【作者】 陈英；

【导师】 袁耀宗；

【作者基本信息】 上海交通大学 ， 内科学（消化系病）， 2015， 博士

【摘要】 背景与目的:急性胰腺炎(AP)为常见的急腹症,合并多器官功能障碍综合征(MODS)的重症急性胰腺炎(SAP)患者中死亡率高达20-30%1,其中尤以急性胰腺炎继发急性肺损伤(APALI)最为常见及严重,约占SAP的14.2-35%2。若能早期从所有AP患者中筛选出存在APALI患者,对其及早进行必要的医疗干预,对有效提高医疗资源利用率、改善患者预后及提高生存率有重大意义。抵抗素样分子-α(RELM-α)是从诱导过敏性肺部炎症的小鼠支气管肺泡灌洗液中新发现的一个分泌蛋白,既往研究发现RELM-α具有维护肺泡上皮细胞功能、抵抗炎症、调控肺血管重构、诱导肺血管平滑肌细胞增殖、缩血管、抗凋亡及刺激肌纤维母细胞分化等功能3。本研究拟揭示RELM-α与APALI疾病严重程度的相关性,证实RELM-α可能是SAP疾病预后的早期标志物之一,并探讨其在APALI中的作用。方法:(1)采用牛磺胆酸钠诱导急性坏死性胰腺炎(ANP)动物模型,通过测定血清淀粉酶、C反应蛋白(CRP)水平及胰腺组织病理对其进行评测。(2)通过肺组织病理学评分、肺湿/干重系数及支气管肺泡灌洗液中炎性细胞数量评估APALI的肺组织损伤。(3)通过Western Blot及免疫组化检测肺组织中的RELM-α的表达。(4)化学合成RELM-α基因,酶切目的基因PCR产物及载体DNA并连接,转入感受态细胞克隆后抽提纯化并扩增,利用同源重组将质粒PDC316-m CMVEGFP-Retula和腺病毒骨架载体PPE3-F35共转染293细胞,获得腺病毒重组质粒AD5/F35-RELM-α。通过PCR及基因测序检测RELM-α基因的表达,通过荧光显微镜下绿色荧光蛋白表达观察病毒感染情况,并计算重组腺病毒的滴度;通过Western Blot检测转染细胞中RELM-α的表达,免疫组化检测大鼠肺及胰腺组织中RELM-α的表达。通过观察大鼠胰腺及肺组织病理改变,评估RELM-α过表达对组织的损伤。结果:(1)ANP组血清淀粉酶及CRP水平指标显著高于其它组(p<0.01),ANP组胰腺组织出现典型SAP病理改变,表明大鼠SAP模型造模成功。(2)ANP组病理学评分、大鼠肺湿/干重系数及支气管肺泡灌洗液炎性细胞显著高于其它组(p值均<0.01)。(3)ANP组RELM-α定位于肺泡上皮细胞,蛋白表达显著高于其它(p<0.05)。(4)构建并验证重组RELM-α腺病毒质粒,测定AD5/F35-RELM-α病毒滴度为4×10 e10 pfu/m L,RELM-α组、空载体组、ANP组大鼠及对照组胰腺组织及肺组织光镜下可见RELM-α组病理组织损伤最明显(p<0.05),大鼠胰腺组织及肺组织光镜下分别可见明显的ANP及ALI病理改变。结论:通过牛磺胆酸可成功制备急性坏死性胰腺炎组模型,并得到血清学及组织病理学的验证。RELM-α蛋白表达与肺组织损伤严重程度呈正相关,其过表达可造成胰腺及肺组织损伤,说明其作为促炎性细胞因子,趋化和激活了炎性细胞,介导APALI发生,并可作为早期预测急性胰腺炎严重程度的指标。更多还原

【Abstract】 Background and aims: Acute pancreatitis is a common acute abdomen, while SAP patients with MODS has a high mortality rate of 20-30%1. Acute pancreatitis-associated acute lung injury(APALI) is the most common and serious in MODS, accounting for 14.2-35 %2 of patients with SAP. If we can screen patients with APALI from all AP patients during early phase of the disease, it’s of great significance for improving health care resource utilization, better patient outcomes and improving the survival to take measures with necessary medical intervention.RELM-α is a newly discovered secretory protein which was found in bronchoalveolar lavage fluid of mice that had been induced to allergic lung inflammation. Previous studies have found that RELM-α has the functions of maintenance of alveolar epithelial cell function, resistance to inflammation, regulation of pulmonary vascular remodeling, inducing pulmonary vascular smooth muscle cell proliferation, vasoconstrictor, anti-apoptosis and stimulating myofibroblast differentiation and so on3. Here we try to investigate the relationship between RELM-α and the severity of APALI, confirm that RELM-α may be one of the early biomarker for SAP prognosis and discuss its role in the APALI.Methods:(1) We use sodium taurocholate to induce acute necrotizing pancreatitis(ANP) animal models and we take the serum amylase, CRP and pancreatic pathology histological observation to in evaluate SAP model.(2) We use use lung histopathology score, lung wet / dry weight coefficient and inflammatory cell counting in bronchoalveolar lavage fluid to evaluate the severity of lung injury of APALI.(3) We use Western Blot and immunohistochemistry to test the expression of RELM-α in lung tissue.(4) We chemically synthesized RELM-α gene. We use restriction endonuclease to digest gene PCR products and vector DNA and connect them. Then the connection is cloned into competent cells for cloning. Then we extract and purify and amplification the cloning. PDC316-m CMV-EGFP-Retula and adenovirus backbone vector PPE3-F35 are co-transfected into the 293 cell by homologous recombination to obtain a recombinant adenoviral vector AD5 / F35-RELM-α. We test the expression of RELM-α gene by PCR and gene sequencing and observe viral infection by green fluorescent protein expression under fluorescence microscopy and calculate the titer of recombinant adenovirus. We detect RELM-α expression in transfected cells by Western Blot and detect RELM-α expression in rat lung and pancreatic tissue by immunohistochemistry. We evaluate tissue damage caused by RELM-α overexpression through pathological changes in rat pancreas and lung tissue.Results:(1)ANP serum amylase and CRP is higher than those in other groups with significant difference(p <0.01). The pancreatic tissue in ANP group shows typical pathological changes of SAP, which means our SAP model is successful.(2) ANP group’s lung histopathology score, lung wet / dry weight coefficient and inflammatory cell counting in bronchoalveolar lavage fluid is higher than those in other groups with significant difference(p <0.01).(3) RELM-α is localized in alveolar epithelial cells in ANP group, and its protein expression is significantly higher than the other(p <0.05).(4) We construct and validate the recombinant adenoviral vector RELM-α and AD5/F35-RELM-αvirus titer is 4×10 10 pfu/m L. We observe the pancreatic and lung tissue of RELM-α group, Vector group, ANP group and control group under light microscope, and find out that the injury of RELM-α group is the most severe(p <0.05) and typical ANP and ALI pathological changes can be observed.CONCLUSION: We build the model of acute necrotizing pancreatitis group successfully by injecting sodium taurocholate and get serological and tissue disease verification. The expression of RELM-α protein in lung tissue is positively correlated with the severity of lung damage. Overexpression of RELM-α can cause pancreatic and lung tissue damage, suggesting as a pro-inflammatory cytokines it can promote and activate inflammatory cells to mediate the occurrence of APALI. It can also be used as an early prediction markers of acute pancreatitis severity.更多还原