4).
2. After airway smooth muscle cells stimulating by different concentration CSE, the expression level of ERK1 in different groups: 0.586±0.032(control group), 0.801±0.067(1:32CSE), 0.842±0.067(1:16CSE) and 0.913±0.101(1:8CSE); the expression level of ERK2 in different groups: 0.589±0.061(control group), 0.765±0.046(1:32CSE), 0.836±0.054(1:16CSE) and 0.919±0.086(1:8CSE); the expression level of p-ERK in different groups: 0.579±0.058(control group), 0.773±0.051(1:32CSE), 0.848±0.075(1:16CSE) and 0.941±0.067(1:8CSE); the expression level of NF-κB in different groups: 0.629±0.069(control group), 0.879±0.058(1:32CSE), 0.901±0.066(1:16CSE) and 0.936±0.087(1:8CSE). The results indicated the expression levels of ERKs,p-ERK and NF-КB gradually were increased compared by control group, and it was associated with the rising CSE concentration (P<0.05,n=4).
3. The expression level of NF-κB had strongly positive correlation with the expression level of p-ERK r=0.858 (P<0.05 ,n=4).
Conclusion:
It is suggested that appropriate concentration CSE can induced the proliferation of airway smooth muscle cells, and it was associated with the rising CSE concentration. The expression levels of ERKs, p-ERK, NF-κB were increased. The activated ERKs(p-ERK) may be associated with the activation of NF-κB and regulated the proliferation of airway smooth muscle cells.
Article two:The Role of PI-3K in airway smooth muscle hyperplasia in COPD model rats induced by smoking
Smoking is The most important risk factor for COPD. The airway smooth muscles were hyperplastic in the airway of COPD patients and COPD model of rats, and cigarette smoke extract can induced the proliferation of airway smooth muscle cells. In the effect factors of the cell proliferation, phosphoinositide3-kinases (PI-3K) pathway regulates various cellular processes, such as proliferation, growth, apoptosis and cytoskeletal rearrangement.Specific inositol lipids such as PtdIns(3, 4)P2 and PtdIns(3, 4, 5)P3 which are generated by phosphoinositide 3-kinases are specific activators to the serine/threonine protein kinase Akt that have been implicated in a plethora of cell functions and then Akt stimulates cell proliferation and suppresses apoptosis, and is implicated in cancer progression. The study indicated PI-3K can induce the synthesis of DNA and promoted the cell proliferation in human airway smooth muscle cells.So the rat models of COPD induced by smoking were made to investigated the effect of the PI-3Kexpression in the airway smooth muscle hyperplasia of rats with chronic obstructive pulmonary disease(COPD) induced by smoking.
Methods:
The rat models of COPD induced by smoking were made to investigate the resistance of airway and the total compliance of respiratory system.The expression of PCNA in ASMCs was determined by immuno–histochemistry, the expression of the PI-3K in ASMCs were determined by reverse transcription polymerase chain reaction(RT-PCR).
Results:
1. The detection of respiratory function The resistance of airway (cmH2O/L/s):2.898±0.856 (COPD group); 1.686±0.142 (control group). The result indicated the risistance of airway was increased in the COPD groups compared by control groups (P<0.05, n=8). The total compliance of respiratory system:0.386±0.046(COPD group); 0.426±0.042(control group), the result had no difference between the COPD groups and control groups(P>0.05,n=8).
2. The HE stain results of lung issue in different group rats indicated that the airway smooth muscle layer was thicker in COPD groups than in control groups.
3. The expressions of PCNA in ASMCs of different group rats: 0.773±0.076 (COPD group); 0.573±0.062(control group). The result indicated the expressions of PCNA in COPD groups were significantly increased compared by control groups (P<0.05, n=8).
4. The expressions of PI-3K in ASMCs of different group rats: 0.872±0.068 (COPD group); 0.648±0.066 (control group). The result indicated the expressions of PI-3K in COPD groups were significantly increased compared by control groups (P<0.05, n=8).
5. The expression level of PI-3K had strongly positive correlation with the expression level of PCNA(r=0.816, P<0.05, n=8).
Conclusion:
It is suggested that the smoking can induce the airway smooth muscle hyperplasia of rats, and increase the expression of PI-3K in the airway smooth muscle of rats.The PI-3K signal pathway may be involved in airway smooth muscle hyperplasia in COPD rats induced by smoking.
PartⅡ: Studies on the possible signal pathways and the protective effect of HSP70 in apoptosis of airway smooth muscle cells by smoking
The most important risk factor for COPD is smoking. Higher concentration cigarette smoke extract (CSE) can induce the apoptosis of airway smooth muscle cells(ASMCs). C-J unN-terminal kinase (JNK) is the important member in the apoptosis course. JNK is the important member of mitogen activated protein kinase(MAPK), and it can express in airway smooth muscle cells. The activation of JNK can activate the downstream apoptosis signal pathway.The expressions of heat shock protein 70 (HSP70) were increased in airway smooth muscle cells (ASMCs) induced by the lower concentration, and it can protect the cells. Geranylgeranylacetone(GGA), an acyclic polyisoprenoid, is developed as an antiulcer drug. It has been shown that this compound is effective in protecting the gastric mucosa against several types of insults without affecting gastric acid secretion. As a non-toxic inducer, geranylgeranylacetone can induce the expression of heat shock protein 70 in cells and gastrointestinal issuses in vitro and vivo. So we increased the expressions of HSP70 induced by geranylgeranylacetone (GGA), then investigated the effect of HSP70 on the p-JNK in the apoptosis of airway smooth muscle cells induced by cigarette smoke extract .
Article one: Effect of geranylgeranylacetone on the the expression of HSP70 in airway smooth muscle cells
Geranylgeranylacetone(GGA), an acyclic polyisoprenoid, is developed as an antiulcer drug. It has been shown that this compound is effective in protecting the gastric mucosa against several types of insults without affecting gastric acid secretion. As a non-toxic inducer, geranylgeranylacetone can induce the expression of heat shock protein 70 in cells and gastrointestinal issuses in vitro and vivo. So we investigated the effect of heat shock protein 70 expression in airway smooth muscle cells, (ASMCs) by GGA.
