【作者】 黄波；

【导师】 匡凤梧； 许峰；

【作者基本信息】 重庆医科大学 ， 儿科学， 2009， 博士

【摘要】 低氧血症是危重新生儿常见的临床表现,严重时可危及生命。氧疗是改善新生儿缺氧状态的重要治疗方法,在新生儿特别是早产儿,由于肺发育不成熟,肺间质和肺泡分化不全,肺弹力纤维和结缔组织发育不良,未成熟肺长时间暴露于高氧下会导致肺上皮细胞损伤、死亡、急性肺损伤(Acute lung injury, ALI)甚至呼吸功能衰竭致支气管肺发育不良( bronchopulmonary dysplasion ,BPD)。近年来BPD在全球发病率逐年增高,存活者常伴有明显肺发育障碍和功能低下,目前国际上尚无确定有效的防治方法,但大量临床资料显示高浓度吸氧、长时间氧疗是导致BPD的高危因素,其病因与高氧性肺损伤密切相关。当前国内外研究主要集中在如下几个方面①尝试用肺表面活性物质蛋白(SP-A)弥补损伤的AECⅡ功能,但SP-A生物提成困难,远期效果不尽理想,②也有学者企图通过上皮干细胞定向分化为AECⅡ再移植给肺损伤的病人,但未解决细胞分化、培养、体内生长调控等问题,③利用基因工程技术所生产的生长因子药物如表皮生长因子(EGF),碱性成纤维细胞因子(bFGF),神经生长因子(NGF)虽较好地应用于体表创烧伤患者的修复重建,但目前尚不能在肺损伤的主动修复重建中应用。④虽发现转化生长因子(TGF-β)和干扰素(INF-γ)在肌成纤微细胞的转型异常中分别起正负调控作用,但两者相互拮抗的机制并不清楚。⑤虽发现细胞外基质(ECM)的积聚在肺纤维化发展过程中的作用,然而目前尚无有效方法阻止ECM的积聚⑥由于肺损伤后AECⅡ修复重建的一系列病理生理变化仍不清,因而现今临床上采用的多种治疗方法如:更换机械通气模式、激素、表面活性物质替代疗法、免疫疗法等均收效甚微。回顾以往的研究多集中在如何阻断有害因素对AECⅡ的损害,而对损伤后如何保护和促进AECⅡ修复重建研究甚少,近年的研究发现,肺泡Ⅱ型上皮细胞(Alveolar epithelial typeⅡcells,AECⅡ)存活和凋亡变化可能参与高氧肺损伤的发展和转归,影响肺损伤修复的结局。如果早期干预AECⅡ的凋亡变化就可能减轻肺上皮细胞损伤,从而逆转肺损伤,并阻断后续的肺间质增生和肺组织纤维化。AECⅡ的主动修复理论是近年来研究的热点,寻找促进AECⅡ主动修复的新型调控因子成为高氧性肺损伤防治的新切入点。近年来由肺神经内分泌细胞(Pulmonary neuroendocrine cell,PNECs)分泌的感觉神经肽类递质的作用开始被注意,神经肽类递质(Neuropeptides,Ne )是一种细胞调控因子或细胞外信使分子,是神经、内分泌和免疫共同识别的信号物质之一,“神经?体液?免疫”网络对创伤修复过程起调控作用。神经肽P物质(substance P,SP)是最早发现的速激肽家族神经肽,广泛分布于气道上皮细胞层内、肺血管、气管、支气管平滑肌层内及腺体和支气管神经节周围,新近研究表明SP在修复细胞的增殖、迁移、分化方面有重要作用,SP对角质细胞、平滑肌细胞、成骨细胞、血管内皮细胞均有明显的促增殖分化作用,呼吸器官含有丰富的SP,而其对高氧肺损伤AECⅡ修复作用尚不清楚。细胞凋亡是基因调控下细胞积极死亡的形式,往往涉及一些信号途径的参与,假如SP是AECⅡ新的调控因子,那么它是通过什么分子机制来现实这一调控过程?丝裂素活化蛋白激酶(Mitogen activated protein kinases , MAPKs )途径,包括细胞外信号调节激酶(Extracellular signal-regulated kinase ,ERK),c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)和P38激酶(P38MAPK),是细胞增殖、分化等信息传递途径的交汇点和共同通路。SP能否促进高氧后损伤的AECⅡ的修复,且是否与调控MAPKs有关,目前尚不清楚。综上所述,SP作为重要的感觉神经肽类递质,可能通过MAPK途径参与高氧性肺损伤AECⅡ的修复,从而减轻高氧性损伤,将是未来高氧ALI和BPD防治的可行性途径。本课题以原代培养的早产鼠AECⅡ为研究对象,通过建立体外细胞氧化损伤模型,观察高氧暴露不同时间及SP干预对AECⅡ增殖存活的影响及MAPKs的调控机制。第一部分原代早产大鼠肺泡II型上皮细胞分离、纯化、培养及鉴定背景氧气疗法是临床上治疗新生儿急性呼吸衰竭常用且有效的措施,但长时间高氧暴露可引起氧化应激性肺损伤。高浓度氧气(氧气体积分数>950ml/L,简称高氧)暴露是导致氧化应激性肺损伤的主要致病因素。高氧肺损伤是弥散性肺泡炎和损伤后修复重建的复杂病理生理过程,其治疗的根本难题在于内源性肺泡上皮不能恢复,肺组织修复过程中不是由正常的肺泡上皮替代,而是由成纤维细胞替代导致肺组织修复障碍。肺泡上皮损伤后修复主要依赖于肺泡上皮的干细胞——AECⅡ的增殖和分化以覆盖肺泡表面,完成肺泡的修复。AECⅡ体积较小、呈立方形,占肺泡上皮细胞数的60%左右,能通过增殖、铺展、迁移,并最终转化为肺泡I型上皮细胞(Alveolar type I epithelial cells, AEC I ),恢复肺泡上皮正常的形态和功能。胎肺发育晚期肺组织主要由AECⅡ和肺成纤维细胞(Lung fibroblast ,LF)组成,因此胎肺AECⅡ和LF的分离、纯化和原代培养对体外研究肺发育和早产儿肺部疾病至关重要,但早产大鼠AECⅡ细胞的原代分离、纯化技术难度大,培养条件相对要求高;特别是AECⅡ不能传代培养,分离高纯度的细胞仍有较大困难。本研究的目的是掌握早产大鼠AECⅡ的分离、纯化和培养技术,以获得数量足够、纯度高的AECⅡ,满足下一步实验的需要。目的确定早产大鼠原代AECⅡ分离、纯化、培养的技术方法,为研究高氧刺激下AECⅡ存活、凋亡和信号转导机制提供数量足够和高纯度的AECⅡ,以满足实验的需要。方法成年SD(Sprague-Dawley,SD)大鼠,购自第三军医大学大坪三院实验动物中心。雌雄按1:1合笼交配,找到阴栓日计为妊娠第1天。取胎龄19天的早产鼠分离AECⅡ,步骤如下:水合氯醛麻醉孕19天大鼠,剖宫取出胎鼠,分离胎肺,去除气管、支气管等非肺组织后剪至1mm3大小,加入0.25%胰酶消化25-30分钟,完全培养基终止消化。100目筛网过滤,滤液800rpm离心后弃上清,沉淀加入0.1%的胶原酶Ⅰ消化45分钟后离心去上清,沉淀重悬在含10%胎牛血清、100u/ml青霉素和100μg/ml链霉素的DMEM/F12培养基中,接种至培养瓶,培养30分钟后贴壁的为LF,未贴壁的为AECⅡ,吸出,800rpm离心后接种至新的培养瓶。如此反复贴壁3次,以纯化AECⅡ。最后未贴壁的细胞按浓度1.5×106/ml接种至6孔培养板,培养12小时后弃除未贴壁的细胞,此时贴附在瓶底的细胞即为AECⅡ。继续接种于6孔培养板或96孔培养板,更换新鲜培养液继续培养12h备用。台盼蓝拒染法测定细胞活力、改良巴氏染色法确定细胞纯度、透射电镜(Transmitting electron microscopy, TEM)鉴定细胞;在体外培养细胞不同的时间(12、24、和48h)观察细胞的性状和形态变化。结果1.每3-5只19天胎龄早产鼠经分离培养可获AECⅡ数约为(36±5)×106。2.台盼蓝拒染法测定细胞活力> 90%。3.改良巴氏染色法证实细胞纯度> 90%。4.电镜下观察到AECⅡ典型细胞结构——板层小体。5.体外培养12至24 h时AECⅡ生长良好,细胞质内有较多颗粒;48h以后细胞呈长索形,细胞内颗粒减少,并出现空泡。结论本实验观察到19d胎鼠AECⅡ原代培养12h ,接近融合状态,原代培养第24～48h,增殖代谢旺盛,生长状态最佳,可获得高产量、高纯度、高活力的原代AECⅡ,此时适合做体外研究。第二部分高氧暴露及神经肽P物质干预对早产大鼠肺泡II型上皮细胞的影响背景氧疗是临床上用于提高血氧饱和度、改善组织缺氧状态的一种辅助治疗手段,高浓度氧气(氧气体积分数> 950ml/L,简称高氧)机械通气是治疗严重呼吸衰竭(Respiratory failure,RF),尤其是急性呼吸窘迫综合征(Acute respiratory distress syndrome,ARDS)的常用措施,但常时间暴露在高氧状态下可产生过量的活性氧自由基(Reactive oxygen species ,ROS),引起细胞内氧化/抗氧化体系失衡而导致氧化应激性肺损伤,引起早产儿肺发育受阻,形成早产儿BPD。如何促进高氧损伤后的修复,防治BPD是临床工作者十分关注的问题。高氧肺损伤时,由于AECI较AECII更易受高浓度氧的损伤,首先受到破坏,AECⅡ是肺组织中的重要细胞,在肺泡受到损伤时能转化为AECI修复受损肺泡,由于AECI是高度分化的细胞,不可能再生,所以肺泡的损伤修复完全依赖于AECⅡ的增殖分化。近年研究发现,在肺发育关键阶段,高氧可引起肺的重塑及生长异常,其中一个主要因素是其导致了AECII的凋亡。但细胞凋亡在高氧肺损伤中的作用仍不清楚,多数学者报道高氧诱导的细胞凋亡与肺损伤的程度成正相关。另外,对于细胞凋亡与肺纤维化关系的研究表明,肺泡和细支气管上皮细胞的凋亡可能是导致肺纤维化的一种机制。据此推测如果采取一定措施防止AECII的凋亡,增加AECII增值、移行,可能对高氧肺损伤后的BPD起到防治作用以往的研究多集中在如何阻断有害因素对AECⅡ的损害,更多的重视免疫源性因素如炎症细胞、细胞因子对创伤修复的影响,而对损伤后如何保护和促进AECⅡ修复重建研究甚少,忽视了“神经?