【作者】 明广峰；

【导师】 刘慧霞；

【作者基本信息】 中南大学 ， 老年医学， 2012， 博士

【摘要】 研究背景重症急性胰腺炎(severe acute pancreatitis, SAP)合并急性肺损伤(acute lung injury, ALI)起病急剧,病情进展凶险,常导致约30%-40%的死亡率。细胞因子和炎症介质的过度释放增强肺微血管内皮细胞的通透性而导致肺水肿是ALI主要病理机制之一。前B细胞克隆增强因子(pre-B-cell colony-enhancing factor, PBEF)是一种新发现与肺损伤发生密切相关的肽类激素。既往资料显示,PBEF参与了多种肺损伤的病理过程,主要与增强肺微血管内皮细胞的通透性而导致肺水肿的发生密切相关。但PBEF在SAP并发ALI发病机制中的作用未见报道,本实验在国内外首次对其进行研究。第一部分血清PBEF水平与重症急性胰腺炎并发急性肺损伤的相关性研究目的：检测SAP并发ALI或ARDS患者和轻症急性胰腺炎患者血清样本中PBEF、TNF-α和IL-8表达。方法：收集19例SAP并发ALI、13例SAP并发ARDS及32例轻症急性胰腺炎患者的血清样本,采用双夹心ELISA方法检测血清样本中PBEF、TNF-α和IL-8表达水平。比较分析PBEF、TNF-α和IL-8表达水平与SAP并发ALI进程的相关性。结果： SAP并发ARDS和并发ALI患者血清中的PBEF检测值显著高于轻症急性胰腺炎的患者,SAP并发ARDS患者的PBEF亦高于并发ALI患者的PBEF水平,差异均具有统计学意义(P<0.05)。同时检测TNF-α和IL-8可以得到相似的结果,并且血清PBEF与TNF-α和IL-8水平具有正相关性(r=3.215,P=0.012；r=4.247,P=0.019)。结论：SAP并发ALI或ARDS的患者血清中PBEF、TNF-α和IL-8的浓度较轻症急性胰腺炎患者血清中高；且血清中PBEF的浓度与TNF-α和IL-8的浓度分别呈正相关。第二部分PBEF在重症急性胰腺炎并发急性肺损伤大鼠模型肺组织中的表达目的：检测SAP并发急性肺损伤的大鼠模型肺组织中PBEF、 TNF-α和IL-8中的表达水平。方法：采用去氧胆酸钠逆行注入大鼠胰胆管方法诱发构建SAP并发ALI的动物模型。实验大鼠随机分为2组：假手术组(SHAM组),胰腺炎并发肺损伤组(ALI组),每组24只。通过检测大鼠模型的血清胰淀粉酶、血气分析和局部病理改变,确定SAP并发ALI的动物模型构建是否成功；应用肺损伤评分对SAP并发ALI的动物模型的肺脏进行病理组织学损伤评分；采用实时荧光定量PCR和Western blot方法检测SAP并发ALI发生时肺脏组织中PBEF、TNF-α和IL-8mRNA和蛋白的表达水平。结果：通过对大鼠模型的胰腺病理改变、胰淀粉酶和血气分析结果分析提示成功建立的大鼠动物模型符合重症急性胰腺炎的病理改变。分别在造模后4小时、8小时、12小时取标本观察相应数据。我们在光镜下初步比较了SHAM组和ALI组实验大鼠的肺组织病理改变结果发现,SAP并急性肺损伤组的肺损伤评分在造模后4小时即可见升高,12小时内逐渐升高差异有统计学意义(P<0.05)；SAP并急性肺损伤组的肺湿/干比值明显增加,并随时间延长逐渐加重,较SHAM组差异具有统计学意义(P<0.05)。我们运用荧光实时定量PCR检测了PBEF、TNF-α和IL-8mRNA在相应时间点SAP并发ALI模型肺脏组织中的表达情况。结果显示：各时间点ALI组PBEF mRNA、TNF-α mRNA和IL-8mRNA表达较SHAM组显著上调,差异有统计学意义(P<0.05)。采用Western blot方法检测了同一批标本中相同时间点的PBEF、TNF-α和IL-8蛋白的表达,结果显示蛋白表达亦明显增高,差异有统计学意义(P<0.05)。结论：从动物模型的肺组织水平观察到了PBEF、TNF-α和IL-8三者在SAP并发急性肺损伤时表达增高。第三部分PBEF介导人肺微血管内皮细胞急性损伤的分子机制目的：通过体外培养肺细胞肺微血管内皮细胞(PMVEC),采用基因重组和转染技术,在PBEF过表达或抑制并细胞因子TNF-a刺激的条件下,观察IL-1p、IL-6和IL-8表达水平的改变；同时,检测在特异性抑制P38、ERK1/2、JNK和PI3K信号通路的条件下的表达改变,探讨PBEF在SAP并发ALI中的作用分子机制。方法：构建pEGFP-N1-PBEF-EGFP过表达重组质粒和慢病毒pLKO.1-PBEF-SiRNA干扰质粒,转染体外培养的PMVEC细胞,采用实时荧光定量PCR和Western blot方法检测PBEFmRNA和蛋白表达水平；流式细胞仪检测PBEF过表达对PMVEC细胞凋亡的影响。细胞因子TNF-α分别刺激pEGFP-N1-PBEF和pLKO.1-PBEF-SiRNA转染的PMVEC细胞,采用实时荧光定量PCR和Western blot方法检测IL-1β、IL-6和IL-8表达水平。进一步在分别阻断P38(阻断剂为U0126)、ERK(阻断剂为SB203580)、JNK(阻断剂为SP600125)和PI3K信号通路(阻断剂为LY294002)条件下,TNF-a诱导PBEF过表达的PMVEC细胞,荧光定量PCR和Western Blot检测IL-1β、IL-6和IL-8表达水平。结果：(1)重组质粒pEGFP-N1-PBEF酶切和测序鉴定结果显示符合后续实验的要求。转染PBEF过表达载体后,PBEF表达水平明显提高,其中mRNA水平为对照的10倍左右,较对照组差异具有统计学意义(P<0.05)。流式细胞仪检测结果显示,PBEF过表达可以促进HPMEC发生细胞凋亡,较对照组差异具有统计学意义(P<0.05)。(2)TNF-α诱导和PBEF过表达载体转染时,IL-1β、IL-6和IL-8表达明显增强,且以10ng/mL浓度的TNF-α诱导后,IL-1β、IL-6和IL-8表达增强更明显(P<0.05)。(3)pLKO.1-PBEF-SiRNA转染细胞后PBEF表达抑制率约为85%,PBEF siRNA能明显抑制PBEF的表达,TNF-α诱导同时进行PBEF siRNA转染后,PBEF可以对抗TNF-α的诱导作用,差异具有统计学意义(P<0.05)。(4)用SB203580抑制P38通路后,IL-1β、IL-6和IL-8表达下降具有统计学意义(P<0.05)；PD98059抑制ERK通路后,IL-1β、IL-6和IL-8表达下降亦具有统计学意义(P<0.05)。用SP600125抑制JNK通路后仅IL-8表达水平下降比较明显,差异具有统计学意义(P<0.05)；用LY294002抑制PI3K通路后,IL-1β、IL-6和IL-8表达变化差异不具有统计学意义(P>0.05)。结论：1,在PMVEC中,TNF-α和PBEF有协同作用；2,本研究首次发现在PMVEC中PBEF可能主要通过P38和ERK通路进行信号转导而促进IL-1β、IL-6和IL-8表达。更多还原

