【作者】 张雪梅；

【导师】 陈海龙；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2008， 博士

【摘要】 目的:探讨重症急性胰腺炎（severe acute pancreatitis,SAP）肺损伤（acute lung injury,ALI）时肺表面活性蛋白A（surfactant protein A,SP-A）、分泌型磷脂酶A2-Ⅱ（secreted phospholipase A2,sPLA2-Ⅱ）的表达,及其在ALI发病中的作用,同时观察肺泡Ⅱ型上皮细胞（alveolar typeⅡcells,ATⅡ）在急性胰腺炎肺损伤时的形态及功能改变,并观察清胰汤对SP-A、sPLA2-Ⅱ表达及病情转归的影响。急性重症胰腺炎时增高的sPLA2-Ⅱ对ATⅡ细胞损伤作用是引起急性胰腺炎肺损伤的重要因素,急性胰腺炎相关性腹水中sPLA2含量明显增高。本研究进一步行ATⅡ细胞原代培养,用大鼠SAP模型腹水干预细胞,观察细胞增殖状况和生存率改变;并分别应用sPLA2-Ⅱ抑制剂（KH064）、Emodin干预,观察其对腹水预处理ATⅡ细胞增殖、SP-A表达的影响,及对细胞形态功能的影响。进一步揭示SP-A、sPLA2Ⅱ及ATⅡ细胞在急性胰腺炎肺损伤发病中的作用,为临床治疗急性肺损伤寻求更为合适的药物作用靶点。方法:实验一:采用胆胰管内逆行注入1.5%去氧胆酸钠建立大鼠SAP时ALI模型。将SD大鼠随机分为假手术组（SO组,n=10）、模型组（SAP组,n=10）、清胰汤组（QYT组,n=10）、地塞米松组（DEX组,n=10）、善宁组（SS组,n=10）和分泌型磷脂酶A2-Ⅱ抑制剂组（KH064,n=10）。SO组仅行剖腹术,翻动胰腺。QYT组在建立SAP模型后30 min、12 h清胰汤灌胃（10 mL/kg）。DEX组于造模后30min、12h静脉注射地塞米松（10mg/kg）。SS组在造模后30min、12h皮下注射善宁（50μg/kg）。KH064组于造模后30min、12h给予KH064灌胃（5mg/kg）。各组动物在术后24 h测PaO₂、PaCO₂、pH和血淀粉酶、sPLA₂、肺湿/干比。应用逆转录聚合酶链式反应（RT-PCR）检测肺组织SP-A、sPLA2-ⅡmRNA的表达强度,采用Western-blot检测肺组织SP-A、sPLA2-Ⅱ表达,免疫组化观察肺组织SP-A表达,并观察胰、肺病理变化及ATⅡ细胞的电镜下变化。实验二:采用Dobbs方法提取ATⅡ细胞,首先肺动脉灌洗减少肺内红细胞,肺离体后经气管灌洗除去肺泡腔内的白细胞,将胰蛋白酶、胶原酶灌入肺内消化分离细胞;采用免疫贴附法纯化细胞,巨噬细胞、淋巴细胞和中性粒细胞等细胞表面有IgG的Fc段受体,将细胞悬液培养于覆被IgG的平皿中,有Fc段受体的细胞被粘附,而无Fc段受体的ATⅡ细胞得以纯化。ATⅡ细胞的鉴定采用电子显微镜和SP-A免疫组化染色,其在电子显微镜下有特征性板层小体,免疫组化染色见细胞浆有SP-A表达。实验三:10只雄性SPF SD大鼠,采用胆胰管内逆行注入1.5%去氧胆酸钠建立大鼠SAP时ALI模型,留取腹水,检测腹水sPLA2活性后分装,-80℃保存。按前述方法分离、提取、培养ATⅡ细胞,腹水预处理按腹水终浓度为12.5μl/ml、25μl/ml、50μl/ml和100μl/ml加入培养液,分6、12、24h三个时点,采用MTT法测定细胞增殖情况和细胞生存率。根据细胞增殖情况,选用腹水预处理浓度为25μl/ml,预处理时间24h,培养孔KH064干预终浓度为:0μg /ml、0.5μg/ml、1μg/ml、2μg/ml、4μg/ml,培养孔Emodin干预终浓度为:0μg/ml,10μg/ml,20μg/ml,40μg/ml,80μg/ml,用MTT法测定细胞增殖情况,根据实验结果选择药物干预剂量为:KH064 1μg/ml、2μg/ml,Emodin 10μg/ml,20μg/ml。腹水预处理组腹水剂量分别为:12.5、25、50、100μl/ml培养液,预处理时间为24h。腹水预处理组和药物干预组分别提取RNA和蛋白,检测SP-A mRNA和蛋白的表达。电子显微镜观察ATⅡ细胞。结果:SAP组血淀粉酶显著高于SO组和各治疗组（P<0.05）。SAP组PaO₂、pH显著低于SO组和治疗组（P<0.05）, SAP组PaCO₂、肺W/T显著高于SO组和治疗组（P<0.05）。SAP组血清sPLA2显著高于SO组（P<0.05）,QYT、DEX、KH064组血sPLA₂与SAP组比较有显著降低（P<0.05）,SS组血sPLA₂与SAP组比较差别无显著性。SAP组SP-A mRNA表达较SO组显著降低（P<0.05）,QYT、DEX、KH064组SP-A mRNA表达较SAP组显著增高（P<0.05）,SS组SP-A mRNA表达与SAP组比较无显著变化。各组SP-A mRNA表达与肺损伤评分呈负相关（P<0.05）。SO组SP-A蛋白有明显表达,与SO组比较,SAP组SP-A表达显著降低,治疗组与SAP组比较SP-A表达增高,其中QYT、DEX、KH064组增高明显。肺组织免疫组化SAP组SP-A表达较SO组显著降低（P<0.05）,各治疗组SP-A表达较SAP组有显著增高（P<0.05）。与SO组比较SAP组sPLA2-ⅡmRNA、蛋白表达显著增高,除SS组外其他各治疗组sPLA2-ⅡmRNA、蛋白表达较SAP组显著降低。治疗组肺组织病理及电镜改变较SAP组明显好转,以QYT、KH064组好转明显。纯化前可得约5×10⁸个ATⅡ细胞/只鼠,IgG免疫粘附纯化后细胞数约为1.6×10⁷个/只。台盼蓝染色显示细胞活力为95%以上。通过SP-A免疫组化染色判定,纯化后ATⅡ细胞纯度可达92%。光学显微镜检查见随预处理腹水剂量增加,细胞生长状态变差,并出现细胞死亡。KH064、Emodin干预组细胞生长良好。MTT结果显示,腹水呈剂量依赖方式抑制大鼠ATⅡ细胞生长,细胞生存率随培养时间的延长降低。KH064浓度为0.5-2μg /ml时,细胞增殖较对照组明显增高,并呈剂量依赖性;Emodin浓度为10-40μg/ml时,细胞增殖较对照组有显著增高,并呈剂量依赖性。