Methods:
The airway smooth muscle cells in Wistar rats were cultured in vitro and divided in four groups: (1) group A, control group; (2) group B, the ethanol with 2μg/mlα-tocopherol group; (3) group C, 10-6MGGA group; (4) group D, 10-5MGGA group. Western bloting was used to detect the expression level of HSP70.
Results:
After airway smooth muscle cells stimulating by different concentration GGA, the expression level of HSP70 in different groups: 0.252±0.018 (group A); 0.264±0.082 (group B); 0.601±0.092 (group C); 0.842±0.062 (group D). The results indicated the expression levels of HSP70 gradually were increased compared by control group, and it was associated with the rising GGA concentration (P<0.05,n=4),and it had no difference between group A and group B(P>0.05,n=4).
Conclusion:
It is suggested that appropriate concentration GGA can increased the expression levels of HSP70 of airway smooth muscle cells and it was associated with the rising GGA concentration.
Article two: Effect of cigarette smoke extract on the p-JNK in the apoptosis of airway smooth muscle cells
The most important risk factor for COPD is smoking. Higher concentration cigarette smoke extract (CSE) can induce the apoptosis of airway smooth muscle cells(ASMCs). C-J unN-terminal kinase (JNK) is the important member in the apoptosis course. JNK is the important member of mitogen activated protein kinase(MAPK), and it can express in airway smooth muscle cells. It remains unknowed if JNK pathways participate in the course of apoptosis of airway smooth muscle cells by cigarette smoke extract. So we study the effect of cigarette smoke extract on the p-JNK in the apoptosis of airway smooth muscle cells in this part.
Methods:
The airway smooth muscle cells in Wistar rats were cultured in vitro and divided in four groups: (1) control group; (2) 15%CSE group; (3) 30% CSE group; (4) 45% CSE group. Flowcytometry was used to detect the apoptosis of airway smooth muscle cells and western bloting was used to detect the expression level of p-JNK.
Results:
1. After airway smooth muscle cells stimulating by different concentration CSE, flowcytometry showed the apoptosis rate of airway smooth muscle cells in different groups: 5.33±0.27 (control group), 17.67±1.24 (15%CSE), 26.38±3.08 (30%CSE) and 30.42±4.28 (45%CSE).Cell apoptosis rates were gradually increased with 15%, 30% and 45% of CSE compared by control group, and it was associated with the rising CSE concentration(P<0.05, n=4).
2. After airway smooth muscle cells stimulating by different concentration CSE, the expression level of p-JNK in different groups: 0.451±0.035 (control group), 0.624±0.059 (15%CSE), 0.736±0.075 (30%CSE) and 0.886±0.079 (45%CSE). The results indicated the expression levels of p-JNK gradually were increased compared by control group, and it was associated with the rising CSE concentration (P<0.05, n=4).
Conclusion:
Higher concentration CSE can induced the apoptosis of airway smooth muscle cells, and it was associated with the rising CSE concentration. And higher concentration CSE can increased the expression levels of p-JNK of airway smooth muscle cells and it was associated with the rising CSE concentration. It is suggested that CSE may induced the apoptosis of airway smooth muscle cells by increasing the expression levels of p-JNK.
Article three: Effect of HSP70 on the p-JNK in the apoptosis of airway smooth muscle cells induced by cigarette smoke extract
The expressions of heat shock protein 70 (HSP70) were increased in airway smooth muscle cells (ASMCs) induced by the lower concentration, and it can protect the cells. The apoptosis of ASMCs were increased in ASMCs induced by the higher concentration CSE. C-J unN-terminal kinase (JNK) is the important member of mitogen activated protein kinase(MAPK), and it can express in airway smooth muscle cells. The activation of JNK can activate the downstream apoptosis signal pathway. So we increased the expressions of HSP70 induced by geranylgeranylacetone (GGA), then investigated the effect of HSP70 on the p-JNK in the apoptosis of airway smooth muscle cells induced by cigarette smoke extract .
Methods:
The airway smooth muscle cells in Wistar rats were cultured in vitro and divided in four groups: (1) group A, control group; (2) group B, 10-5MGGA group; (3) group C, 30%CSE group; (4) group D, 10-5MGGA and 30%CSE group. Flowcytometry was used to detect the apoptosis of airway smooth muscle cells and western bloting was used to detect the expression level of HSP70 and p-JNK.
Results:
1. After airway smooth muscle cells stimulating by different concentration CSE, flowcytometry showed the apoptosis rate of airway smooth muscle cells in different groups: 5.33±0.27 (A group), 4.15±0.23 (B group), 26.38±3.08 (C group), 14.49±1.48 (D group). Cell apoptosis rates were increased in C group compared by A group (P<0.05, n=4); cell apoptosis rates in B group and D group were decreased compared by A group (P<0.05, n=4). Cell apoptosis rates in B group were lower than that in D group (P<0.05, n=4).
2. The expression level of HSP70 in different groups: 0.601±0.092 (A group), 0.873±0.078 (B group), 0.434±0.082 (C group), 0.784±0.072 (D group). The results indicated the expression levels of HSP70 in C group were decreased compared by A group (P<0.05,n=4); the expression levels of HSP70 in B group and D group were increased compared by A group (P<0.05, n=4). The expression levels of HSP70 in B group were higher than that in D group (P<0.05, n=4).
3. The expression level of p-JNK in different groups: 0.216±0.026 (A group), 0.157±0.018 (B group), 0.736±0.075 (C group), 0.468±0.058 (D group). The results indicated the expression levels of p-JNK in C group were increased compared by A group (P<0.05,n=4); the expression levels of p-JNK in B group and D group were decreased compared by A group (P<0.05,n=4). The expression levels of p-JNK in B group were lower than that in D group (P<0.05,n=4).
Conclusion:
Our study indicated HSP70 can decrease the apoptosis of cells induced by higher concentration CSE, and down-regulate the expression levels of p-JNK in cells when the expression levels of HSP70 were increased in airway smooth muscle cells. It is suggested that the increased expression levels of HSP70 may decreased the apoptosis of cells by down-regulated the expression levels of p-JNK in airway smooth muscle cells.
