【作者】 徐淑凤；

【导师】 陈良安；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2008， 博士

【摘要】 研究背景和目的急性肺损伤(Acute lung injury,ALI)/急性呼吸窘迫综合征(Acute respiratorydistress syndrome,ARDS)是指由心源性以外的各种肺内外致病因素如严重感染、创伤、休克等导致的以炎症反应和肺泡毛细血管内膜损伤为主要病理学特征的急性、进行性缺氧性呼吸衰竭。该症病情凶险,死亡率高。感染尤其是革兰氏阴性菌脓毒症是该病的主要病因,作为革兰氏阴性菌外膜上主要成分的细菌脂多糖(Bacterial lipopolysaccharide LPS)是一种强烈的炎症启动因子,可致细胞组织发生过度的炎性反应、功能障碍、结构破坏乃至凋亡坏死等一系列病理生理改变。在LPS诱导的信号转导通路中,Toll样受体-4(Toll-1ike receptor-4,TLR-4)是控制着LPS炎症信号进入细胞内进而启动炎症反应和细胞凋亡坏死的“门户”蛋白,是LPS信号转导最上游的关键分子,完整地介导了脂多糖的信号转导通路,在半胱天冬蛋白酶(cysteinyl aspartate-specific proteinase,caspase)的序贯激活和核转录因子-κB(nuclear factor-κB,NF-κB)的激活中起着不可或缺的作用。caspases尤其是其中的caspase-3被称作凋亡的“执行者”,而NF-κB能够促进许多与炎症反应相关的基因产物的表达,二者分别是LPS引发细胞凋亡损伤和诱导细胞产生炎性反应的关键中间环节。在肺组织中,数量庞大的肺实质细胞如肺微血管内皮细胞(pulmonarymicrovascular endothelial cells PMVECs)和肺泡上皮细胞(alveolar epithelial cells,AECs)不仅是炎性细胞和炎性介质作用的靶细胞,而且也是活跃的炎症细胞和效应细胞,对急性肺损伤的发生、发展同样起着不可忽视的作用。但以往对此两种细胞在ALI/ARDS发病中的作用研究的相对较少。尽管在不断探索,但ALI/ARDS的临床治疗尚未取得突破性进展,呼吸支持仍是主要的治疗手段之一。全氟化碳(perfluorocarbon,PFC)作为液态呼吸介质,应用于ALI/ARDs的治疗有着充满希望的前景。研究表明,除具有高携氧和二氧化碳的良好气体溶解运载特性外,PFC在体内体外尚显示出广泛的非特异性生物学效应。但其作用机制尚未完全阐明,而且PFC对肺内实质细胞如PMVECs和AECs的保护作用及可能的机制有待进一步的深入研究探讨。基于上述认识,本课题的研究目的为:①从细胞凋亡损伤和炎性反应两方面观察LPS对PMVECs和AECs两种细胞的损伤作用和LPS/TLR-4信号通路在LPS激活PMVECs和AECs并引起损伤中所起的作用。②探讨PFC对LPS诱导的PMVECs和AECs损伤的保护效应和对LPS/TLR-4信号通路功能的影响,即研究PFC对LPS引起的此两种细胞损伤的保护效应及可能的效应机制。通过上述研究可望对PFC的生物学保护效应及可能的机制有更深入的理解和阐释,为临床应用PFC治疗ALI/ARDS提供较为客观和更充分的理论依据。方法1.PFC对LPS诱导的大鼠PMVECs损伤的保护作用和机制研究1.1 SD大鼠PMVECs的分离培养、纯化和鉴定:联合应用胶原酶和中性蛋白酶消化肺边缘组织,分离细胞,血清诱导内皮细胞聚集的方法分离原代细胞,通过传代培养进行纯化。根据内皮细胞典型的铺路石样的形态学特征、免疫细胞化学染色检测细胞浆内的Ⅷ因子抗原、透射电镜观察细胞的超微结构对分离培养的内皮细胞进行鉴定。1.2 PFC对LPS诱导的PMVECs凋亡损伤的保护作用:应用流式细胞仪(FCM)检测细胞凋亡率和透射电镜(TEM)观察细胞凋亡的形态变化。将分离培养的第三、四代SD大鼠PMVECs分为四组:①对照组(control group):不作任何干预;②PFC(全氟辛烷)组:按30%体积比(PFC:培养液)加入全氟辛烷;③LPS(脂多糖)组:干预时按10 ng/ml,100ng/ml,1μg/ml,10μg/ml的浓度加入相应剂量的LPS;④PFC+LPS组(共培养组):按上述比例分别加入PFC和LPS。FCM不同LPS浓度干预取12h一个时间点的样本,不同处理组取6,12h两个时间点的样本,TEM取干预后6h的样本进行相关检测。1.3 PFC对LPS诱导PMVECs凋亡损伤的TLR-4信号通路的影响:采用实时荧光定量PCR(real time PCR)和蛋白质免疫印迹法(western blotting)检测TLR-4和caspase-3基因表达和蛋白水平的变化。细胞分组和干预方法同前。唯LPS组选定给予的LPS浓度为10μg/ml。基因表达观察2,6,12h三个时间点,蛋白水平观察0.5,2,6,12h四个时间点上述参数的变化。1.4 PFC对LPS激活PMVECs炎性反应的影响的研究1.4.1应用ELISA和放射免疫法检测细胞培养上清液促炎介质ICAM-1,TNF-α,和IL-8等的水平;1.4.2 PFC对LPS诱导PMVECs炎性反应的TLR-4信号通路的影响:包括采用real time PCR和western blotting检测TLR-4的基因表达蛋白水平变化;和NF-κB活性的检测即应用western blotting检测P65和IκB-α蛋白水平变化以及细胞免疫化学双染色检测此二蛋白在细胞内的表达情况。细胞分组和干预方法同前。2.PFC对LPS诱导的AECs损伤的保护作用和机制研究作为平行研究,本课题同时进行了PFC对LPS诱导AECs损伤的保护作用和机制研究,实验细胞分组和处理及观测指标同上述PMVECs中的研究。实验细胞为A549细胞株。结果1.PFC对LPS诱导的PMVECs损伤的保护作用和机制研究结果1.1大鼠PMVECs的分离培养纯化和鉴定:本研究建立了分离纯化大鼠PMVECs的方法。分离的PMVECs表现为典型的铺路石样,长至融合状态后呈现单层生长和接触抑制的特性;免疫细胞化学染色检测到细胞内有Ⅷ因子的表达;透射电镜观察到典型的内皮细胞超微结构特征,包括:细胞浆内有W-P小体,胞质中有丰富的吞饮小泡,内皮细胞腔面有大小不一的微绒毛。综合上述特征鉴定所分离的细胞为肺微血管内皮细胞。1.2细胞凋亡的FCM和TEM结果①FCM结果:LPS以10 ng/ml,100ng/m1,1μg/ml,10μg/ml作用于PMVECs12h后的早期细胞凋亡率分别为6.23%,6.07%,9.51%和23.48%;晚期凋亡和坏死率分别为3.93%,6.03%,5.84%和10.89%;细胞存活率分别为88.91%,87.33%,84.19%和64.86%,即随着LPS浓度的增加,细胞凋亡坏死率逐渐上升,而细胞存活率逐渐下降。与对照组、PFC组和LPS+PFC共培养组比较,LPS作用于PMVECs后的6,12h,PMvECs的早期细胞凋亡率、晚期细胞凋亡和坏死率以及存活率(唯LPS干预后6h的晚期凋亡率与PFc组的比较除外,p=0.066)均显著增加,且12h明显重于6小时;与对照组比较,PFC组的细胞凋亡率和存活率的差异无统计学意义,且PFc两组之间差异也无统计学意义;与对照组和全氟化碳组比较,共培养组的细胞早期凋亡率升高,存活率下降,即PFC能明显拮抗LPS的诱导凋亡作用,但不能完全阻断LPS对PMVECs的促凋亡效应。