体液?免疫”网络对修复过程的调控。故寻找促进AECⅡ主动修复的新型调控因子成为高氧性肺损伤防治的新切入点。神经肽类递质(Neuropeptides,Ne )是一种细胞调控因子或细胞外信使分子,是神经、内分泌和免疫共同识别的信号物质之一。新近研究表明感觉神经肽SP在修复细胞的增殖、迁移、分化方面有重要作用,在成体皮肤创伤愈合中已证实SP不仅启动愈合早期的神经源性炎性反应,同时对修复细胞增殖、再生和瘢痕形成密切相;气道SP主要来自感觉神经c-纤维末端,分布于气道上皮细胞层内、肺血管、气管、支气管平滑肌层内及腺体和支气管神经节周围。气道神经内分泌细胞、平滑肌细胞、嗜酸性粒细胞、淋巴细胞和肺泡巨噬细胞都能合成和分泌SP;SP特异性受体可见于气道平滑肌、粘膜下腺、血管内皮,一些炎性细胞也表达SP受体。呼吸器官含有丰富的SP,但其在肺损伤修复中的地位和作用国内外尚无相关报道。本部分研究高氧暴露不同时间及SP干预对AECⅡ增殖存活的影响和高氧暴露不同时间早产鼠肺组织SP含量的动态变化。目的1.采用高氧和无血清培养基建立原代早产大鼠AECⅡ的氧化损伤模型。2.研究高氧暴露不同时间及SP干预对AECⅡ形态、膜脂质过氧化程度指标丙二醛(MDA)、总抗氧化能力(TAOC)、存活和凋亡的影响,探讨AECⅡ损伤与高氧作用的时间效应关系及SP干预对AECⅡ损伤的作用。3.早产鼠高氧肺损伤动物模型的制备及SP含量的测定。方法1.分离、纯化清洁级Spraque-Dawley早产大鼠的AECⅡ,台盼兰拒染法鉴定细胞活性,改良巴氏染色法鉴定细胞纯度。将AECⅡ随机分为:空气暴露组、高氧暴露组、SP干预空气暴露组、SP干预高氧暴露组,空气暴露组氧体积分数为210ml/L,高氧暴露组氧体积分数为950ml/L),SP干预组于暴露前加入SP 1×10-6mol/L,在置于氧体积分数为210ml/L和950ml/L中各组分别暴露12、24、和48h,电镜观察AECⅡ的形态变化;采用分光光度计测定各组丙二醛(MDA)、总抗氧化能力(TOAC)浓度;噻唑兰检测法(MTT法)及流式细胞仪测定其增殖率和凋亡率,以明确细胞损伤与高氧作用的时间效应关系及SP干预对AECⅡ损伤的作用。2.剖宫取出SD大鼠孕2l d(足月为22 d)早产鼠,随机分为空气暴露组和高氧暴露组,空气暴露组氧体积分数为210ml/L,高氧暴露组氧体积分数为950ml/L,分别于暴露3,7,14 d后,取肺组织测定其SP含量。结果1.与空气暴露组相比,AECⅡ高氧暴露后12h,细胞间隙稍增宽,部分细胞内可见反光增强的空泡;高氧暴露后24h,细胞间隙增宽,细胞体积减小,胞内空泡增多,部分细胞核浓缩变小,细胞内颗粒(板层小体)减少;高氧暴露后48h,大量细胞变圆、皱缩明显,细胞脱壁,培养上清液中可见漂浮的细胞和细胞碎片;透射电镜下可观察到典型的凋亡细胞(细胞体积变小,细胞质浓缩,胞膜内陷,细胞核固缩,核染色质浓缩并凝结成块)和凋亡小体围绕。与空气暴露组比较,发现随高氧暴露时间的延长,细胞的形态损害随之加重。而SP干预后高氧暴露12、24及48h,较高氧暴露组同时间点比较细胞器变化减轻。2.高氧暴露及SP干预高氧暴露12、24及48h对AECⅡ氧化损伤的影响:与空气组比较,高氧组12、24及48h TAOC显著下降,MDA明显升高,而与SP干预高氧暴露组比较,其TAOC仍低于空气组,但较高氧组明显升高,同时MDA虽高于空气组,但较高氧组明显降低。3. MTT实验结果表明:与空气暴露组比较,高氧暴露组12、24及48h AECⅡ增殖活性显著下降,AECⅡ存活率呈下降明显(P < 0.05)。随作用时间的延长,细胞存活率逐渐下降,各组间比较有显著差异(P < 0.05)。而与SP干预高氧暴露组比较,其增殖活性仍低于空气暴露组,但较高氧暴露组明显升高。4.流式细胞仪检测细胞周期检测AECⅡ凋亡结果显示:与空气暴露组比较,高氧暴露组12、24及48h,随高氧作用时间的延长, AECⅡ凋亡率呈明显上升趋势(P < 0.05)。而与SP干预高氧暴露组比较,同时间点凋亡率虽高于空气暴露组,但较高氧暴露组明显降低。5.与空气暴露组比较,高氧暴露3、7、14 d早产鼠肺组织SP含量显著降低(P<0.01),且随高氧暴露的延长,SP含量降低明显(高氧暴露组间比较,P<0.01)。结论1.高氧刺激可诱导AECⅡ发生凋亡,并以时间依赖的方式加重AECⅡ损伤。2. SP干预后可减少MDA产生,提高TAOC水平,提示SP干预后可增加机体AOE的活性,使氧化和抗氧化失衡状态得到纠正,有助于减轻和耐受高氧肺损伤。3. SP干预后可减少高氧所致AECⅡ的凋亡率增加存活率,提示SP可以减轻AECⅡ的氧化损伤,对氧化应激状态下的AECⅡ可能起到保护作用。4.高氧暴露可导致SP含量的动态变化,提示高氧暴露后导致气道感觉神经c-纤维末端SP分泌不足,局部SP含量的降低可能与AECⅡ的损伤程度有关。第三部分高氧暴露及神经肽P物质干预调控早产大鼠肺泡II型上皮细胞修复的MAPKs信号机制背景氧气疗法是临床上治疗急性呼吸衰竭常用且有效的措施,但长时间吸人高浓度氧,肺直接暴露于高氧中,引起肺泡毛细血管通透性增高、肺泡液渗出增多、炎性损伤、纤维蛋白沉积及肺表面活性物质活性降低等非特异性改变,引起氧化应激性肺损伤致BPD,BPD是早产儿长时间吸入高体积分数氧(高氧)治疗的常见并发症。第二部分证实了高氧以时间依赖的方式诱导AECⅡ损伤,高氧刺激可诱导AECⅡ发生凋亡;SP干预后可减少高氧所致AECⅡ的凋亡率增加存活率,对氧化应激状态下的AECⅡ可能起到保护作用。细胞凋亡是基因调控下细胞积极死亡的形式,往往涉及一些信号途径的参与,SP是AECⅡ新的调控因子,那么它是通过什么分子机制来实现这一调控过程?丝裂原活化蛋白激酶(mitogen.activated protein kinases,MAPKs)信号传递通路在介导高氧肺损伤过程中扮演重要角色,MAPKs主要包括3个家族成员:胞外信号调节激酶(extracellular si gna 1-regulated kinases,ERKs),C-JUN氨基端激(C-JUN-NH2.terminal kinases,JNKs)和p38激酶。目前认为,MAPK信号通路是细胞外的各种信号从细胞表面传导到细胞核内部的重要通道,能被多种刺激因素激活而介导细胞的凋亡。MAPKs是细胞应激反应、增殖、分化和凋亡等信息传递途径的交汇点和共同通路,细胞外各种刺激信号可通过细胞内不同的信息传递通路,共同交汇于MAPKs。已有研究证实,暴露于95%氧气以及H2O2应激可触发转录因子AP-1及MAPK家族p38、JNK成员的持续激活,从而介导高氧所致肿胀性细胞死亡以及细胞凋亡,且细胞的存活率在特异性抑制剂抑制MAPK活化的同时得到明显的改善;维甲酸通过调控MAPK途径减轻早产大鼠高氧肺损伤。MAPKs通路参与了高氧氧化应激下AECⅡ凋亡信号转导,并对AECⅡ起促凋亡的作用。SP干预是否也可以通过MAPKs通路,对AECⅡ的凋亡存活产生影响?其调控机制尚不清楚。本部分进一步研究高氧暴露及SP干预对原代培养的AECⅡ的损伤及保护中是否有MAPK信号的参与及MAPK信号的调控机制。目的1.采用高氧和无血清培养基建立原代早产大鼠AECⅡ的氧化损伤性细胞模型。2.研究高氧暴露不同时间及SP干预对AECⅡ形态、存活和凋亡的影响,探讨AECⅡ损伤与高氧作用的时间效应关系及SP干预对AECⅡ损伤的作用。3.研究高氧氧化应激及SP干预下AECⅡ凋亡过程中MAPKs的活化情况,从而研究MAPKs对AECⅡ凋亡的作用及SP干预后对MAPKs信号机制的调控。方法分离、纯化清洁级Spraque-Dawley早产大鼠的AECⅡ,台盼兰拒染法鉴定细胞活性,改良巴氏染色法鉴定细胞纯度。将AECⅡ随机分为:空气暴露组、高氧暴露组、SP干预空气暴露组、SP干预高氧暴露组,空气暴露组氧体积分数为210ml/L,高氧暴露组氧体积分数为950ml/L ,SP干预组于暴露前加入SP 1×10-6mol/L,在置于氧体积分数为210ml/L和950ml/L中各组分别暴露12、24、和48h,蛋白质免疫印迹法(Western blot)检测各组各时间点磷酸化总ERKs、JNKs或p38表达。结果1.高氧暴露后AECⅡ12、24及48h ,发现高氧暴露刺激后很快诱导p-ERK表达,12、24及48h时p-ERK表达与空气暴露组相比显著增加,其中48h时p-ERK蛋白条带表达最强, SP干预后高氧暴露12、24及48h,较高氧组同时间点比较p-ERK表达更明显。2.高氧暴露后AECⅡ12、24及48h ,发现高氧暴露刺激后很快诱导p- p38表达,12、24及48h时p- p38表达与空气暴露组相比显著增加,其中48h时p- p38蛋白条带表达最强, SP干预后高氧暴露12、24及48h,较高氧组同时间点比较p- p38表达减弱。3.高氧暴露后AECⅡ12、24及48h ,发现高氧暴露刺激后很快诱导p-JNK表达,12、24及48h时p-JNK表达与空气暴露组相比显著增加,其中48h时p-JNK蛋白条带表达最强,SP干预后高氧暴露12、24及48h,较高氧暴露组同时间点比较p-JNK表达减弱。结论本研究发现高氧暴露刺激AECⅡ后能很快诱导MAPKs表达,在48h左右达到峰值,使用SP干预MAPKs信号通路后,在高氧刺激下SP干预组细胞的凋亡率明显低于高氧组的凋亡率,而其细胞存活率明显高于高氧组的存活率,说明MAPKs信号介导了AECⅡ的凋亡,SP干预后进一步激活胞外信号调节激酶并抑制C-JUN氨基端激酶和p38激酶信号激活对氧化应激状态下的AECⅡ有保护作用。更多还原