Auxiliay: Effect of smoking on pulmonary function and the expression of IL-2mRNA in human peripheral blood T lymphocytes
The most important risk factor for COPD is smoking. Studies indicated there were chronic inflammation in the airway, lung parenchyma and pulmonary vascular vessel of COPD patients.T lymphocytes and cytokines play a important role in the course of inflammation induced by smoking.Interleukine-2 (IL-2) is a important number of T lymphocyte cytokines. It play a important role in development, activation of T lymphocyte and maintains immune response. It is unknowed the effects on pulmonary function and IL- 2mRNA by smoking. We study the effect of smoking on pulmonary function and IL- 2mRNA in human peripheral blood T lymphocytes.
Methods:
We randomly selected 24 healthy smokers as smoking group and 24 healthy nonsmokers as control group. To detected their pulmonary function by portable spirometers and the expression of the IL-2mRNA in T lymphocytes were determined by reverse transcription polymerase chain reaction(RT-PCR).
Results:
The FEV1、FEV1/ FVC % and MMEF75/25 in smoking group had significantly decreased compared with control group(P<0.05,n=24); the expression of the IL-2mRNA in T lymphocytes from smoking group had significantly increased compared with control group(P<0.05, n=24).The pulmonary function had strongly negative correlation with the expression level of IL-2mRNA(r=-0.785, P<0.05,n=24).
Conclusion:
Although smokers did not show clinical symptoms, their pulmonary function had been reduced; It is suggested that smoking may increased lung inflammatory response by inducing the expression level of IL-2mRNA.
气道平滑肌细胞(airway smooth muscle cells,ASMCs)不仅在气道重建中起着决定性的作用,而且在气道慢性炎症的形成和调节方面也发挥重要的作用。吸烟可以导致小气道的炎症、纤维化、平滑肌增生,使气道壁增厚、气道狭窄。在气道平滑肌细胞增殖的过程中有许多的信号通路被涉及到。在气道平滑肌细胞有丝分裂的信号通路中,细胞外信号调节蛋白激酶(extracellular signal regulated kinase,ERK)和磷酰肌醇3激酶(phosphatidylinositol 3-kinase, PI-3K)通路是气道平滑肌有丝分裂的主要的正向调节者。然而,细胞外信号调节蛋白激酶和磷酰肌醇3激酶通路是否参与了吸烟引起的气道平滑肌细胞增殖的过程中尚不清楚。因而,本课题部分主要探讨在体内外引起细胞增殖的可能的信号通路及其它们之间的联系。
论文一:香烟提取物对气道平滑肌细胞增殖中ERKs及NF-κB的影响
在吸烟的慢性阻塞性肺疾病患者以及熏香烟复制的大鼠慢性阻塞性肺疾病模型中,气道平滑肌都显著增生。而且香烟提取物(cigarette smoke extract,CSE)能刺激体外气道平滑肌细胞(airway smooth muscle cells,ASMCs)增殖。在影响细胞增殖的因素中,细胞外信号调节蛋白激酶(extracellular regulated protein,ERK)为丝裂原激活蛋白激酶(mitogen activated protein kinase,MAPK)家族重要成员,ERK1/2信号通路是多数生长因子、细胞因子调控细胞增殖的重要途径。研究显示ERK1/2通路是调节有丝分裂原诱导气道平滑肌增殖事件中的一个关键信号。另外,核因子-κB(nuclearfactor-kappaB,NF-κB)能被多种因素激活,激活后的NF-кB不仅参与机体的免疫应答和炎症反应,而且还可调控细胞增殖和凋亡相关基因,在熏香烟复制的大鼠COPD模型中肺泡巨噬细胞及气道上皮细胞中NF-кB表达增加,而且NF-кB在正常平滑肌细胞增殖中也发挥重要作用。本研究探讨香烟提取物对气道平滑肌细胞细胞增殖中ERKs及NF-κB的影响。
方法:
体外培养Wistar大鼠的气道平滑肌细胞,给予不同浓度的香烟提取物刺激24小时后,MTT法检测增殖情况,采用Western blot的方法检测细胞外信号调节蛋白激酶以及磷酸化的细胞外信号调节蛋白激酶和核因子-κB的表达。
结果:
1.在不同浓度香烟提取物对气道平滑肌细胞刺激后,气道平滑肌细胞的A值分别为0.247±0.027(对照组)、0.298±0.038(1/32CSE组)、0.353±0.017(1/16CSE组)和0.376±0.024(1/8CSE组)。随CSE浓度的增高细胞增殖程度逐渐增加,与对照组比较均有显著性增高(P<0.05,n=4)。
2.在不同浓度香烟提取物对气道平滑肌细胞刺激后:ERK1的表达分别为0.586±0.032(对照组)、0.801±0.067(1/32CSE组)、0.842±0.067(1/16CSE组)及0.913±0.101(1/8CSE组);ERK2的表达分别为0.589±0.061(对照组)、0.765±0.046(1/32CSECSE组)、0.836±0.054(1/16组)及0.919±0.086(1/8CSE组);p-ERK的表达分别为0.579±0.058(对照组)、0.773±0.051(1/32CSE组)、0.848±0.075(1/16CSE组)及0.941±0.067(1/8CSE组);NF-κB的表达分别为0.629±0.069(对照组)、0.879±0.058(1/32CSE组)、0.901±0.066(1/16CSE组)及0.936±0.087(1/8CSE组)。经过对数据统计分析后显示,ERKs、p-ERK、NF-κB的表达均随CSE浓度的增高而逐渐增高,且与对照组相比有显著性差异(P<0.05,n=4)。