随着干预腹水浓度的增高, ATⅡ细胞SP-AmRNA的表达降低,表现出明显的剂量依赖性。KH064和Emodin干预组SP-AmRNA的表达水平显著高于对照组（P<0.05）,各干预组不同剂量之间SP-AmRNA的表达水平无显著差别（P>0.05）。腹水预处理组ATⅡ细胞SP-A蛋白表达显著低于对照组,并随腹水预处理剂量的增高SP-A的表达降低,呈现剂量依赖性。KH064和Emodin干预组ATⅡ细胞SP-A蛋白的表达水平显著高于对照组。KH064和Emodin干预组ATⅡ细胞电子显微镜下改变较腹水预处理组明显减轻。结论:急性胰腺炎肺损伤时,ATⅡ细胞结构破坏,导致SP-AmRNA表达减少,进而使肺表面活性物质中SP-A表达降低,其对sPLA2-II抑制作用降低,使后者表达增高,从而加速肺泡表面活性物质磷脂水解,导致肺泡表面张力增加,肺泡萎陷,最终导致ARDS。SP-A和sPLA2- II的平衡在急性胰腺炎肺损伤发生中起关键作用。中药清胰汤在急性胰腺炎肺损伤时起保护ATⅡ细胞的作用,增加肺组织SP-A表达,同时降低肺组织sPLA2- II表达,具有维持肺泡表面活性物质的功能,从而保持良好肺泡表面张力,防止ALI。地塞米松可能通过减轻炎症反应,间接增加肺组织SP-A的表达,降低肺组织sPLA2-II的表达,实现保护肺功能的作用。善宁明显降低急性胰腺炎血淀粉酶水平,对SP-A、sPLA2- II表达无显著影响。分离、纯化大鼠ATⅡ细胞时,合适的胰蛋白酶浓度及作用时间对细胞活性有重要作用,大鼠IgG粘附纯化可以得到高纯度的ATⅡ细胞,可以通过电子显微镜观察板层小体和免疫组化染色检测细胞SP-A表达鉴定ATⅡ细胞。胰腺炎腹水抑制ATⅡ细胞增殖,随着腹水浓度增高、作用时间延长其对细胞增殖的抑制作用增强,表现出明显剂量、时间依赖性。sPLA₂-Ⅱ抑制剂可以减轻腹水对ATⅡ细胞的损伤,表明腹水中对细胞损伤起主要作用的物质可能是sPLA₂-Ⅱ。Emodin也有减轻腹水对ATⅡ细胞的损伤作用,其可能主要通过抑制sPLA₂-Ⅱ活性,减轻腹水中sPLA₂-Ⅱ对ATⅡ细胞的损伤作用而保护细胞功能。腹水预处理使ATⅡ细胞SP-AmRNA、蛋白表达较对照组减少,表明腹水中有抑制SP–A表达物质,应用sPLA₂-Ⅱ抑制剂KH064、Emodin可以使SP–A表达增高,表明这两种药物可减轻细胞损伤,通过增加ATⅡ细胞SP-A表达而起防止肺损伤的作用。更多还原

【Abstract】 Objectives:The purpose of our study is to investigate the expression of surfactant protein A （SP-A） and secreted phospholipase A₂-II （sPLA₂-II） in rats with acute lung injury （ALI） caused by severe acute pancreatitis （SAP）, observe the morphological and functional alteration of alveolar type II cells （ATII cells） and the therapeutic effect of Qingyitang. In this study we also created an ALI model of cultured AT II cells in vitro, which is generated by treating AT II cells with pancreatitis-associated ascitic fluid （PAAF）. Based on this model, we investigated the secretary function alteration in AT II cells. We use Emodin, one of major compounds of traditional medicine pieplant, to interfere the injury process in vitro to evaluate its therapeutic effect.Methods:Part 1:Sixty male SD rats were randomly divided into 6 groups: sham operation group （SO, n=10）,SAP model group （SAP, n=10）, Qingyitang group （QYT, n=10） , Dexamethasone group （DEX, n=10）, Sandostatin group （SS, n=10） and 5-（4-benzyloxyphenyl）- 4S- （phenyl-heptanoylamino） - pentanoic acid group （KH064, n=10）. Severe acute pancreatitis was induced by inversely injecting 1.5% sodium deoxycholate into the common bile-pancreatic duct of rats for SAP and interference groups. Sham operation group was performed operation only. Qingyitang （10 ml/kg） was intragastric administrated 30 minutes and 12 hours after SAP was induced in QYT group.Dexamethasone （10mg/kg） was injected intravenously 30 minutes and 12 hours after SAP was induced in DEX group. Octreotide （50μg/kg） was subcutaneously injected 30 minutes and 12 hours after SAP was induced in SS group. KH064（5mg/kg） was