【Abstract】 Hypoxemia is a common clinical feature of critical neonates and severe hypoxemia can threat their lives. Oxygen therapy is an important method to improve hypoxia condition of neonate. In neonate especially preterm infant, pulmonary developmental immaturity leads to pulmonary interstitial and al-veolus differential insufficency and pulmonary elastic fibrous and connective tissular dysplasia. Long-time exposure to hyperxia will result in alveolar epithelial cells damage, death, acute lung injury (ALI) and even to bron-chopulmonary dysplasion (BPD) caused by respiratory function failure. In the past few years, the incidence of BPD raised yearly in the world. Usually, pulmonary maldevelopment and hypofunction are accompanying with the survivors. Up to now, there is no definited effective prevention and cure approach globally. However, vast of clinical data indicate hypoxemia and prolonged oxygen therapy are high risk factors for BPD. Furthermore, the etiopathogenisis of BPD is closely related to hyperxia-induced lung injury. Currently, the domestic and overseas studies focus the following aspects: Ⅰ.To attempt to retrieve impaired AECⅡfunction by pulmonary surfat-cant-associated protein A (SP-A). However, the biologic extract of SP-A is a difficult task and the long-term effectiveness is not satisfied.Ⅱ. Some scholars tried to differentiate epithelium stem cell into AECⅡand transplant the AECⅡinto patients with lung injury. Unfortunately, some key tech-niques such as cell differentiation, culture and growth regulation in vivo could not be solved successfully.Ⅲ. To utilize growth factor medicines produced by genetic engineering technology. Although epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) were successfully used in body surface repairing and reconstruction of patients suffered from wound and burn, these growth factors have not been applied in the active repairing and reconstruction during lung injury.Ⅳ. Transforming growth factorβ(TGF-β) and interferonγ(INF-γ) plays a positive and negative role in abnormal transconformation of myofibroblast, respectively. However, the rivalry mechanism of the both factors is still unknown.Ⅴ. Although the role of extracellular matrix (ECM) accumulation in the development of pulmonary fibrosis was confirmed, there is still no effective method to prevent ECM accumulation.Ⅵ. For the a series of un-certain pathophysiological change in AECⅡrepairing and reconstruction after lung injury, many therapy methods such as changing mechanical ven-tilation pattern, hormone, surface active substance replacement therapy and immunotherapy achieved little. We reviewed the past studies and found the studies mainly focused on how to block the damage of harmful factors to AECⅡ. However, the studies on how to protect and promote AECⅡre-pairing and reconstruction are very few. Recent study indicated that survival and apoptosis change of alveolar epithelial typeⅡcells (AECⅡ) might involve in the development and turnover of hyperxia-induced lung injury, which affected the repairing after lung injury. Pulmonary epithelial cells injury may be alleviated if the change of AECⅡapoptosis is intervented at early stage, which can reverse lung injury and block subsequent pulmonary interstitial hyperplasia and pulmonary fibrosis. Now, active repairing theory on AECⅡis a hot spot. Looking for new regulatory factors for AECⅡac-tive repairing is becoming an new point for preventing hyperxia-induced lung injury. Recently, the role of sensor neuropeptides (Ne) transmitters that are secreted by pulmonary neuroendocrine cell (PNECs) has been focused. Ne is a cellular regulatory factor or extracellular messenger molecular. It is one of the signal substances recognized by nerve, endocrine and immunity systems jointly. Meanwhile, Nerv e-Hu mor-Im munity network regulates the process of wound repairing. SP was first found to be a neuropeptide in tachykinin family, and it distributes widely in airway endothelial