3.将对照组及不同浓度的CSE组中气道平滑肌细胞p-ERK和NF-κB免疫印迹的表达的A值与相应β- actin的A值比较后所得的数据进行相关分析,发现NF-κB的表达水平随p-ERK的表达的增加呈增加趋势,其r=0.858(P<0.05, n=4)。
结论:
一定浓度的CSE作用气道平滑肌细胞后可引起其增殖,而且随CSE浓度的增高细胞增殖程度逐渐增加;同时ERKs、p-ERK及NF-κB的表达增加,磷酸化激活后的ERKs可能通过NF-κB的活化参与气道平滑肌细胞的增殖。
论文二: PI-3K在吸烟大鼠COPD模型气道平滑肌增生中的表达的作用
吸烟是引起慢性阻塞性肺疾病(COPD)的最重要的环境危险因素。在吸烟的慢性阻塞性肺疾病患者以及熏香烟复制的大鼠慢性阻塞性肺疾病模型中,气道平滑肌都显著增生。而且香烟提取物(cigarette smoke extract,CSE)能刺激体外气道平滑肌细胞(airway smooth muscle cells , ASMCs)增殖。磷脂酰肌醇3激酶( phosphoinositide3-kinases, PI-3K)是多磷脂酰肌醇通路中一个重要的效应器和涉及信号转导、细胞转化的关键酶。PI-3K产生的特异肌醇脂质3,4,5-三磷酸肌醇和3,4-二磷酸肌醇是调节Akt活性的重要作用分子,与多种细胞功能有关。研究发现PI-3K通过效应分子Akt能促进细胞增殖、抑制细胞凋亡,而且与癌症的发展有关。目前研究表明人的气道平滑肌细胞中,PI-3K能够诱导其DNA的合成,从而诱导气道平滑肌细胞的加速生长。本研究通过建立吸烟大鼠COPD模型来探讨PI -3K在COPD气道平滑肌增生中的作用机制。
方法:
制作大鼠吸入香烟烟雾后的COPD模型,测定大鼠气道阻力及呼吸系统总顺应性,对气道平滑肌进行HE染色及PCNA免疫组化观察,并且逆转录聚合酶链反应(RT-PCR)检测气道平滑肌中PI-3KmRNA表达。
结果:
1.呼吸功能检测两组的呼吸功能检测显示:在气道阻力(cmH2O/L/s)方面CODP组和对照组分别为2.898±0.856和1.686±0.142,COPD组比对照组有显著性增高(P<0.05,n=8);而在肺顺应性(L/cmH2O)方面COPD组和对照组分别为0.386±0.046和0.426±0.042,两者相比无统计学差异(P>0.05,n=8)。
2.肺组织的HE染色在吸烟4月组,见大鼠支气管气道平滑肌增厚,部分肺泡壁变薄、断裂、肺泡融合、肺泡腔扩大以及炎症细胞侵润;而对照组的支气管及肺组织结构正常。
3.气道平滑肌PCNA的表达平滑肌细胞核棕黄色为提示PCNA阳性。采用HPIAS - 100高清晰度彩色病理图文分析系统测定PCNA的表达。结果表明:COPD组和对照组大鼠气道平滑肌PCNA表达分别为0.773±0.076和0.573±0.062,两者相比有显著性差异(P<0.05,n=8)。
4. COPD大鼠组和对照组PI-3KmRNA的表达将PI-3KmRNA的A值与β-actinA值比较后,结果发现:COPD后大鼠和对照组气道平滑肌PI-3KmRNA的表达分别为0.872±0.068和0.648±0.066,两者相比有显著性差异(P<0.05,n=8)。
5.将对照组及COPD大鼠组中气道平滑肌中PI-3KmRNA表达的A值与相应β- actin的A值比较后所得的值和PCNA表达的值进行相关分析后发现,PI-3KmRNA的表达水平与PCNA的表达水平的呈明显正相关(r=0.816,P<0.05, n=8)。
结论:
香烟烟雾可能通过PI-3K信号途径在COPD大鼠气道平滑肌增生中发挥作用。
第二部分:吸烟对气道平滑肌细胞凋亡中p-JNK的影响及HSP70在凋亡中保护作用的研究
吸烟是引起慢性阻塞性肺疾病发生的主要危险因素。较高浓度的香烟提取物(cigarette smoke extract ,CSE)刺激气道平滑肌细胞(airway smooth muscle cells,ASMCs)后可导致其凋亡。凋亡程序牵涉到几个主要的执行者,应激活化蛋白激酶(C-J unN-terminal kinase,JNKs)是其中之一。JNK是丝裂原激活蛋白激酶(mitogen activated protein kinase,MAPK)家族重要成员之一,它在气道平滑肌细胞中也有表达。JNK信号途径的活化可激活下游的细胞凋亡信号通路。较低浓度的香烟提取物刺激气道平滑肌细胞后,可以引起气道平滑肌细胞HSP70表达的增加,而增加表达的HSP70能对细胞起保护作用。施维舒(Geranylgeranylacetone,GGA)是一种树木香气和树汁中含有的萜类物质,具有组织修复作用,特别能强化抗溃疡作用。作为一种非毒性的诱导剂,无论是在体内还是在体外都能诱导胃肠道组织和细胞HSP70的表达。因而我们通过GGA增加气道平滑肌细胞中HSP70的表达,探讨HSP70表达的增高对较高浓度CSE所致气道平滑肌细胞凋亡中p-JNK的影响。
论文一:施维舒对气道平滑肌细胞HSP70表达的影响
施维舒(Geranylgeranylacetone,GGA)是一种树木香气和树汁中含有的萜类物质,具有组织修复作用,特别能强化抗溃疡作用。作为一种非毒性的诱导剂,无论是在体内还是在体外都能诱导胃肠道组织和细胞HSP70的表达。本研究探讨施维舒对气道平滑肌细胞(airway smooth muscle cells,ASMCs)中的热休克蛋白70(heat shock protein 70, HSP70)表达的影响。
方法:
将体外培养Wistar大鼠的气道平滑肌细胞进行分组:(1)A组,空白对照组:为未加任何处理的气道平滑肌细胞;(2)B组,含2μg/mlα-tocopherol的乙醇处理的气道平滑肌细胞组;(3)C组,10-6MGGA处理组;(4)D组,10-5MGGA处理组。用Western blot的方法检测气道平滑肌细胞中HSP70的表达情况。
结果:
各组气道平滑肌细胞HSP70的表达免疫印迹的表达:A组,0.252±0.018;B组,0.264±0.082;C组,0.601±0.092;D组,0.842±0.062。将所得数据进行统计学分析发现:与A组比较,C组和D组HSP70的表达均增加,而且随GGA浓度增高而增加(P<0.05,n=4);A组与B组之间HSP70的表达无显著差异(P>0.05, n=4)。结论:
一定浓度的GGA能诱导气道平滑肌细胞HSP70表达的增加,而且随GGA浓度增高而增加。
论文二:香烟提取物对气道平滑肌细胞凋亡中p-JNK的影响
吸烟是引起慢性阻塞性肺疾病发生的主要危险因素。较高浓度香烟提取物(cigarette smoke extract ,CSE)的刺激可导致气道平滑肌细胞的凋亡。凋亡程序牵涉到几个主要的执行者,应激活化蛋白激酶(C-J unN-terminal kinase,JNKs)是其中之一。JNK是丝裂原激活蛋白激酶(mitogen activated protein kinase,MAPK)家族重要成员之
一,它在气道平滑肌细胞中也有表达。目前,JNK是否参与了香烟提取物引起的气道平滑肌细胞的凋亡过程尚不清楚。因此,我们通过给予不同浓度的香烟提取物刺激气道平滑肌细胞后,观察香烟提取物对气道平滑肌细胞凋亡中p-JNK的表达的影响。
方法:
将体外培养Wistar大鼠的气道平滑肌细胞分组:(1)对照组;(2)15%香烟提取物处理组;(3)30%香烟提取物处理组;(4)45%香烟提取物处理组。给予不同浓度的香烟提取物刺激3小时后,流式细胞仪检测凋亡细胞的发生率法;用Western blot的方法检测气道平滑肌细胞中p-JNK的表达。
结果:
1.给予不同浓度的香烟提取物刺激后,各组的平滑肌细胞凋亡情况如下:对照组为5.33±0.27;15%香烟提取物处理组为17.67±1.24; 30%香烟提取物处理组为26.38±3.08; 45%香烟提取物处理组30.42±4.28。将所得的数据经统计学分析后发现:与对照组相比,不同浓度的香烟提取物组的凋亡均显著的升高,而且随香烟提取物浓度的增加升高越明显(P<0.05,n=4)。