②TEM观察细胞凋亡形态的结果:于对照组可见细胞核膜清晰,细胞核内常染色质和异染色质分布均匀,PFC孵育后的PMVECs结构与对照组比较基本无差别,两者均未见细胞凋亡的特征性表现;而在LPS作用6h的细胞中可见异染色质浓集成团块并边集于核膜下,核膜不清,线粒体肿胀,细胞接近崩解等早、中期凋亡的超微结构变化;在共培养组的PMVEcs中可见细胞核膜也是清晰的,染色质均匀,异染色质稍多但无凝聚边集现象,即无明显的凋亡结构改变。因此再次从形态学上证实了LPS的诱导PMVECs凋亡和PFC对抗LPS诱导凋亡的作用。1.3启动分子TLR-4和关键的效应分子caspase-3的基因表达和蛋白水平变化的结果①real time PCR结果:LPS作用于PMVECs,使TLR4和caspase-3的基因表达明显增加,前者于2,6,12h,后者于6,12h表达显著增加;PFC单独作用对此两条基因的表达无明显影响;当PFC和LPS共培养细胞时,则此二基因的表达显著下降。②western blotting结果:LPS作用于PMVECs 0.5,2,6h可显著增加TLR-4蛋白水平,LPS作用后0.5,2,6,12h均可使caspase-3前体蛋白激活降解为17和11 kDa的两个活性片段,即LPS通过激活LPS/TLR-4—caspases通路而诱导了细胞的凋亡;PFC单独作用于PMVECs细胞,对此二蛋白的活性和水平无明显影响;当PFC和LPS共培养细胞时,则见TLR-4和caspase-3的活性和蛋白水平显著下降。1.4 PFC对LPS激活PMVECs炎性反应的影响的研究结果1.4.1细胞因子水平的测定结果:LPS作用于PMVECs可使促炎细胞因子显著增加,其中ICAM-1,TNF-α,于2,6和12h三个时间点均显著增加,分泌高峰均在2h,IL-8于LPS刺激后6h开始增加,12h达高峰;PFc单独作用于PMVECs对上述细胞因子的产生均无明显影响,而与LPS共培养时可使LPS诱导的上述细胞因子的分泌显著下降,从而显示出PFC的抗炎效应。1.4.2 LPS/TLR-4炎性反应信号通路的变化①启动分子TLR-4的基因和蛋白水平变化:LPS作用于PMVECs可使TLR-4的基因表达明显增加,以2h上调最显著并持续12h;而共培养组的TLR4基因表达显著下降;LPS并使TLR4的蛋白水平明显增加,以0.5h增加最显著并持续6h;全氟化碳能明显下调LPS诱导的PMVECs TLR-4蛋白水平表达。②NF-κB活性的检测结果:1)western boltting检测结果:LPS作用于PMVECs,可使NF-κB抑制蛋白IκB-α蛋白0.5h即发生降解,使NF-κB P65游离恢复活性并转入细胞核内,IκB-α并在2,6,12h均有显著降解,P65蛋白除2h外进入核内的水平显著增加,IκB-α和NF-κB P65两蛋白的变化时相基本同步;PFC单独干预对NF-κBP65和IκB-α的水平无明显影响;于共培养组,则IκB-α的降解显著下降,细胞核内的NF-κB P65显著减少,提示PFC能够抑制NF-κB的激活,从而阻断LPS促进炎性反应的效应。2)NF-κB的免疫细胞化学双染色结果:正常对照组的细胞浆内IκB-α染色阳性,而细胞核内基本无P65存在,故无着色;LPS刺激后的30min-6h,细胞浆内IκB-α染色明显变淡,细胞核内的P65染色呈强阳性,说明LPS刺激后IκB-α降解,P65恢复活性转入核内。PFC单独干预的细胞与对照组比较基本相似。提示PFC单独干预对NF-κB的活性无明显影响。共培养组的染色结果与正常对照组比较,于30min-2h,主要是细胞浆内IκB-α染色阳性,而细胞核内基本无P65存在,故无着色;在6h的图片中,也以细胞浆内IκB-α染色阳性为主,少数细胞核内见因P65存在而着色。再一次从形态学方面证实PFC可抑制LPS诱导PMVECs NF-κB活性增加的效应。2.PFC对LPS诱导的AECs损伤的保护作用和机制研究结果2.1细胞凋亡的FCM和TEM结果①FCM结果:LPS以10ng/ml,100ng/ml,1μg/ml,10μg/ml作用于A549细胞12h后的早期细胞凋亡率分别为4.12%,6.42%,7.53%和16.12%;晚期凋亡和坏死率分别为3.49%,5.22%,4.31%和5.67%;细胞存活率分别为91.34%,87.87%,87.78%,和77.74%,即随着LPS浓度的增加,细胞凋亡坏死率逐渐上升,而其细胞存活率逐渐下降。与对照组、PFC组和共培养组比较,LPS作用于A549细胞后的6,12h,其早期细胞凋亡率、晚期细胞凋亡和坏死率显著升高,存活率显著下降,且12h明显重于6小时;与对照组比较,PFC两个时相组的细胞凋亡率和存活率的差异无统计学意义,且两组之间差异也无统计学意义;与对照组和全氟化碳组比较,共培养组A549细胞的12h早期凋亡率升高,存活率下降,即PFC能明显拮抗LPS的诱导凋亡作用,但不能完全阻断LPS对细胞的促凋亡效应。②TEM检测细胞凋亡形态的结果:正常和PFC干预组的A549细胞的超微结构基本无差别,两者均未见细胞凋亡的特征性表现;而在LPS作用于6h的A549细胞中可见异染色质浓集成团块并边集于核膜下,并见典型的呈马蹄形聚集于核膜下的染色质,核膜不清等早、中期凋亡的超微结构变化;在共培养组的A549细胞未见明显的凋亡结构改变,因此从形态学上证实了LPS的诱导A549细胞凋亡和PFC对抗LPS诱导凋亡的效应。2.2启动分子TLR-4和关键的效应分子caspase-3的基因表达和蛋白水平变化的结果①real time PCR结果:LPS作用于A549细胞,使TLR4和caspase-3的基因表达明显增加,前者于2,6,12h,后者于6,12h表达显著增加;PFC单独作用对此两条基因的表达无明显影响;当PFC和LPS共培养细胞时,此二基因的表达显著下降。②western bloning结果:LPS作用于A549细胞0.5,2,6,12h可显著增加TLR-4蛋白水平,并见caspase-3前体降解的17 kDa和11 kDa两个活性片段;PFC单独作用于A549细胞,对此二蛋白的活性和水平无明显影响;于共培养组,TLR-4蛋白水平和caspase-3的活性显著下降,提示PFC能够显著抑制LPS诱导的此二蛋白的激活进而阻断LPS的诱导细胞凋亡的效应。2.3 PFC对LPS激活AECs细胞炎性反应的影响的研究结果2.3.1细胞因子水平的测定结果:LPS作用于A549细胞使促炎因子ICAM-1,TNF-α和IL-8于2h,6h和12h三个时间点均显著增加,前二者的分泌高峰均在2h,而IL-8的分泌高峰位于12h,PFC单独作用于A549细胞对上述细胞因子的产生均无明显影响,于共培养组可见LPS诱导的上述细胞因子的分泌显著下降,提示PFC能抑制LPS诱导A549细胞激活和炎性因子分泌的效应。2.3.2 LPS/TLR-4炎性反应信号通路的变化①TLR-4的基因和蛋白水平变化:LPS作用于A549细胞可使TLR4的基因表达明显增加,以2h上调最显著,持续12h;LPS并使TLR4的蛋白水平明显增加,于0.5h增加最显著并于6h内保持较高水平,至12h仍高于正常;而共培养组的TLR4基因表达和蛋白水平显著下降;②NF-κB活性测定结果:1)western boltting检测结果:LPS作用于A549细胞,可使IκB-α蛋白迅速降解并持续12h,NF-κB P65游离恢复活性并转入细胞核内,即使NF-κB活性增加,但是只有2h和6h有统计学意义,IκB-α和NF-κB P65两蛋白的变化时相基本同步;PFc单独干预对NF-κB和其抑制蛋白IκB-α的活性无明显影响;PFC与LPS共同孵育细胞,IκB-α的降解显著减少,细胞核内的NF-κB P65显著下降。