cell layer, pulmonary vessel, trachea, bronchus smooth muscle and around gland and bronchus ganglion. Recent studies suggest SP plays a key role in prolifera-tion, migration and differentiation of impaired cells. Meanwhile, SP has significant proliferation and differentiation effect on keratinocyte, smooth muscle cell, osteoblast and endothelium. SP expresses abundant in respiratory or-gans. However, the repairing effect of SP on hypoxia-induced lung injury is still unknown.Apoptosis is a cellular active death manner under gene regulation, which frequently involves some signal pathways. If SP is an new regulatory factor for AECⅡ, then what is the regulatory mechanism? Mitogen acti-vated protein kinases (MAPKs) pathway including Extracellular sig-nal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and P38 kinase is a junction and common access of information transfer pathway such as cell proliferation and differentiation. However, whether SP can promote AECⅡrepairing after hypoxia-induced lung injury and the regulatory mechanism is related to MAPK pathway or not is still not understood.In this context, as an important sense neuropeptides transmitter, SP may alleviate hypoxia-induced lung injury via MAPK pathway. It is a feasible approach to prevent and cure hypoxia-induced acute lung injury(ALI) and BPD. In present study, primary cultured premature rats AECⅡwas selected for the following study. First, we established cellular oxidative damage patternl in vitro, and then observed the effect of SP on proliferation and survival of AECⅡat different time. Finally, the related MAPKs regulatory mechanism was investigated. PartⅠ. Isolation, purification, culture and identifica-tion of primary AECⅡof preterm ratsBackgroundOxygen therapy is a commonly and effective measure for neonate acute respiratory failure(ARF). However, long-time hypoxia exposure leads to oxidative stress- induced lung injury. Hypoxia (>95% oxygen) exposure is a main pathogenic factor that causes oxidative stress-induced lung injury. Hypoxia-induced lung injury is a complicated pathophysiological event involving disseminated alveolitis, repairing and reconstruction after lung injury. Fibroblast not normal alveolar epithelium replaces the impaired ep-ithelium in the process of lung repairing, which results in the disorder of endogenous alveolar epithelial restoration after injury.Alveolar epithelial repairing dependents on the proliferation and differentiation of alveolar ep-ithelial stem cell- AECⅡ. AECⅡcan cover the surface of alveolus and completes the repairing. AECⅡis comparatively smaller and cuboidal. The amount is about 60% of total alveolar epithelial cells. It can transform into alveolar type I epithelial cells (AECI) by proliferation, spreading and mi-gration, which restores normal morphology and function of alveolar epithe-lium. Lung tissue is mainly consist of AECⅡand lung fibroblast (LF) in the late stage of fetal lung development. Therefore, isolation, purification and primary culture of AECⅡand LF in fetal lung is crucial to study pulmonary disease of preterm infant in vitro. Nevertheless, it is difficult to isolate and purify primary AECⅡfrom preterm rats. Meanwhile, AECⅡcan not be passage cultured and the culture condition is comparatively more strict. In present study, we managed to obtain enough and pure AECⅡfor next needs by isolation, purification, culture of primary AECⅡof preterm rats. ObjectiveTo establish the isolation, purification, culture methods for primary AECⅡof preterm rats and provide enough and pure AECⅡto investigate related survival, apoptosis and signal transduction mechanism under hy-poxia exposure.MethodsThe adult specific-pathogen-free (SPF) Sprague-Dawley( SD ) rats used in this study were obtained from the Experimental Animal Center of Third Affiliated Hospital of Third Military Medical University (Chongqing, China). Rats were raised in a cage with an appropriate proportion (1:1) between female and male. Gravidity was confirmed if vaginal plug was seen at the second morning. Pick out the premature rats from the pregnant rats at 19 day and isolated AECⅡquickly. Briefly, the pregnant rats were anesthe-tized by chloral hydrate at 19 day. Uterine-incision delivery was used to pick out the infant rats and isolated infant lung. Non-lung tissues such as trachea and bronchus were cut into a 1mm3-size. The cells were digested by 0.25% trypsogen for 25-30 min.Complete medium was added to suspend the di- gestion. Then, the cell suspension was filtered by a 100 screen openings-grit. Centrifuged at 800 rpm for 10 min and discarded the supernatant. 