【Abstract】 BackgroundSevere acute pancreatitis (SAP) has multiple complications and a hazardous prognosis. It results in mortality rates ranging from30to40%. Acute lung injury (ALI)and acute respiratory distress syndrome (ARDS) is the most common and severe complication in SAP. It is also one of the important death reasons in clinical patients with SAP. The main pathological features and cause of ALI is excessive release of cytokines and inflammatory mediators that affect the permeability of the lung microvascular endothelial cells and lead to pulmonary edema. Pre-B cell clones enhancement factor (PBEF) is a novel peptide hormone, which is closely related lung injury. As current research data shown, PBEF is involved in a variety pathological process of lung injury. However, the role of PBEF in SAP induced ALI remains larger unknown and this study is the first to investigated it in the world.Chapter I study of the relationship between the serum PBEF and SAP with ALI/ARDSObjective:The expression level of PBEF, TNF-α and IL-8in serum samples were detected by ELISA method.Method:19serum samples were collected from SAP patients with ALI,and13samples from patients with ARDS while32serum samples were collected from mild acute pancreatitis as control. The expression level of PBEF, TNF-a and IL-8were detected by double sandwich ELISA method. The relationship of PBEF, TNF-α and IL-8expression level with the development of SAP patients with ALI were comparative analysis.Results:The serum PBEF of SAP patients complicated by ARDS was significantly higher than that of SAP complicated by ALI and mild acute pancreatitis, the difference was significant (P<0.05). Further analysis showed that serum TNF-α and IL-8level were higher as PBEF and three of them were positive correlated (r=3.215, P=0.012; r=4.247,P=0.019,P<0.01).Conclusion:We found the correlation of the level of serum PBEF and the level of serum TNF-a and IL-8in patient with SAP complicated by ALI or ARDS and mild acute pancreatitis.Chapter Ⅱ The expression of PBEF in the lung of animal model of SAP with ALI/ARDSObjective:The mRNA and protein of PBEF, TNF-α and IL-8were examined in lung tissue of SAP complicated ALI rat model.Method:Total of48rats were randomly divided into sham operation group (SHAM group, n=24) and pancreatitis with acute lung injury group (ALI group, n=24). SAP complicated ALI in animal models was constructed by sodium deoxycholate retrograde injected into the rat pancreatic bile duct method. To determine the success of SAP complicated ALI animal model, Pancreatic amylase, blood gas analysis,lung wet/dry ratio and the lung histology injury scoring system were detect at4h,8h,12h after the animal models were constructed. And then,the expression of PBEF, TNF-α and IL-8mRNA and protein were detected by real-time quantitative PCR and Western blot at the same time.Results:The pathological changes of the pancreas, pancreatic amylase, and blood gas analysis results suggested that animal models met the pathological changes of severe acute pancreatitis. In the light microscope, we made a preliminary comparison of the lung pathological change of experimental animals of the SHAM group and the ALI group,and found pulmonary edema, inflammatory cell infiltration, and hemorrhage in the SAP with ALI group.Total lung tissue injury score increased four hours after the modeling, deteriorated gradually within12hours,the difference was statistically significant (P<0.05);In the SAP with ALI group,lung wet to dry ratio was significantly increased, and gradually increased with time, compared with the SHAM group, the differences were significant(P<0.05)We used real-time PCR to detect the expression of PBEF, TNF-α and IL-8mRNA in the lung tissues of SAP model complicated by ALI at4h,8h,12h after the animal models were constructed. The expression level of PBEF mRNA as well as TNF-a mRNA and IL-8mRNA in ALI group was significant higher than control group; We tested the