内质网应激介导COPD肺泡上皮细胞凋亡及GRP78抗凋亡作用

发布时间：2018-03-29 17:40

本文选题：慢性阻塞性肺疾病　切入点：内质网应激　出处：《中南大学》2010年博士论文

【摘要】： 研究背景肺泡上皮细胞凋亡是COPD发病的重要机制之一。内质网应激(ERS)介导的细胞凋亡途径是一条新的细胞凋亡途径。有关ERS标志性分子GRP78的生物学功能的研究已经引起生物学家们的广泛重视,GRP78在ERS时表达增加,被认为可以减轻ERS,从而减少细胞凋亡。GRP78表达上调的信号传导通路目前不完全清楚,有研究提示p38MAPK途径可以上调GRP78的表达。吸烟是COPD最重要的发病因素。香烟烟雾中富含氧自由基和多种毒性成分,氧化应激和毒性物质都是ERS的诱发因素,因此吸烟很可能触发ERS。国内外研究均证实,在COPD中,香烟烟雾可以引起p38MAPK的活化。结合上述研究,我们推测在COPD发生过程中,吸烟可能通过诱导ERS导致肺泡上皮细胞凋亡从而参与COPD肺气肿的形成,在此过程中GRP78表达上调,发挥抗凋亡作用,p38MAPK途径在香烟烟雾诱导肺泡上皮细胞GRP78表达过程中起重要的调控作用。本研究拟以COPD大鼠及香烟烟雾提取物(CSE)干预的A549细胞为主要模型,探讨香烟烟雾诱导ERS的发生、GRP78的抗凋亡作用以及p38MAPK途径在GRP78表达过程中的调控作用。第一章COPD大鼠肺组织内质网应激与细胞凋亡目的观察COPD大鼠肺组织内质网应激的发生及肺泡上皮细胞的凋亡情况。方法24只Wistar大鼠随机均分为2组：正常对照组和COPD模型组。采用被动吸烟加气管内注射脂多糖法建立COPD大鼠模型。造模完成后,测定各组大鼠的肺功能、观察肺组织病理学变化、免疫组化检测GRP78在肺组织中的表达和分布,RT-PCR检测GRP78、CHOP mRNA水平,Western blot检测GRP78、CHOP、active caspase-12蛋白水平。TUNEL法检测肺泡上皮细胞凋亡结果COPD模型组大鼠FEV0.3/FVC (%)、动态肺顺应性(Cdyn)均较对照组明显降低,而气道阻力(RI)明显增高；免疫组化结果显示GRP78表达于肺泡上皮细胞、支气管上皮细胞、血管内皮细胞等细胞的细胞浆中,以肺泡上皮细胞明显。对照组GRP78呈弱阳性表达,阳性产物为棕黄色颗粒；模型组GRP78呈强阳性表达,阳性产物为棕褐色颗粒。与对照组相比,模型组肺组织GRP78表达显著增高；RT-PCR结果显示COPD模型组大鼠支气管肺组织中GRP78 mRNA和CHOP mRNA表达量较对照组显著增高；Western blot结果显示COPD模型组大鼠肺组织中GRP78、CHOP、active caspase-12蛋白表达较正常对照组显著增高；TUNEL法结果显示COPD模型组肺泡上皮细胞凋亡指数较正常对照组显著增高。结论采用被动吸烟加气管内注射脂多糖的方法,成功复制出了COPD大鼠模型;COPD大鼠肺组织发生了内质网应激,尤以肺泡上皮细胞明显；COPD大鼠肺泡上皮细胞凋亡增加,内质网应激可能是介导肺泡上皮细胞发生凋亡的重要途径。第二章香烟烟雾提取物诱导肺泡上皮细胞GRP78的表达及其抗凋亡作用目的观察CSE对A549细胞GRP78表达的影响,并探讨GRP78抗细胞凋亡作用。方法第一部分,体外培养A549细胞,给予不同浓度(0%-10%)和不同时间(0h-24h)的CSE干预后,以RT-PCR、Western blot分别检测GRP78 mRNA和蛋白表达；第二部分,将A549细胞分为四个处理组：对照组,5%CSE组,GRP78siRNA+5%CSE组,control siRNA+5%CSE组。以RT-PCR、Western blot分别检测GRP78 mRNA和蛋白的表达水平,Western blot检测active caspase-3蛋白表达水平,TUNEL检测A549细胞凋亡指数(AI)的变化。结果随着CSE浓度的增加或干预时间的延长,A549细胞GRP78 mRNA及GRP78蛋白表达量逐渐增加,以5%CSE、干预12h组增加最明显,但继续增加CSE浓度或延长干预时间,GRP78表达并不增加,反而下降；小干扰GRP78后,RT-PCR、Western blot检测小干扰组GRP78表达显著下降,GRP78siRNA成功下调了GRP78基因表达；GRP78水平下调后,A549细胞active caspase-3的蛋白表达水平和细胞凋亡指数与单纯的CSE刺激组比较显著升高。结论低浓度、短时间CSE刺激后,A549细胞启动了保护性的非折叠蛋白反应,GRP78表达上调。但高浓度、长时间CSE刺激,内质网出现功能障碍,其保护机制失代偿,GRP78表达反而下降；GRP78抵抗香烟烟雾提取物诱导的A549细胞凋亡。第三章p38MAPK在香烟烟雾提取物诱导肺泡上皮细胞GRP78表达过程中的调控目的明确p38MAPK在CSE诱导肺泡上皮细胞GRP78表达过程中的调控作用。方法第一部分,24只Wistar大鼠随机均分为2组：正常对照组和COPD模型组。采用被动吸烟加气管内注射脂多糖法建立大鼠COPD模型。造模完成后,Western blot检测各组大鼠肺组织内P-p38、GRP78蛋白表达水平,并将二者进行相关性分析；第二部分,将A549细胞分为三个处理组：对照组,5%CSE组,SB203580+5%CSE组,以RT-PCR、Western blot分别检测GRP78 mRNA和蛋白的表达水平变化。结果COPD模型组大鼠支气管肺组织中P-p38蛋白表达水平较对照组显著升高,且P-p38蛋白水平与GRP78蛋白表达呈正相关(r=0.848, P0.01); p38MAPK抑制剂SB203580干预后,P-p38蛋白表达水平显著降低。抑制了p38MAPK通路活化后再给予CSE干预,GRP78 mRNA及GRP78蛋白表达水平较单纯的CSE刺激组显著降低。结论COPD中p38MAPK途径发生了磷酸化激活；p38MAPK途径能上调香烟烟雾提取物诱导的GRP78表达水平。
[Abstract]:The study suggests that smoking is the most important factor in COPD . The study suggests that smoking may induce apoptosis of alveolar epithelial cells by inducing ERS . The study suggests that smoking may play an important role in the pathogenesis of COPD . The study suggests that smoking may induce the apoptosis of alveolar epithelial cells by inducing ERS .