0.1% collagenase I was added to digest the precipitation for 45 min. Centrifuged at 800 rpm for another 10 min and removed the supernatant. The precipitation was resuspended in dulbecco’s modified eagle’s medium(DMEM/F12) complemented with 10% fetal calf serum, 100 U/ml penicillin and 100μg/ml streptomycin. The cells were inoculated in a plastic flask. The adherent cells were LF, and non-adherent ones were AECⅡ. Removed AECⅡand centrifuged at 800 rpm for 10 min. Then inoculated in an new plastic flask. The ma-nipulation was repeated three times to purify AECⅡ. Finally, the non-adherent cells were inoculated in a 6-well plate (1.5×106 /ml). Cultured for 12 h and discarded the non-adherent cells. Inoculated AECⅡin another 6-well plate or 96 well plate. Replaced fresh culture medium and cultured for another 12 h for use. Cells viability was assessed by trypan blue exclusion. Modified papanicolaou staining was employed to evaluate cells purity. The cells were identified under transmitting electron microscopy (TEM). Meanwhile, we observed cellular character and morphologic change at 12, 24 and 48 h after the inoculation.ResultsI. (36±5)×106 AECⅡcould be obtained from every 3-5 preterm rats (19 d).II. Trypan blue exclusion assay showed the cells viability was more than 90%.III. Cells purity was confirmed to be more than 90% by modified papani-colaou staining.IV. Typical structure of AECⅡ, lamellar body, was observed under TEM.V. AECⅡgrew well at 12-24 h. More particles appeared in cytoplasm. Cells began to elongate at 48 h along with decreased particles and emergence of vacuolus.ConclusionIn present study, we observed primary AECⅡfrom preterm rats(19d) was in a fusion status approximately at 12 h. The cellular proliferation and metabolism was enhanced at 24-48 h after the primary culture, and the growth status is best. Therefore, productive, pure and active primary AECⅡcould be obtained for study in vitro in this period.PartⅡ. Effect of hypoxia exposure and intervention of SP on typeⅡalveolar epithelial cells of preterm ratsBackgroundAs an adjunctive therapy, oxygen therapy is employed to enhance saturation of blood oxygen and improve tissular hypoxia condition. Me- chanical ventilation of high concentration oxygen(>95% oxygen, hyperxia) is a common therapy for the treatment of respiratory failure(RF) especially acute respiratory distress syndrome(ARDS). However, prolonged exposure to hyperxia will result in pulmonary oxidative stress-injury. It will cause impaired pulmonary development and injury of preterm infants, which lead to BPD in preterm infants. Therefore, how to promote repairing after hy-perxia-induced lung injury and prevent and cure BPD is a conspicuous issue in clinic. Compared with AECⅡ, AECⅠis more susceptible to suffer from hyperxia-induced injury. For AECⅡis an important cell in lung tissue, it can transform into AECⅠand repair the impaired alveolus when alveolus are damaged. At this moment, proliferation and transformation of AECⅡis needed to repair the impaired alveolus structure. Recently, studies indicated that hyperxia is responsible for repatternling and abnormal growth in key stage of pulmonary development. Among it, hyperxia-induced apoptosis is a primary factor. However, the role of apoptosis in hyperxia-induced lung injury is still not known. Meanwhile, majority of the studies indicated hy-perxia-induced apoptosis apoptosis was positively related to lung injury degrees. In addition, study