2)NF-κB的免疫细胞化学双染色结果:对照组和PFC组主要是细胞浆内IκB-α染色阳性,而细胞核内P65很少存在,仅个别着色;LPS作用6h后细胞浆内IκB-α染色很少,几乎所有的细胞核内均可见P65着色;在共培养组可见双染色的细胞和以胞浆IκB-α或胞核P65着色为主的细胞。再一次从形态学方面证实PFC可抑制LPS诱导AECs NF-κB活性增加的效应。但总的染色结果不如PMVECs典型,其0.5和2h均未能染色出来。结论1.LPS作用于PMVECs和A549细胞可诱导其发生凋亡的损伤改变,这种效应呈剂量和时间依赖性;LPS并使细胞凋亡信号通路的始动分子TLR-4和凋亡关键效应分子caspase-3的基因表达和蛋白水平显著增加,提示LPS通过LPS/TLR-4→caspases序贯激活→细胞凋亡通路介导了PMVECs和A549细胞的凋亡效应。2.PFC单独作用于PMVECs和A549细胞,不诱导其凋亡损伤的发生,并对LPS/TLR-4→caspases通路无明显影响,当PFC与LPS共同孵育PMVECs和A549细胞时,则可以显著减轻LPS诱导的PMVECs和A549细胞凋亡的损伤,增加细胞的存活率,并且显著下调TLR-4和caspase-3的基因表达和蛋白水平,提示全氟化碳通过阻断TLR-4分子的激活和基因表达来阻止LPS信号的启动进而拮抗LPS的诱导此两种细胞凋亡的作用。3.LPS作用于PMVECs和A549细胞可激活此两种细胞群使其释放炎性介质显著增加,并使LPS的炎性反应信号通路的始动分子TLR-4的基因表达和蛋白水平显著增加,同时其下游的关键效应分子NF-κB的活性显著增强,提示LPS通过LPS/TLR-4→NF-κB通路诱导了PMVECs和A549细胞的炎性反应。4.PFC单独作用于PMVECs和A549细胞,不诱导炎性因子的释放增加,并对LPS/TLR-4→NF-κB通路无明显影响,当PFC与LPS共同孵育此两种细胞时,则可以显著减轻LPS诱导的细胞因子的分泌释放,并且显著下调TLR-4的基因表达和蛋白水平,以及NF-κB的活化核内转位,提示PFC通过阻断TLR-4分子的激活和基因表达来阻止LPS信号的启动进而减轻LPS的促进PMVECs和A549细胞炎性反应的作用。更多还原

【Abstract】 Study background and objectiveThe acute lung injury and the respiratory distress syndrome (ALI/ARDS) is a criticalclinical syndrome with progressive dyspnea, refractory hypoxemia and higy mortality, which pathological features mainly include inflammatory reaction and alveolar-capillary membrane injury resulted from severve infection, trauma, shock, etc. The difference of the both lies in the pathogenetic condition, the latter is more severe than the former. The essence of ALI/ARDS is a vascular leak syndrome induced by excessive inflammatory response in lung tissue. Infection especially gram-negative bacterial sepsis is a key etiological factor for ALI/ARDS. Bacterial lipopolysaccharide (LPS) or endotoxin, being of a component of the outer envelope of all gram-negative bacteria is a highly proinflammatory molecule. LPS may induce excessive inflammatory response of the body tissue and cells, which results in a series of pathophysiological changes including functional disorder, disorganization, apoptosis and even necrosis.Among the LPS signal transduction pathway, Toll-like receptor-4 (TLR-4) is known as ’the door protein’, which controls the transduction of LPS inflammatory signal into the cell and subsequently initiates inflammatory response and induces a series of pathophysiological changes. TLR-4 is integrally involved in LPS signaling and has a requisite role in the activation of caspases (eg. caspase-3) and nuclear transcription factor-κB (NF-κB), both of which are the key signaling events that mediates an array of cell responses. Caspase-3 is known as ’the apotosis executioner’ and NF-κB promotes the expression of proinflammatory gene products.Among the enormous lung parenchymal cells, pulmonary microvascular endothelial cells (PMVECs) and alveolar epithelial cells (AECs) are two important cell groups. They are not only the target cells of inflammatory cells and mediators, but are active inflammatory cells and effector cells as well. They play important roles in the occurance and progress of ALI/ARDS. Regrettably however, the roles of the two cell groups in the pathogenesis of ALI/ARDS have been paid little attention to in the past.Though unceasing researches have been being done by scholars all over the world, the therapeutic methods for treating ALI/ARDS have not been