2.加入不同浓度的香烟提取物处理的气道平滑肌细胞后p-JNK的表达结果:对照组为0.451±0.035;15%香烟提取物处理组为0.624±0.059; 30%香烟提取物处理组为0.736±0.075; 45%香烟提取物处理组0.886±0.079。所得数据经统计学分析后显示与对照组相比,不同浓度的香烟提取物处理组的p-JNK的表达均显著的升高,而且随香烟提取物浓度的增加升高越明显(P<0.05,n=4)。
结论:
通过我们的研究表明较高浓度的香烟提取物作用气道平滑肌细胞后引起气道平滑肌细胞的的凋亡增加,而且香烟提取物浓度越高气道平滑肌细胞的凋亡率越高;同时,气道平滑肌细胞中p-JNK表达的增加,而且其表达也随着香烟提取物浓度的增高而增加。较高浓度的香烟提取物作用气道平滑肌细胞后可能通过JNK的活化启动细胞凋亡程序导致细胞凋亡。
论文三: HSP70对CSE引起的气道平滑肌细胞凋亡中p-JNK的影响
较低浓度的香烟提取物(cigarette smoke extract ,CSE)刺激气道平滑肌细胞(airway smooth muscle cells,ASMCs)后,可以引起气道平滑肌细胞HSP70表达的增加,而增加表达的HSP70能对细胞起保护作用。较高浓度的香烟提取物刺激气道平滑肌细胞后可导致其凋亡。JNK是丝裂原激活蛋白激酶(mitogen activated protein kinase,MAPK)家族重要成员之一,它在气道平滑肌细胞中也有表达。JNK信号途径的活化可激活下游的细胞凋亡信号通路。因而我们通过GGA增加气道平滑肌细胞中HSP70的表达,探讨HSP70表达的增高对较高浓度CSE所致气道平滑肌细胞凋亡中p-JNK的影响。
方法:
将体外培养Wistar大鼠的气道平滑肌细胞分组:(1)A组,空白对照组;(2)B组,10-5MGGA处理组;(3)C组,30%CSE处理组,CSE的终浓度为30%;(4)D组,预处理10-5MGGA后加30%CSE处理组,CSE的终浓度为30%。给予不同刺激3小时后,流式细胞仪检测凋亡细胞的发生率法;用Western blot的方法检测气道平滑肌细胞中HSP70及p-JNK的表达。
结果:
1.各组中气道平滑肌细胞的凋亡情况气道平滑肌细胞的凋亡率:在A组中为5.33±0.27;在B组中为4.15±0.23;在C组中为26.38±3.08;在D中为14.49±1.48。将各组的数据做统计学分析后显示:与A组相比较,C组气道平滑肌细胞的凋亡率明显升高(P<0.05,n=4);B组和D组中气道平滑肌细胞的凋亡率明显降低(P<0.05,n=4);B组气道平滑肌细胞的凋亡率比D组中的低(P<0.05,n=4)。
2.各组中气道平滑肌细胞HSP70的表达气道平滑肌细胞的HSP70的表达免疫印迹的表达,经与β- actin的A值比较后结果分别为:在A组中为0.601±0.092;在B组中为0.873±0.078;在C组中为0.434±0.082;在D组中为0.784±0.072。将各组的数据做统计学分析后显示:与A组相比较,C组气道平滑肌细胞的HSP70的表达明显降低(P<0.05,n=4);B组和D组中气道平滑肌细胞的HSP70的表达明显增多(P<0.05,n=4);B组中的气道平滑肌细胞HSP70的表达较D组中的高(P<0.05,n=4)。
3.各组中气道平滑肌细胞p-JNK的表达气道平滑肌细胞的p-JNK的表达免疫印迹的表达,经与β- actin的A值比较后结果分别为:在A组中为0.216±0.026;在B组中为0.157±0.018;在C组中为0.736±0.075;在D组中为0.468±0.058。将各组的数据做统计学分析后显示:与A组相比较,C组气道平滑肌细胞p-JNK的表达明显增加(P<0.05,n=4);B组和D组中气道平滑肌细胞p-JNK的表达明显降低(P<0.05,n=4);B组中的气道平滑肌细胞p-JNK的表达较D组中的少(P<0.05,n=4)。
结论:
我们的研究表明,在气道平滑肌细胞中HSP70表达增加时,一方面可以降低较高浓度的CSE诱导的凋亡,另一方面可下调气道平滑肌细胞中p-JNK的表达。我们推测增加表达的HSP70可能通过下调细胞中p-JNK的表达而降低细胞的凋亡。
附属部分:吸烟对肺功能及外周血T淋巴细胞IL-2mRNA的影响
吸烟是慢性阻塞性肺疾病(COPD)发生的危险因素[1]。大量的研究表明,在COPD患者的气道、肺实质和肺血管中均存在着慢性炎症,并且影响肺功能。T淋巴细胞及其分泌的细胞因子等参与吸烟引起的炎症过程。白介素-2(IL-2)是一种有效的T淋巴因子,IL-2在淋巴细胞发育、淋巴细胞激活和维持免疫反应中都发挥着重要的作用。目前,吸烟对肺功能及外周血T淋巴细胞中IL-2mRNA表达的影响尚不清楚。本研究通过检测吸烟者外周血T淋巴细胞IL-2mRNA表达的变化,以及吸烟对肺功能的影响,探讨吸烟在COPD发病中的作用。
方法:
随机选择健康吸烟者和非吸烟者各24名,检测肺功能指标、检测外周血T淋巴细胞中IL-2mRNA表达。
结果:
吸烟组中FEV1、FEV1/ FVC %、MMEF75/25较对照组有明显下降(P<0.05,n=24);吸烟组中IL-2mRNA较对照组有显著性增高(P<0.05,n=24)。肺功能中FEV1随IL-2mRNA的表达水平增加呈降低趋势,其r=-0.758(P<0.05,n=24)。
结论:
吸烟者在尚无临床症状时,肺功能已经受损。吸烟可能通过增加T淋巴细胞IL-2mRNA的表达促进炎症反应导致肺功能损害。
PartⅠ: Studies on the effect of possible signal pathways in the proliferation of airway smooth muscle cells by smoking
Airway smooth muscle cells (ASMCs) have an important role not only on airway remodeling but also on chronic airway inflammation. Smoking can cause the inflammation, fibrosi, the hyperplasis of airway smooth muscle, the thicknesses of airway wall and the stenosis of airway in the small airway There are lots of signal pathways involved in the course of proliferation of airway smooth muscle cells. In the mitogen-activated signal pathways of airway smooth muscle cells, the extracellular signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI-3K) pathways appear to be key positive regulators of airway smooth muscle mitogenesis.But it remains unknown if ERK and PI-3K pathways participate in the course of proliferation of airway smooth muscle cells by smoking. So we study the effect of possible signal pathways in the proliferation of airway smooth muscle cells by smoking and their relations in vitro and vivo in this part.