on the relationship between apoptosis and pul-monary fibrosis indicated that apoptosis of alveolus and bronchiole epithe-lium may be responsible for pulmonary fibrosis. In view of this, we specu-late that BPD after hyperxia-induced lung injury can be prevented and cured if some measures are adopted to suppress the apoptosis of AECⅡ. Studies in the past mostly focused on to how to block the damage of harmful factors to AECⅡ. They included the effect of immunogenic factors such as inflammatory cells and cytokines on wound repairing. However, few studies are pay attention to on how to protect and promote repairing and reconstruction of AECⅡ, which ignores the regulation of Nerve- Hu-mour-Inmmunity network to the process of repairing. Therefore, searching new regulatory factors for AECⅡactive repairing is becoming an new point for preventing hyperxia-induced lung injury. Ne is a cellular regulatory factor or extracellular messenger molecular. It is one of the signal substances recognized by nerve, endocrine and immunity systems jointly. Meanwhile, Nerve-Humor-Immunity network regulates the process of wound repairing. Recently, study suggested that sensory neuropeptide SP plays an important role in proliferation, migration and differentiation of impaired cells. In the study on skin wound healing, SP was not only confirmed to initiate nerve source inflammatory reaction in the early stage of healing but closely related to proliferation, regeneration and scarring of impaired cells. SP in airway is mainly from the erminatio of sensory nerve c- fiber. It distributes widely in airway endothelial cell layer, pulmonary vessel, trachea, bronchus smooth muscle and around gland and bronchus ganglion. Neuroendocrine cell, smooth muscle cell, eosinophile granulocyte, lymphocyte and alveolar macrophage in airway all can synthesize and secrete SP. Special receptor for SP distributes in smooth muscle, submucosal gland and blood vessel endo- thelium. Some inflammatory cells also can express SP receptor. SP is rich in respiratory organs. However, the role of SP in lung injury is seldom reported in domestic and oversea study. In present study, we investigated the effect of hyperxia exposure and SP intervention on proliferation and survival of AECⅡat different time. Meanwhile, we observed dynamic change of SP when exposed to hyperxia at different time.ObjectiveⅠ. To establish oxidative damage patternl for primary AECⅡof preterm rats in serum-free medium by hyperxia exposure.Ⅱ. To observe effect of hyperxia exposure and SP intervention on mor-phology, survival and apoptosis of AECⅡat different time and inves-tigate time-effect relationships between hyperxia exposure, SP inter-vention and AECⅡinjury degrees.Ⅲ. To establish animal patternl for hyperxi- induced lung injury and assay SP.MethodsI. Isolated and purified AECⅡfrom preterm specific-pathogen free(SPF) Spraque-Dawley(SD) rats. Trypan blue exclusion assay and modified papanicolaou staining was used to identify the cells viability and purity, respectively. The AECⅡwere seperated into the following groups: air group (21% oxygen), hyperxia group (95% oxygen), SP + air group and SP + hyperxia group. For SP groups, 1×10-6 mol/L SP was added before the exposure to air and hyperxia. Then, the cells were exposed to air and hyperxia for 12, 24 and 48 h, respectively. The morphologic changes of AECⅡwere observed under TEM. Respectively, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay and flow cytometry(FCM) was used to determine the proliferation and apoptosis rate to confirm the time-effect relationship between cell damage and hyperxia exposure time and the effect of SP intervention on the injury of AECⅡII. Uterine-incision delivery was used to pick out the preterm rats from the pregnant rats at 21 day (22 d for full term ) and isolated AECⅡquickly. The isolated AECⅡwere randomly divided into air group(21% oxygen) and hyperxia group(95% oxygen). The cells were exposed to air and hyperxia for 3, 7 and 14 d, respectively. Then, SP in lung tissue