got a breakthrough. And respiratory support is still one of the major methods for treating the disease. Being as a kind of liquid breathing medium, perfluorocarbon (PFC) is a promising method in treating ALI/ARDS. In addition to its high ability at carrying and dissolving oxygen and carbon dioxide, PFC has shown a wide non-specific biological effects both in vivo and in vitro. But the mechanism of the biological effects of PFC is little known. Besides, the probable protective roles of PFC in PMVECs and AECs and the mechanism underlined are needed to research.In view of the above-mentioned, the study objective is:①To investigate the apoptosis injury and inflammatory resopnses of PMVECs and AECs induced by LPS. And to detect the activation of LPS/TLR-4 signal pathway so as to elucidate the role of the signal pathway in mediating the apoptosis injury and inflammatory responses of the both cells.②To study and research the protective effects of PFC on the injury of PMVECs and AECs induced by LPS and the effects of PFC on LPS/TLR-4 signal pathway, namely to search for the mechanism of the protective effects of PFC.Mehtods1. Methods investigating the protective effects of PFC on the injury of PMVECs induced by LPS and the mechanism of PFC’s protective effects1.1 Method of isolation, culture, purification and identification of PMVECs from rat lung: Primary cells of PMVECs were obtained after isolated peripheral lung tissues of SD rat, digested with type IV collagenase and dispase and aggregated by serum stimulation. The purified was done by serial subcultivation. According to typical cobble morphology of endothelial cells, VIII factor (also known as von willebrand factor, vWF) in endothelial cells detected by immunocytochemical staining, and ultrastructure characteristics of PMVECs observed under transmission electronic microscopy, PMVECs were identified.1.2 Methods investiagting the protective effects of PFC on the apoptosis injury induced by LPS: cells apoptosis rate was detected by Flow cytometry (FCM) and the ultrastructure changes of apoptosis were observed under TEMThe third and/or fourth generation PMVECs were used in our research. The cells were divided into four groups:①control group: cells did not receive any intervention,②PFC group: PFC was added to the cell culture medium to a final volume concentration [vol/vol, PFC: culture media] of 30%. After vortexing, the mixed liquor containing PFC and culture media was transfered into the culture flask. As PFC is not miscible with the medium, cells exposed to PFC were constantly shaken (60 times/min).③LPS group: cells were incubated with LPS at a final concentration of 10 ng/ml, 100ng/ml, 1μg/ml, 10μg/ml, and④LPS+PFC group (coculture group): cells were incubated with both LPS and PFC according to the above-mentioned percentage. The samples were collected at 6,12h after intervention for FCM and 6h for TEM.1.3 The expression of TLR-4 and caspase-3 mRNA were detected by real time PCR and the protein level of the two were detected by western blotting. The groups and the intervention methods were identical with those in FCM detection. The samples were collected at 2, 6,12h after intervention for real time PCR and 0.5, 2, 6, 12h for western blotting. LPS concentration