Article one: Effect of cigarette smoke extract on the ERKs and NF-κB in the proliferation of airway smooth muscle cells
The airway smooth muscles were hyperplastic in the airway of COPD patients and COPD model of rats, and cigarette smoke extract can induced the proliferation of airway smooth muscle cells. In the effect factors of the cell proliferation, extracellular regulated protein(ERK) is the important member of mitogen activated protein kinase(MAPK). ERK pathway is the important approach in regulating the cell proliferation by grow factors and cytokines. The study indicated ERK pathway was the key signal in regulating the cell proliferation by mitogen. The transcription factor nuclear factor- kappaB(NF-κB) regulates various genes involved in immunoresponses, cell proliferation and apoptosis. The expression levels of NF-κB were increased in the alveolar macrophages and airway primary epithelial cells of COPD model of rats induced by smoking. NF-κB play the important role in the proliferation of airway smooth muscle cells. We study the effect of cigarette smoke extract on the ERKs and NF-κB in the proliferation of airway smooth muscle cells.
Methods:
The airway smooth muscle cells in Wistar rats were cultured in vitro and stimulated with CSE for 24h. Cell proliferation was determined with mitochondrial dehydrogenase activty (MTT assay). Western bloting was used to detect the expression level of ERKs, p-ERK and NF-κB.
Results:
1. After airway smooth muscle cells stimulating by different concentration CSE, the absorbency results in different groups: 0.247±0.027(control group), 0.298±0.038(1:32CSE), 0.353±0.017(1:16CSE) and 0.376±0.024(1:8CSE).Cell proliferation was gradually increased with 1:32, 1:16 and 1:8 of CSE compared by control group, and it was associated with the rising CSE concentration(P<0.05, n=4).
2. After airway smooth muscle cells stimulating by different concentration CSE, the expression level of ERK1 in different groups: 0.586±0.032(control group), 0.801±0.067(1:32CSE), 0.842±0.067(1:16CSE) and 0.913±0.101(1:8CSE); the expression level of ERK2 in different groups: 0.589±0.061(control group), 0.765±0.046(1:32CSE), 0.836±0.054(1:16CSE) and 0.919±0.086(1:8CSE); the expression level of p-ERK in different groups: 0.579±0.058(control group), 0.773±0.051(1:32CSE), 0.848±0.075(1:16CSE) and 0.941±0.067(1:8CSE); the expression level of NF-κB in different groups: 0.629±0.069(control group), 0.879±0.058(1:32CSE), 0.901±0.066(1:16CSE) and 0.936±0.087(1:8CSE). The results indicated the expression levels of ERKs,p-ERK and NF-КB gradually were increased compared by control group, and it was associated with the rising CSE concentration (P<0.05,n=4).
3. The expression level of NF-κB had strongly positive correlation with the expression level of p-ERK r=0.858 (P<0.05 ,n=4).
Conclusion:
It is suggested that appropriate concentration CSE can induced the proliferation of airway smooth muscle cells, and it was associated with the rising CSE concentration. The expression levels of ERKs, p-ERK, NF-κB were increased. The activated ERKs(p-ERK) may be associated with the activation of NF-κB and regulated the proliferation of airway smooth muscle cells.