Chapter 1 Stress and Apoptosis in the Lung Tissue of COPD Rats

Objective To investigate the occurrence of endoplasmic reticulum stress and the apoptosis of alveolar epithelial cells in COPD rats .

Methods 24 Wistar rats were randomly divided into 2 groups : normal control group and COPD model group .

Results In COPD model group , FEV0.3 / FVC ( % ) and dynamic lung compliance were significantly lower than those in the control group , while the resistance to airway resistance ( RI ) was significantly increased .
The results showed that GRP78 was expressed in alveolar epithelial cells , bronchial epithelial cells , vascular endothelial cells and other cells . The results showed that GRP78 was weakly positive in alveolar epithelial cells .
Compared with the control group , the expression of GRP78 in the lung tissue of the model group was significantly higher than that of the control group .
RT - PCR showed that the expression of GRP78 mRNA and CHOP mRNA in broncho - lung tissues of COPD model group was significantly higher than that in the control group .
Western blot showed that the expression of GRP78 , CHOP and active caspase - 12 in lung tissues of COPD model group was significantly higher than that in the control group .
TUNEL method showed that the apoptosis index of alveolar epithelial cells in COPD model group was significantly higher than that in control group .

Conclusion The model of COPD rats was successfully reproduced by the method of passive smoking and intratracheal injection of lipopolysaccharide . The lung tissue of COPD rats had an endoplasmic reticulum stress , especially in the alveolar epithelial cells .
In COPD rats , the apoptosis of alveolar epithelial cells is increased , and the endoplasmic reticulum stress may be an important way to mediate the apoptosis of alveolar epithelial cells .

Study on the expression of GRP78 in alveolar epithelial cells induced by cigarette smoke extract and its anti - apoptotic effect

Objective To investigate the effect on the expression of GRP78 in A549 cells and to explore the anti - apoptotic effect of GRP78 .

Methods The expression of GRP78 mRNA and protein was detected by RT - PCR and Western blot in the first part of the cell culture A549 cells , different concentrations ( 0 % -10 % ) and different time ( 0h - 24h ) .
The expression level of GRP78 mRNA and protein was detected by Western blot , and the expression level of active caspase - 3 protein was detected by Western blot , and the apoptosis index ( AI ) of A549 cells was detected by TUNEL .

Results The expression of GRP78 mRNA and GRP78 protein in A549 cells increased gradually with the increase of the concentration or the time of intervention . The expression of GRP78 mRNA and GRP78 protein in A549 cells increased most obviously , but the expression of GRP78 was not increased , but the expression of GRP78 decreased .
The expression of GRP78 was significantly decreased by RT - PCR and Western blot after GRP78 , and GRP78 siRNA was successfully downregulated by GRP78 siRNA .
After downregulation of GRP78 , the expression level of active caspase - 3 and the apoptosis index of A549 cells were significantly increased compared with that of the simple ones .

Conclusion The expression of GRP78 is up - regulated in A549 cells after stimulation of low - concentration and short - term , but the expression of GRP78 is decreased .
GRP78 is resistant to the apoptosis of A549 cells induced by cigarette smoke extract .

The regulation of p38MAPK in the expression of GRP78 in alveolar epithelial cells induced by cigarette smoke extract

Objective To clarify the role of p38MAPK in the expression of GRP78 in alveolar epithelial cells .

Methods In the first part , 24 Wistar rats were randomly divided into 2 groups : normal control group and COPD model group .
In the second part , A549 cells were divided into three treatment groups : control group , 5 % sse group , SB203580 + 5 % se group , RT - PCR and Western blot were used to respectively detect the expression level of GRP78 mRNA and protein .

Results The expression level of P - p38 protein in the lung tissues of rats with COPD was significantly higher than that in the control group ( r = 0.848 , P0.01 ) . The level of P - p38 protein was significantly decreased after the intervention of p38MAPK inhibitor SB203580 .

Conclusion The p38MAPK pathway in COPD is activated by phosphorylation .
The p38MAPK pathway can regulate the expression level of GRP78 induced by cigarette smoke extract .

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

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