was 10μg/ml in the experiment.1.4 Methods researching for the effects of PFC on the inflammatory response of PMVECs induced by LPS1.4.1 The release of proinflammatory mediators of ICAM-1, TNF-αand IL-8 were tested by ELISA or radioimmunity method;1.4.2 The expression of TLR-4 mRNA and TLR-4 protein were detected by real time PCR and western blotting respectively;1.4.3 the activities of NF-κB were determined by western blotting and by immuno-cytochemical double staining.The groups and the intervention methods were identical with those in FCM detection. LPS concentration was 10μg/ml in the experiment.2. Methods investigating the protective effects of PFC on the injury of AECs induced by LPS and the mechanism of PFC’s protective effectsBeing as a parallel study, we investigated the protective effects of PFC on the injury of AECs induced by LPS and the mechanism of PFC’s protective effects. The cell groups, intervention methods and the observing parameters were all identical to that in PMVECs experiment. The only difference was that A549 cell line was used in this test.Results1. results of the protective effects of PFC on the injury of PMVECs induced by LPS and the mechanism of PFC’s protective roles1.1 Isolation, culture, purification and identification of PMVECs from rats lung: Methods of isolation and identification of PMVECs from rat lung were established. And the purified PMVECs from rats presented a typical cobblestone morphology with monolayer growth and contact inhibition. vWF were expressed in cytoplasm of the purified cells, which was shown by immunocytochemical staining. Purified cells presented some typical ultrastructure characteristics of endothelial cell, including Weibel-Palade (W-P) bodies and abundant heterophagic vacuoles in cytoplasm, abundant mirovilli on PMVECs plasma membrane, etc..1.2 apoptosis results detected by FCM and TEM①results of FCM: The early apoptosis rates of PMVECs induced by LPS 10 ng/ml, 100ng/ml, lμg/ml, 10μg/ml, 12h after stimulated, were 6.23%, 6.07%, 9.51% and 23.48% respectively. The late apoptosis and necrosis rates were 3.93%, 6.03%, 5.84% and 10.89% respectively; and the viable cell rates were 88.91%, 87.33%, 84.19% and 64.86% respectively. This indicated that apoptosis damage of PMVECs induced by LPS was elevated at dose dependent, whereas the viable cell rate was decreased at dose dependent.Compared with Control, PFC and LPS+PFC groups, the early apoptosis rate and the late and necrosis rate in LPS groups were significantly increased (only there was no difference of late apoptosis rate at 6h between LPS and PFC group, p=0.066), the viable rate was markedly decreased. And the injury degree at 12h was more severe than that at 6h group. At 6 and 12h, there was no statistical difference of apoptosis and viable cell rate between the control and PFC groups and PFC groups each other. Compared with control and PFC groups, the early apoptosis rate of coculture group (LPS + PFC) at 6, 12h was raised, and the viable rate was decreased.