was assayed at different time, respectively.ResultsI. Compared with air exposure group, slightly widened intercellular space and reflect augmented vacuolus appeared at 12 h after hyperxia expo-sure. Widened intercellular space, smaller cells, increased intracellular vacuolus, part shrinked cell nucleus and decreased intracellular parti-cles-lamellar bodies presented at 24 h after hyperxia exposure. At 48 h, plenty of rounding and shrinked cells, non-adherent cells, floating cells and debris in the supernate could be seen. Typical apoptotic cell (smaller volume, shrinked cytoplasm, invaginated membrane and ka-ryopyknosis) and apoptotic body surrounding were observed under TEM. With the prolong of hyperxia exposure time, the cellular mor-phologic damage was aggravate compared with air group. Interestingly, the organs damage was alleviated by the intervention of SP at the same hyperxia exposure time compared with hyperxia group.II. MTT assay indicated that the proliferation activity of AECⅡwas sig-nificantly decreased at 12, 24 and 48 h after hyperxia exposure com-pared with air group. The survival of AECⅡdecreased remarkably(P < 0.05). The survival rate was decreased gradually along with the expo-sure time. There were significant differences among groups(P < 0.05). However, the proliferation activity of AECⅡin SP+ hyperxia group was lower than that in air group, whereas the activity was obviously enhanced compared with simple hyperxia group.III. FCM results suggested that the apoptosis of AECⅡin hyperxia group increased gradually along with the prolong of hyperxia exposure time compared with air group(P < 0.05). Although apoptosis rate in SP group was higher than that in air group, it was obviously lower than that in hyperxia group.IV. The SP product in lung tissue was significantly decreased compared with air group at 3, 7 and 14 d after hyperxia exposure, respectively(P < 0.01). Furthermore, SP level decreased remarkably along with hyperxia exposure time. There was a statistical difference between SP+hyperxia group and simple hyperxia group(P < 0.01).ConclusionI. Hyperxia induced the damage of AECⅡin a time-dependent manner.II. It was confirmed that hyperxia induced the apoptosis of AECⅡby FCM and TEM.III. SP intervention could decrease hyperxia-induced AECⅡapoptosis and enhance the cells viability, suggesting SP could attenuate the oxidative stress on AECⅡand might have a protective effect on AECⅡunder oxidative stress.IV. Hyperxia could result in dynamic change of SP product, suggesting hyperxia exposure led to SP loss at the end of c-fiber of airway sensory nerve and regional decreased SP might be associated with the injury degrees of AECⅡ.PartⅢ. The signal mechanism for hyperxia exposure and SP intervention regulating the repair-ing of AECⅡfrom preterm ratsBackgroundAfter long-time hyperxia uptake, non-specific changes including in-creased alveolar capillary permeability, increased alveolus effusion, in-flammatory damage, fibrin deposition and decreased pulmonary surfactant activity will lead to oxidative stress-induced lung injury. Meanwhile, BPD is a common complication after hyperxia therapy in preterm infant.We have confirmed that hyperxia induced the injury of AECⅡin a time-dependent manner in PartⅡ. Hyperxia exposure could induce the apoptosis of AECⅡ. SP intervention could decrease the cells apoptosis rate and increase survival rate after hyperxia exposure, which might have a protective effect on AECⅡunder oxidative stress. Apoptosis is a cellular active death pattern involving some signal pathways frequently. SP is an new regulatory factor for AECⅡ, and then what is related molecular me-chanism? MAPK pathway plays a key role in mediating hyperxia-induced lung injury. There are 3 members in MAPKs including ERKs, JNKs and p38 kinase. MAPK is an important channel by which different extracellular signals transduct from cellular surface into nucleus. It can mediate cell apoptosis induced by the activation under stimulation. MAPK is a junction and common access of