【作者】 李洁；

【导师】 陈海龙；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2010， 硕士

【摘要】 目的本实验将通过观察前B细胞克隆增强因子(Pre-B-cell colony-enhancing factor, PBEF)在重症急性胰腺炎(severe acute pancreatitis, SAP)肺损伤(acute lung injury, ALI)模型大鼠肺组织中的表达及功能,并观察清胰汤对SAP大鼠肺脏PBEF表达的影响。从新的角度揭示重症急性胰腺炎相关肺损伤的发病机制,同时探讨清胰汤的治疗作用机理,为临床上中西医结合治疗重症急性胰腺炎提供进一步理论基础。方法健康Spraghe-Dawley(SD)大鼠144只,体重200-220g,雄性,随机分为6组(SAP模型组,假手术组,地塞米松组,铃兰欣组,善宁组,清胰汤治疗组)每组24只,每组随机分为造模6h,12h,24h三个时相典亚组,各时相点亚组8只。SAP组和药物干预组开腹后用无损伤动脉夹夹闭胰胆管入肝门处,逆行胰胆管缓慢注入1.5%的去氧胆酸钠溶液(1ml/kg),取出动脉夹,关腹,建立SAP时ALI模型。地塞米松组于造模后立即静脉注射一次及造模后12h再次静脉注射一次,剂量：1 ml/kg。善宁组于造模后立即静脉注射一次及造模后12h再次静脉注射一次,剂量20μg/kg。铃兰欣组于造模后立即静脉注射一次及造模后1 2h再次静脉注射一次,剂量2mg/kg。清胰汤组造模后胃管注入中药煎液及造模后12h再次灌胃一次,剂量：10ml/kg。假手术组常规剖腹后翻动胰腺,关腹。分别于造模后6h、12h、24h处死相应时相点大鼠,取肺组织及动静脉血做病理检查和各项指标检测。采用双抗体夹心(enzyme linked immunosorbent assay, ELISA)法测定大鼠血清中PBEF含量,免疫组化检测其在肺组织中的表达及组织定位；RT-PCR法检测肺组织中PBEFmRNA表达；Western Blotting法检测肺组织中PBEF蛋白含量；鲎试剂偶氮基质显色法定量检测血清内毒素含量；放免法检测TNF-a, IL-1 p,IL-8在肺组织中的表达；真空干燥法测定肺湿干重(W／D)比值；全自动生化分析仪检测动脉血气；酶法测血清淀粉酶(AMY),大体及镜下观察肺脏病理学改变。结果1.模型组血清PBEF含量升高、PBEFmRNA及其蛋白出现高表达,TNF-α、IL-1β和IL-8均明显高于假手术组。同时,模型组病理学表现为明显的肺损伤,肺W／D比值、血中内毒素、血中AMY等指标均较假手术组显著升高(p<0.01)。清胰汤、地塞米松、善宁可以下调血清PBEF、PBEFmRNA及其蛋白表达,减轻肺损伤程度,而铃兰欣作用不明显。2.清胰汤组血中内毒素、TNF-α、IL-1 p、IL-8等指标下降显著(p<0.05)。善宁组抑制血中AMY作用最强((p<0.05)。地塞米松组对改善肺W／D比值、PaCO2、PaO2作用明显。结论1.应用1.5%的去氧胆酸钠逆行胰胆管内注射,大鼠的胰腺充血水肿明显,血清AMY、肺W/D、PaCO2的指标明显升高,随着时间的推移逐渐增高,较相同时相点假手术组显著增高Pa02呈现相反变化。还可见肺组织水肿、充血、炎细胞浸润,表明重症急性胰腺炎合并肺损伤的模型复制成功。2.重症急性胰腺炎相关肺损伤时,内毒素水平升高,PBEF表达过度上调,进而启动TNF-α、IL-1 p和IL-8等炎性细胞因子的大量表达,在SAP相关ALl发病中起着十分重要的作用。3.中药清胰汤通过下调PBEF的表达,减缓肺泡巨噬细胞的过度激活,减少TNF-α、IL-1 p和IL-8炎性细胞因子的大量表达,减少中性粒细胞(polymorphonuclear neutrophil, PMN)聚集,降低血中高水平的内毒素和AMY浓度、降低肺泡通透性等多个方面机制对肺组织起到保护作用。4.善宁、地塞米松可以不同程度下调PBEF的表达,通过不同途径对不同指标产生影响,从而实现对ALl某种程度的保护作用。5.中、西药物在SAP相关ALl发病的不同环节中,起着十分重要的作用,各有所长,中西医结合理论指导下的中西药联合应用,将会为临床SAP相关ALl的防治提供新的有效途径。更多还原