②resuls of TEM: There were no apoptotic ultrastructure signs were observed under TEM in both cotrol and PFC groups. In LPS group, heterochromatin condensation, nuclear membrane unclear, mitochondrion swollen, etc., which representing the signs of early and intermediate stage of apoptosis were observed under TEM. And in coculture group, the nuclear membrane was clear, chromatin was even. Although heterochromatin was much more but no condensation, which indicated that there was no apoptotic ultrastructure signs were observed in the coculture group.1.3 results of the expression of TLR-4 and caspase-3 mRNA detected by real time PCR and the protein level of the two detected by western blotting:①results of real time PCR: the expression of TLR-4 and caspase-3 mRNA in LPS groups was markedly increased, the former increased at 2,6,12h, and the latter increased at 6,12h. There was no significant effect of PFC alone on the expression of the two genes. Whereas the expression of the two genes in the coculture groups was significantly decreased.②results of western blotting: In LPS groups, TLR-4 protein was markedly elevated at 0.5, 2, 6h, and caspase-3 precursor was significantly degraded and activated caspase-3 (17 kDa and 11 kDa subunits) was significantly increased at 0.5, 2, 6, 12h; There was no obvious effect of PFC alone on the two proteins. But in the coculture groups, the proteins of TLR-4 and activated caspase-3 were markedly decreased.1.4 Results of the protective effects of PFC on the inflammatory responses of PMVECs induced by LPS1.4.1 results of cytokines tested: The proinflammatory mediators were significantly increased in LPS groups. ICAM-1 and TNF-αwere increased at 2, 6, 12h, the secrete peaks were at 2h, IL-8 increased at 6h, and the secrete peak at 12h. There was no obvious impact of PFC alone on the above mediators. In the coculture groups, however, all the above mediators were markedly decreased.1.4.2 results of the expression of TLR-4 mRNA and TLR-4 protein: read the result of 1.3 please.1.4.3 results of activeties of NF-κB①results of activeties of NF-κB detected by western boltting: Being as the inhibitor of NF-κB, IkB-αwas significantly degraded at 0.5h, NF-κB P65 was released and translocated into the nucleus. Except for 2h, IκB-α. was degraded markedly and the level of P65 in nucleus was increased. The changes of the two proteins were approximately in-synchronism. There was no obviously effect of PFC alone on the activeties of NF-κB. But in coculture groups, the activeties of NF-κB were obviously decreased, which indicated by the degradation of IicB-αmarkedly decreased and the reduction of NF-κB P65 in the nucleus.(2) results of immunocytochemical double staining of NF-κB: In control group, IκB-αwas observed in cytoplasm and there was nearly no P65 protein in the nuclus, similar results were observed in PFC groups. In LPS groups, from 30min to 6h after stimulated, the coloration of IκB-αwas obviously thinningz and even no, whereas the heavy nuclear staining of P65 was observed, which indicated P65 was activated and translocated into the nucleus. In coculture groups, cytoplasmic immunocytochemical staining of IκB-αwas mainly observed and there was nearly no coloration of NF-κB P65 was observed in nucleus at 0.5 and 2h. At 6h, in addition to the coloration of IKB-αin cytoplasm, NF-κB P65 protein in nucleus was also observed in a few cells.2. results of the protective effects of PFC on the injury of AECs induced by LPS