intragastric administrated 30 minutes and 12 hours after SAP was induced in KH064 group. Serum amylase （AMY）、sPLA₂ levels, PaO₂、PaCO₂、pH and lung wet/dry ratio （W/D） were recorded. The SP-A and sPLA₂ mRNA expression levels in lung tissue were detected by reverse transcription polymerase chain reaction （RT-PCR）. The SP-A protein level in lung tissue was detected by both Western-blot and immunohistochemical methods. The pathological changes of pancreas, lung and AT II cells were observed 24 hours after the SAP model was established.Part 2: AT II cells were isolated by Dobbs’s method which had been proved to be effective. Then, cells were purified by using rat IgG coated dishes and were characterized by using electronic microscope and SP-A immunohistochemical staining. The cellular ultra-structure of AT II cells was observed by using electronic microscope.Part3: 1.5% sodium deoxycholate （1ml/kg）was inversely injected into the common bile-pancreatic duct of SAP rat model. Pancreatitis-associated ascitic fluid （PAAF） was obtained from the SAP rats. The activity of sPLA₂ in ascites was detected by using sPLA₂ kits. AT II cells were previously cultured in vitro and exposed to medium with different PAAF concentrations at 12.5μl/ml, 25μl/ml, 50μl/ml and 100μl/ml. The morphology and activity of those cells were measured by using inverted microscope and MTT method at 6、12 and 24 hours post treatment.Cultured ATII cells were treated by KH064 with the final concentration at 0μg /ml,0.5μg/ml,1μg/ml, 2μg/ml and 4μg/ml at the presence of 25μl/ml PAAF for 24 hours. Another group of ATII cells were exposed to Emodin with the final concentration at 0μg/ml,10μg/ml,20μg/ml,40μg/ml and 80μg/ml at the presence of 25μl/ml PAAF for 24 hours. Cell survival rate was detected with MTT assay. Then Cultured AT II cells were exposed to medium containing PAAF at final concentrations of 12.5μl/ml, 25μl/ml, 50μl/ml and 100μl/ml for 24 hours, and cells were exposed to 25μl/ml PAAF with KH064 （1μg/ml、2μg/ml） or Emodin （10μg/m , 20μg/ml）. Cells were harvested and extracted for total RNA isolation with Trizol reagent, mRNA level of SP-A was detected by using reverse transcription polymerase chain reaction （RT-PCR）. The expression of SP-A protein was detected by Western blot. AT II cells were observed by using electron microscope.Results: Serum AMY level, PaCO₂, W/D of lung in SAP group were found significantly higher than other groups （P<0.05）. Serum sPLA₂ level in SAP was significantly higher than all other groups （P<0.05）, and PaO₂ level in SAP was lower than SO and interference group （P<0.05）. The SP-A mRNA and SP-A expression level in lung tissue of