【Abstract】 Objective:To observe the expression and function of pre-B cell cloning factor (PBEF) of the rat’s blood and lung tissue in severe acute pancreatitis (SAP) associated acute lung injury (ALI) model, and the effects of Qingyitang on PBEF expression. To reveal a new perspective pathogenesis of severe acute pancreatitis associated with lung injury.Chinese medicine Qingyitang is used and the results may give us some new ideas for severe acute pancreatitis treatment.Methods:One hundred and fortyfour health Spraghe-Dawley (SD) rats weight (200-220) g, male, were randomly divided into 6 groups (SAP model group, Sham operation group, and Dexamethasone group, Linglanxin group, Sandostatin group, Qing Yitang group). These 24 animals in each group were randomly divided into Oh,6h,12h,24h subgroup,8 rats were at each subgroup. All modles were induced by retrograde infusion of 1.5% sodium deoxycholate (1 ml/kg) into biliopancreatic duct of Spraghe-Dawley rats except sham operation group. Dexamethasone group, Sandostatin group, Linglanxin group were done with intravenous injection immediately after modeling the first and 12h once again, respectively intravenous dose: 1 ml/kg,20μg/kg,2mg/kg. Qingyitang was given in Qingyitang group with gastric canal injection immediately after modeling the first and 12h in dose:10ml/kg. In Sham group, the pancreas was only flipped after laparotomy. All the rats were sacrificed at 6 h,12 h,24 h respectively after operation, the lung tissue and arterial and venous blood were taken off for pathological examination and some indexes measurement. The level of PBEF in lung was measured by enzyme linked immunosorbent assay(ELISA),the expression and location of PBEF were measured by immunohistochemistry. RT-PCR technique was used to detect the expression of mRNA levels of PBEF and Western Blotting was used to detect the expression of protein levels of PBEF, respectively. Quantitative chromogenic tachypleus amebocyte was used to detect the endotoxin. TNF-α, IL-1βand IL-8 were measured by radioimmunoassay respectively. Wet/dry weight ratio of lung were calculated. Blood gas analysis and serum amylase (AMY) was determined by biochemical method. Histopathological changes in lung were observed under light microscope.Results:The expression of PBEF mRNA and protein in lung tissue, the levels of serum PBEF, TNF-α, IL-1β, IL-8, endotoxin,amyIase, lung W/D ratio, PaCO2 in arterial blood of model group were significantly higher in the same point than those of sham operated control group, meanwhile PaO2 in arterial blood decreased significantly (p<0.01). Some other indexes of ALI showed obvious pathogenic changes in model group and so did the pathogenic examination of lung tissue. Qingyitang Dexamethasone, Santonstadine could decrease the levels of serum PBEF, the expression of PBEF mRNA and its protein in lung tissue, eliminate the extent of lung injury, but Linglanxin could not do that well.The decline of endotoxin, TNF-α, IL-1β, IL-8 in Qingyitang group was more significant(p<0.05). Santonstadine could reduce amylase content obviously. And Dexamethasone showed superiority in ameliorating lung W/D ratio、PaCO2 and PaO2.Conclusions:Infusion of 1.5% sodium deoxycholate into biliopancreatic duct of Spraghe-Dawley rats, the pathologic changes of lung were edema and congestive with large of neutrial cells infiltration and focal atelectasis, the levels of serum AMY, PaCO2 and W/D increased obviously, meanwhile PaO2 decreased significantly. All indexes showed that the APALI model is welldone.During acute lung injury induced by severe acute pancreatitis, many factors have very important effects on pathogenisis of ALI, such as elevated level of endotoxin,overexpression of PBEF which activated overexpression of TNF-a, IL-1β, IL-8.Qingyitang could exert protecting effects through decreasing PBEF expression,reducing the excessive activation of alveolar macrophages, expression of TNF-a, IL-1βand IL-8 in a large number of inflammatory factors, aggregation of PMN, the level of serum endotoxin and amylase and decreasing alveolar permeability and other aspects of mechanism.Dexamethasone and Santonstadine could play their available role in ALI by eliminating PBEF of different levels. Both of them could impact different indicators through different ways.The effects of Chinese and Western medicines on SAP were related to different aspects of the pathogenesis of ALI, and play an important role by their own strengths. Combining Chinese and Western medicine under the guidance of the integration of traditional and western medicine theory, will provide a new effective way to prevent and cure SAP and SAP associated ALI in clinic.更多还原