and the mechanism of PFC’s protective roles2.1 apoptosis results detected by FCM and TEM①results of FCM: The early apoptosis rates of A549 cells induced by LPS 10 ng/ml, 100ng/ml, lμg/ml, 10μg/ml, 12h after stimulated, were 4.12%, 6.42%, 7.53% and 16.12% respectively. The late apoptosis and necrosis rates were 3.49%, 5.22%, 4.31% and 5.67% respectively, and the viable cell rates were 91.34%, 87.87%, 87.78%, and 77.74% respectively. This indicated that the apoptosis injury of A549 cells induced by LPS was elevated at dose dependent, whereas the viable cell rate was decreased at dose dependent.Compared with control group, PFC and LPS+PFC groups, the early apoptosis rate and the late and necrosis rate in LPS groups were significantly increased at 6 and 12h, the viable cell rates were markedly reduced. And the injury degree at 12h was more severe than that at 6h group. There was no statistical difference of apoptosis and viable cell rate between the control and PFC groups at both 6 and 12h and PFC groups each other. Compared with control and PFC group, the early apoptosis rate of coculture group at 12h was obviously increased and the viable rate was decreased.②results of TEM: There were no apoptotic ultrastructure signs were observed under TEM in both cotrol and PFC groups. In LPS group, heterochromatin condensation, especially a typical horseshoe shape of chromatin condensation, nuclear membrane unclear, etc., which representing the signs of early and intermediate stage of apoptosis were observed under TEM. And there was no obviously apoptotic ultrastructure signs were observed in the coculture group.2.2 results of the expression of TLR-4 and caspase-3 mRNA detected by real time PCR and the protein level of the two detected by western blotting.①results of real time PCR: After stimulated by LPS, the expression of TLR-4 and caspase-3 mRNA was markedly increased, the former increased at 2, 6,12h, and the latter increased at 6,12h. There was no effect of PFC alone on the expression of the two genes. Whereas the expression of the two genes in the coculture group was significantly decreased.②results of western blotting: After stimulated by LPS, TLR-4 protein was markedly elevated at 0.5, 2, 6,12h, caspase-3 precursor was significantly degraded and activated caspase-3 (17 kDa and 11 kDa subunits) was synchronicly increased at 0.5, 2, 6 and 12h. There was no effect of PFC alone on the two proteins. In the coculture group, however, the proteins of TLR-4 and activated caspase-3 were markedly decreased.2.3 Results of the effects of PFC on the inflammatory responses of A549 cells induced by LPS2.3.1 results of cytokines tested: The proinflammatory mediators were significantly increased in LPS groups. ICAM-1, TNF-αand IL-8 were increased at 2, 6,12h; and the secrete peaks of ICAM-1, TNF-αwere all at 2h, and IL-8 was at 12h; There was no effect of PFC alone on the above mediators. In the coculture groups, however, all the above mediators were markedly decreased.2.3.2 results of the expression of TLR-4 mRNA and TLR-4 protein: read the result of 2.2 please.2.3.3 results of activeties of NF-κB①detected by western boltting: IκB-αwas significantly degraded at 0.5,2, 6 and 12h. Being as the in-synchronism response, NF-κB P65 was released and translocated into the nucleus. But there was statistical significance only at 2 and 6h. There was no effect of PFC alone on the activeties of NF-κB. But in coculture groups, the activeties of NF-κB were obviously decreased, indicated by the degradation of IκB-αmarkedly decreased and the reduction of NF-κB P65 in the nucleus.