SAP group was significantly decreased than both SO and interference groups （P<0.05）. The mRNA levels of sPLA₂-II and SP-A in lung tissue of SAP group were significantly lower than SO and interference groups （P<0.05）. There were milder pathological changes of pancreas, lung tissue and AT II cells in either interference group and SO group than SAP group.There were collected 5×10⁸ cells isolated from ascitic fluid and only 1.6×10⁷ cells were harvested after purification. The trypan blue staining showed the cell viability is above 95 percent. Immunohistochemical staining using anti-SP–A demonstrated that the purity of AT II cell is 92%. Morphologic manifestation of AT II cell was changed with PAAF concentrations and time points. PAAF can induce AT II cells damage measured by morphology and death detected by cell viability. The cell viability detected with MTT in PAAF groups were significantly lower than in control group. SP-A expression decreased at both transcription and translation levels in PAAF intervention groups.Emodin and KH064 can effectively prevent the damage induced by PAAF on AT II cells and increase SP–A expression in both transcription and translation levels. The pathological changes of AT II cells induced by KH064 and Emodin were milder than PAAF group.Conclusion: The cellular structure disorganization of AT II cells was observed in lung tissue of SAP rats with lung injury. SP-A decreased and sPLA₂-II increased significantly during ALI induced by SAP. Qingyitang can lessen the lung injury by protecting AT II cells, up regulating SP-A expression and down regulating sPLA₂-II expression. The balance between SP-A and sPLA₂-II is crucial in acute pancreatitis-associated lung injury （APALI）. Dexamethasone can up regulate SP–A expression and down regulate sPLA₂-II expression in injured lung. This effect might involve alleviating inflammatory reaction. The content of hemodiastase was much higher in SS group, while the change of SP-A and sPLA₂-II expression in lung of this group was not found as significant as in SO group.In AT II cells isolation, purification and identification procedures, optimizing the concentration of digest enzyme and time can improve the cell vitality. Purification of AT II cell with IgG coated plate is a better method than other reported methods. AT II cell could be characterized through detection of the lamellar bodies with electronic microscope.PAAF can damage AT II cell in vitro. It might be due to induce cell necrosis and decrease expression of SP-A. However, both KH064 and Emodin can prevent PAAF induced damage in ATII cell culture system through up-regulation of SP-A expression level.更多还原