Article two:The Role of PI-3K in airway smooth muscle hyperplasia in COPD model rats induced by smoking
Smoking is The most important risk factor for COPD. The airway smooth muscles were hyperplastic in the airway of COPD patients and COPD model of rats, and cigarette smoke extract can induced the proliferation of airway smooth muscle cells. In the effect factors of the cell proliferation, phosphoinositide3-kinases (PI-3K) pathway regulates various cellular processes, such as proliferation, growth, apoptosis and cytoskeletal rearrangement.Specific inositol lipids such as PtdIns(3, 4)P2 and PtdIns(3, 4, 5)P3 which are generated by phosphoinositide 3-kinases are specific activators to the serine/threonine protein kinase Akt that have been implicated in a plethora of cell functions and then Akt stimulates cell proliferation and suppresses apoptosis, and is implicated in cancer progression. The study indicated PI-3K can induce the synthesis of DNA and promoted the cell proliferation in human airway smooth muscle cells.So the rat models of COPD induced by smoking were made to investigated the effect of the PI-3Kexpression in the airway smooth muscle hyperplasia of rats with chronic obstructive pulmonary disease(COPD) induced by smoking.
Methods:
The rat models of COPD induced by smoking were made to investigate the resistance of airway and the total compliance of respiratory system.The expression of PCNA in ASMCs was determined by immuno–histochemistry, the expression of the PI-3K in ASMCs were determined by reverse transcription polymerase chain reaction(RT-PCR).
Results:
1. The detection of respiratory function The resistance of airway (cmH2O/L/s):2.898±0.856 (COPD group); 1.686±0.142 (control group). The result indicated the risistance of airway was increased in the COPD groups compared by control groups (P<0.05, n=8). The total compliance of respiratory system:0.386±0.046(COPD group); 0.426±0.042(control group), the result had no difference between the COPD groups and control groups(P>0.05,n=8).
2. The HE stain results of lung issue in different group rats indicated that the airway smooth muscle layer was thicker in COPD groups than in control groups.
3. The expressions of PCNA in ASMCs of different group rats: 0.773±0.076 (COPD group); 0.573±0.062(control group). The result indicated the expressions of PCNA in COPD groups were significantly increased compared by control groups (P<0.05, n=8).
4. The expressions of PI-3K in ASMCs of different group rats: 0.872±0.068 (COPD group); 0.648±0.066 (control group). The result indicated the expressions of PI-3K in COPD groups were significantly increased compared by control groups (P<0.05, n=8).
5. The expression level of PI-3K had strongly positive correlation with the expression level of PCNA(r=0.816, P<0.05, n=8).
Conclusion:
It is suggested that the smoking can induce the airway smooth muscle hyperplasia of rats, and increase the expression of PI-3K in the airway smooth muscle of rats.The PI-3K signal pathway may be involved in airway smooth muscle hyperplasia in COPD rats induced by smoking.
PartⅡ: Studies on the possible signal pathways and the protective effect of HSP70 in apoptosis of airway smooth muscle cells by smoking
The most important risk factor for COPD is smoking. Higher concentration cigarette smoke extract (CSE) can induce the apoptosis of airway smooth muscle cells(ASMCs). C-J unN-terminal kinase (JNK) is the important member in the apoptosis course. JNK is the important member of mitogen activated protein kinase(MAPK), and it can express in airway smooth muscle cells. The activation of JNK can activate the downstream apoptosis signal pathway.The expressions of heat shock protein 70 (HSP70) were increased in airway smooth muscle cells (ASMCs) induced by the lower concentration, and it can protect the cells. Geranylgeranylacetone(GGA), an acyclic polyisoprenoid, is developed as an antiulcer drug. It has been shown that this compound is effective in protecting the gastric mucosa against several types of insults without affecting gastric acid secretion. As a non-toxic inducer, geranylgeranylacetone can induce the expression of heat shock protein 70 in cells and gastrointestinal issuses in vitro and vivo. So we increased the expressions of HSP70 induced by geranylgeranylacetone (GGA), then investigated the effect of HSP70 on the p-JNK in the apoptosis of airway smooth muscle cells induced by cigarette smoke extract .
Article one: Effect of geranylgeranylacetone on the the expression of HSP70 in airway smooth muscle cells
Geranylgeranylacetone(GGA), an acyclic polyisoprenoid, is developed as an antiulcer drug. It has been shown that this compound is effective in protecting the gastric mucosa against several types of insults without affecting gastric acid secretion. As a non-toxic inducer, geranylgeranylacetone can induce the expression of heat shock protein 70 in cells and gastrointestinal issuses in vitro and vivo. So we investigated the effect of heat shock protein 70 expression in airway smooth muscle cells, (ASMCs) by GGA.
Methods:
The airway smooth muscle cells in Wistar rats were cultured in vitro and divided in four groups: (1) group A, control group; (2) group B, the ethanol with 2μg/mlα-tocopherol group; (3) group C, 10-6MGGA group; (4) group D, 10-5MGGA group. Western bloting was used to detect the expression level of HSP70.
Results:
After airway smooth muscle cells stimulating by different concentration GGA, the expression level of HSP70 in different groups: 0.252±0.018 (group A); 0.264±0.082 (group B); 0.601±0.092 (group C); 0.842±0.062 (group D). The results indicated the expression levels of HSP70 gradually were increased compared by control group, and it was associated with the rising GGA concentration (P<0.05,n=4),and it had no difference between group A and group B(P>0.05,n=4).
Conclusion:
It is suggested that appropriate concentration GGA can increased the expression levels of HSP70 of airway smooth muscle cells and it was associated with the rising GGA concentration.
Article two: Effect of cigarette smoke extract on the p-JNK in the apoptosis of airway smooth muscle cells
The most important risk factor for COPD is smoking. Higher concentration cigarette smoke extract (CSE) can induce the apoptosis of airway smooth muscle cells(ASMCs). C-J unN-terminal kinase (JNK) is the important member in the apoptosis course. JNK is the important member of mitogen activated protein kinase(MAPK), and it can express in airway smooth muscle cells. It remains unknowed if JNK pathways participate in the course of apoptosis of airway smooth muscle cells by cigarette smoke extract. So we study the effect of cigarette smoke extract on the p-JNK in the apoptosis of airway smooth muscle cells in this part.