(2) results of the immunocytochemical double staining of NF-κB: IκB-αwas observed in cytoplasm and there was nearly no P65 protein in nuclus in control and PFC groups. In LPS group, the heavy nuclear staining of P65 was observed at 6h. In coculture group, double coloration cells , IκB-αpositive staining cells and P65 cells were all observed. It should be pint out that staining result of NF-kB in A549 cells was not as good as that in PMVECs, and we failed to succeed at staining A549 cells at 0.5 and 2h. The result was maybe caused by some reasons we still unable to definite.Conclusions1. LPS can induce apoptosis injury in both PMVECs and ECs in vitro, and the injury degree is at dose- and time-dependant. The expression of TLR-4 and caspase-3 mRNA and protein level were markedly increased after stimlated by LPS. As TLR-4 is the initiator of the LPS/ TLR-4 signal pathway, and caspase-3 is known as the apotosis executioner, which suggests that LPS induces apoptosis in PMVECs and AECs maybe via mediating the pathway of LPS/ TLR-4→caspases activation in proper order→ apoptosis.2. PFC alone does not induce apoptosis injury in both PMVECs and AECs in vitro and has no obviously effects on the expression of TLR-4 and caspase-3 mRNA and the protein level of the both two signal molecules. In the coculture groups, however, the apoptosis rates of the both cell populations were significantly decreased, the viable cells were markedly increased. Meanwhile, the up-regulation of the expression of TLR-4 and caspase-3 mRNA and the protein level induced by LPS were significantly reduced. This means that PFC is able to protect PMVECs and AECs from LPS-induced apoptosis injury via blocking the initiation of LPS signal pathway .3. LPS activates both PMVECs and A549 cells in vitro, make the two cell populations release proinflammatory mediators. Being as the priming signal molecule, the expression of TLR-4 mRNA and protein was markedly increased after stimlated by LPS. Meanwhile, the activities of NF-κB, the key downstream effector molecule of the signal pathway were increased rapidly stimulated by LPS as well. This suggests that LPS induces inflammory responses in PMVECs and AECs via the signal transduction pathway of LPS/ TLR-4→NF-κB activation→proinflammatory mediators production.4. PFC alone does not induce the release of cytokines in PMVECs and AECs in vitro and has no effects on the expression of TLR-4 gene and protein. Meanwhile PFC alone does not affect the activities of NF-κB as well. When the two population cells were incubated with LPS and PFC, however, the release of proinflammatory cytokines was obviously reduced. And the expression of TLR-4 gene and protein as well as the activities of NF-κB were significantly down-regulated. Which suggests that PFC is able to prevent PMVECs and AECs from LPS-induced inflammatory responses via blocking the initiation of LPS signal pathway.更多还原