information transfer pathway such as cell prolifera-tion and differentiation.Studies have confirmed that hyperxia exposure and H2O2 stress could trigger persistent activation of activator protein-1 (AP-1), MAPK family members p38 and JNK, which mediated hyperxia-induced cell swelling death and apoptosis. Moreover, cell survival rate could be significantly im-proved after the adding of specific blocker. Some study suggested that tretinoin could attenuate hyperxia-induced lung injury in preterm rats via regulating MAPK pathway. MAPK pathway was involved in signal transduction of AECⅡapoptosis under hyperxia-induced oxidative stress, duction of AECⅡapoptosis under hyperxia-induced oxidative stress, and it promoted the apoptosis of AECⅡ. Whether SP can affect the survival and apoptosis of AECⅡvia MAPK pathway is still unknown. In this part, we further to investigate the effect of hyperxia and SP intervention on the damage of primary cultured AECⅡ.In view of this, we expected to confirm MAPK pathway participation in lung injury and related regulatory mechanism.ObjectiveI. To establish oxidative damage patternl for primary AECⅡof preterm rats in serum-free medium by hyperxia exposure.II. To observe effect of hyperxia exposure and SP intervention on mor-phology, survival and apoptosis of AECⅡat different time and inves-tigate time-effect relationships between hyperxia exposure, SP inter-vention and AECⅡinjury degrees.III. To understand the activation of MAPK in AECⅡapoptosis under hy-perxia-induced oxidative stress and SP intervention, and investigate the effect of MAPKs on AECⅡapoptosis and the regulation of SP inter-vention to MAPKs pathway.MethodsIsolated and purified AECⅡfrom preterm specific-pathogen free(SPF) Spraque-Dawley(SD) rats. Trypan blue exclusion assay and modified pa-panicolaou staining was used to identify the cells viability and purity, respec- tively. The AECⅡwere divided into the following groups: air group (21% oxygen), hyperxia group (95% oxygen), SP + air group and SP + hyperxia group. For SP groups, 1×10-6 mol/L SP was added before the exposure to air and hyperxia. Then, the cells were exposed to air and hyperxia for 12, 24 and 48 h, respectively. Western blot was used to assay the expressions of total phosphorylated ERKs, JNKs and p38 at different exposure time.ResultsI. The phosphorylated ERK(p-ERK) was induced and expressed rapidly after hyperxia exposure. Respectively, the level of p-ERK in hyperxia group was significantly higher than that in air group at 12, 24 and 48 h after hyperxia exposure. The p-ERK protein was expressed best at 48h. The expression in SP+hyperxia group was more than that in hyperxia group at 12, 24 and 48 h, respectively.II. The phosphorylated p38 (p- p38) was induced and expressed rapidly af-ter hyperxia exposure. Respectively, the level of p- p38 in hyperxia group was significantly higher than that in air group at 12, 24 and 48 h after hyperxia exposure. The p- p38 protein expressed best at 48 h. The expression in SP+hyperxia group was less than that in hyperxia group at 12, 24 and 48 h, respectively.III. The phosphorylated JNK (p- JNK) was induced and expressed rapidly after hyperxia exposure. Respectively, the level of p- JNK in hyperxia group was significantly higher than that in air group at 12, 24 and 48 h after hyperxia exposure. The p- JNK protein expressed best at 48 h. The expression in SP+hyperxia group was less than that in hyperxia group at 12, 24 and 48 h, respectively.ConclusionIn present study, we found hyperxia exposure could induce expressions of MAPKs in AECⅡrapidly. The expressions levels reached peaks at 48 h after hyperxia exposure.Respectively, the cells apoptosis rate and survival rate in SP+hyperxia group was obviously lower and higher than that in hy-perxia group at the same time, suggesting MAPKs signals mediated the apoptosis of AECⅡ. The intervention of SP had a potential protective effect on AECⅡunder oxidative stress via activating extracellular signals and suppressing the activation of JNK and p38.更多还原