Methods:
The airway smooth muscle cells in Wistar rats were cultured in vitro and divided in four groups: (1) control group; (2) 15%CSE group; (3) 30% CSE group; (4) 45% CSE group. Flowcytometry was used to detect the apoptosis of airway smooth muscle cells and western bloting was used to detect the expression level of p-JNK.
Results:
1. After airway smooth muscle cells stimulating by different concentration CSE, flowcytometry showed the apoptosis rate of airway smooth muscle cells in different groups: 5.33±0.27 (control group), 17.67±1.24 (15%CSE), 26.38±3.08 (30%CSE) and 30.42±4.28 (45%CSE).Cell apoptosis rates were gradually increased with 15%, 30% and 45% of CSE compared by control group, and it was associated with the rising CSE concentration(P<0.05, n=4).
2. After airway smooth muscle cells stimulating by different concentration CSE, the expression level of p-JNK in different groups: 0.451±0.035 (control group), 0.624±0.059 (15%CSE), 0.736±0.075 (30%CSE) and 0.886±0.079 (45%CSE). The results indicated the expression levels of p-JNK gradually were increased compared by control group, and it was associated with the rising CSE concentration (P<0.05, n=4).
Conclusion:
Higher concentration CSE can induced the apoptosis of airway smooth muscle cells, and it was associated with the rising CSE concentration. And higher concentration CSE can increased the expression levels of p-JNK of airway smooth muscle cells and it was associated with the rising CSE concentration. It is suggested that CSE may induced the apoptosis of airway smooth muscle cells by increasing the expression levels of p-JNK.
Article three: Effect of HSP70 on the p-JNK in the apoptosis of airway smooth muscle cells induced by cigarette smoke extract
The expressions of heat shock protein 70 (HSP70) were increased in airway smooth muscle cells (ASMCs) induced by the lower concentration, and it can protect the cells. The apoptosis of ASMCs were increased in ASMCs induced by the higher concentration CSE. C-J unN-terminal kinase (JNK) is the important member of mitogen activated protein kinase(MAPK), and it can express in airway smooth muscle cells. The activation of JNK can activate the downstream apoptosis signal pathway. So we increased the expressions of HSP70 induced by geranylgeranylacetone (GGA), then investigated the effect of HSP70 on the p-JNK in the apoptosis of airway smooth muscle cells induced by cigarette smoke extract .
Methods:
The airway smooth muscle cells in Wistar rats were cultured in vitro and divided in four groups: (1) group A, control group; (2) group B, 10-5MGGA group; (3) group C, 30%CSE group; (4) group D, 10-5MGGA and 30%CSE group. Flowcytometry was used to detect the apoptosis of airway smooth muscle cells and western bloting was used to detect the expression level of HSP70 and p-JNK.
Results:
1. After airway smooth muscle cells stimulating by different concentration CSE, flowcytometry showed the apoptosis rate of airway smooth muscle cells in different groups: 5.33±0.27 (A group), 4.15±0.23 (B group), 26.38±3.08 (C group), 14.49±1.48 (D group). Cell apoptosis rates were increased in C group compared by A group (P<0.05, n=4); cell apoptosis rates in B group and D group were decreased compared by A group (P<0.05, n=4). Cell apoptosis rates in B group were lower than that in D group (P<0.05, n=4).
2. The expression level of HSP70 in different groups: 0.601±0.092 (A group), 0.873±0.078 (B group), 0.434±0.082 (C group), 0.784±0.072 (D group). The results indicated the expression levels of HSP70 in C group were decreased compared by A group (P<0.05,n=4); the expression levels of HSP70 in B group and D group were increased compared by A group (P<0.05, n=4). The expression levels of HSP70 in B group were higher than that in D group (P<0.05, n=4).
3. The expression level of p-JNK in different groups: 0.216±0.026 (A group), 0.157±0.018 (B group), 0.736±0.075 (C group), 0.468±0.058 (D group). The results indicated the expression levels of p-JNK in C group were increased compared by A group (P<0.05,n=4); the expression levels of p-JNK in B group and D group were decreased compared by A group (P<0.05,n=4). The expression levels of p-JNK in B group were lower than that in D group (P<0.05,n=4).
Conclusion:
Our study indicated HSP70 can decrease the apoptosis of cells induced by higher concentration CSE, and down-regulate the expression levels of p-JNK in cells when the expression levels of HSP70 were increased in airway smooth muscle cells. It is suggested that the increased expression levels of HSP70 may decreased the apoptosis of cells by down-regulated the expression levels of p-JNK in airway smooth muscle cells.
Auxiliay: Effect of smoking on pulmonary function and the expression of IL-2mRNA in human peripheral blood T lymphocytes
The most important risk factor for COPD is smoking. Studies indicated there were chronic inflammation in the airway, lung parenchyma and pulmonary vascular vessel of COPD patients.T lymphocytes and cytokines play a important role in the course of inflammation induced by smoking.Interleukine-2 (IL-2) is a important number of T lymphocyte cytokines. It play a important role in development, activation of T lymphocyte and maintains immune response. It is unknowed the effects on pulmonary function and IL- 2mRNA by smoking. We study the effect of smoking on pulmonary function and IL- 2mRNA in human peripheral blood T lymphocytes.
Methods:
We randomly selected 24 healthy smokers as smoking group and 24 healthy nonsmokers as control group. To detected their pulmonary function by portable spirometers and the expression of the IL-2mRNA in T lymphocytes were determined by reverse transcription polymerase chain reaction(RT-PCR).
Results:
The FEV1、FEV1/ FVC % and MMEF75/25 in smoking group had significantly decreased compared with control group(P<0.05,n=24); the expression of the IL-2mRNA in T lymphocytes from smoking group had significantly increased compared with control group(P<0.05, n=24).The pulmonary function had strongly negative correlation with the expression level of IL-2mRNA(r=-0.785, P<0.05,n=24).
Conclusion:
Although smokers did not show clinical symptoms, their pulmonary function had been reduced; It is suggested that smoking may increased lung inflammatory response by inducing the expression level of IL-2mRNA.
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