expression of PBEF, TNF-α and IL-8protein by using Western blotting at the same time as above in the same batch of samples, the results showed:the trend of PBEF, TNF-α and IL-8protein were alike as mRNA,and the difference was significant(P<0.05).Conclusion:The expression of PBEF, TNF-alpha and IL-8in lung tissue of SAP complicated ALI rat model are higher than that of sham operation group.CHAPTER ⅢThe molecular mechanism of PBEF mediated acute injury of human pulmonary microvascular endothelial cellObjective:To investigate the molecular mechanism of PBEF in mediating acute injury of human pulmonary microvascular endothelial cell.Method:Human pulmonary microvascular endothelial cells (PMVECs) were transfected with pEGFP-PBEF recombinant plasmid and lentiviral pLKO.1-PBEF-SiRNA interference vector in vitro. The expression of PBEF were detected by real-time quantitative PCR and Western blot. The effect of PBEF over-expression on apoptosis of PMVEC cells were determined by flow cytometry. We treated the PMVECs with pEGFP-PBEF recombinant plasmid and lentiviral pLKO.1-PBEF-SiRNA interference vector with TNF-α, and tested the expression of IL-1β,IL-6and IL-8by real-time quantitative PCR and Western blot. After the PMVECs with PBEF over expression were treated by TNF-α, the P38, ERK, JNK and PI3K signaling pathways were inhibited with corresponding signaling pathway-specific inhibitor blockers (U0126, SB203580, of SP600125, LY294002respectively). The expression of IL-1β,IL-6and IL-8were detected by real-time quantitative PCR and Western blot.Results:(1)The results of digestion and sequencing of Construc-tion of recombinant plasmid pEGFP-N1-PBEF showed the plasmid comply with following experiments., we verified PBEF mRNA and protein expression levels were much higher after transfected with ove r expression vector than the control group, and the the PBEF mRN A was10times of that in the control group, the difference was sig nificant (P<0.05). The test results of flow cytometry showed that PB EF overexpression promotes apoptosis of human HPMEC, the differenc e was significant (P<0.05).(2)Transfected with PBEFover-expression v ector or treated with TNF-α, the expression levels of IL-1β, IL-6an d IL-8were significantly increased, especially after induced by TNF-α, the expression of IL-1β, IL-6and IL-8will be further enhanced, the difference was significant (P<0.05).(3)The inhibiting ratio of PB EF was85%after cells transfected by pLKO.1-PBEF-SiRNA, siRN A of PBEF significantly down regulated the expression of PBEF, Transfection of SiRNA PBEF after induced by TNF-α could still sig nificantly inhibited the expression of PBEF,against the induced effect of SiRNA PBEF,the difference was significant (P<0.05).(4)The expr ession of IL-1β, IL-6and IL-8decreased significantly when using SB203580inhibition of P38pathway,the difference was significant (P<0.05);when using PD98059inhibited ERK pathway, the inflammatory fa ctor expression was also apparent,the difference was significant (P<0.05); when using JNK inhibitor SP600125, only IL-8expression decrease d obviously, the difference was significant (P<0.05); and when using t he LY294002to inhibit the PI3K pathway, the expression of inflamma tory cytokines did not change significantly (P>0.05).Conclusion:1,In PMVEC,TNF-a and PBEF have synergistic reaction;2,It is the first time to find that PBEF promoted the expression of IL-1β, IL-6and IL-8mainly through P38and ERK pathway in PMVEC in vitro更多还原