RELM-α在APALI中病情评估价值的研究

发布时间：2018-04-15 15:08

本文选题：RELM-α + 急性胰腺炎　；参考：《上海交通大学》2015年博士论文

【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(p0.01),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(p0.05)。(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(p0.05),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。
[Abstract]:Background & objective: acute pancreatitis (AP) is a common acute abdomen. The mortality rate of severe acute pancreatitis (SAP) with multiple organ dysfunction syndrome (MODS) is as high as 20-301, especially acute lung injury (APALI) secondary to acute pancreatitis.About 14.2-352.00% of SAP.It is of great significance to improve the utilization of medical resources, improve the prognosis and improve the survival rate of patients with APALI if they can be screened out from all patients with AP early and carry out necessary medical intervention as soon as possible.Resistin like molecule- 伪 luciferin 伪 is a newly found secretory protein from the bronchoalveolar lavage fluid of mice induced by allergic pulmonary inflammation. Previous studies have found that RELM- 伪 can maintain the function of alveolar epithelial cells, resist inflammation and regulate pulmonary vascular remodeling.Induces pulmonary vascular smooth muscle cells proliferation, vasoconstriction, anti-apoptosis and stimulate myofibroblast differentiation.This study aims to reveal the correlation between RELM- 伪 and the severity of APALI disease, to confirm that RELM- 伪 may be one of the early prognostic markers of SAP disease, and to explore its role in APALI.Methods the animal model of acute necrotizing pancreatitis (ANPs) was induced by sodium taurocholate. The levels of serum amylase C-reactive protein (CRP) and pancreatic histopathology were measured.Lung wet / dry weight coefficient and the number of inflammatory cells in bronchoalveolar lavage fluid (BALF) the lung tissue injury of APALI was evaluated. The expression of RELM- 伪 in lung tissue was detected by Western Blot and immunohistochemistry. The RELM- 伪 gene was chemically synthesized.The target gene PCR product and vector DNA were ligated and ligated. The recombinant plasmid PDC316-m CMVEGFP-Retula and adenovirus cytoskeleton PPE3-F35 were co-transfected into 293 cells by homologous recombination. The adenovirus recombinant plasmid AD5 / F35-RELM- 伪 was obtained.The expression of RELM- 伪 gene was detected by PCR and gene sequencing, the viral infection was observed by green fluorescent protein expression under fluorescence microscope, and the titer of recombinant adenovirus was calculated, and the expression of RELM- 伪 in transfected cells was detected by Western Blot.The expression of RELM- 伪 in rat lung and pancreas was detected by immunohistochemistry.The pathological changes of pancreas and lung were observed to evaluate the tissue damage caused by RELM- 伪 overexpression.Results the levels of serum amylase and CRP were significantly higher in the control group than those in the other groups. The pathological changes of pancreatic tissue in the control group were significantly higher than those in the control group, which indicated that the rat model of SAP was successfully established and the pathological score of the group was higher than that of the control group.The lung wet / dry weight coefficient and bronchoalveolar lavage fluid inflammatory cells in 0.01).(3)ANP group were significantly higher than those in other groups. RELM- 伪 was located in alveolar epithelial cells, and the protein expression was significantly higher than that in other groups.The titer of AD5 / F35-RELM- 伪 virus was determined to be 4 脳 10 e10 pfu/m / L,The histopathological damage of RELM- 伪 group was the most obvious under light microscope, and the pathological changes of ANP and ALI in pancreatic tissue and lung tissue of rats were obvious under light microscope.Conclusion: the model of acute necrotizing pancreatitis can be successfully established by taurocholic acid, and the positive correlation between the expression of .RELM- 伪 protein and the severity of lung injury can be obtained by serological and histopathological analysis, and the overexpression of taurocholic acid can cause pancreatic and lung injury.It is suggested that as a pro-inflammatory cytokine, it chemotaxis and activates inflammatory cells, mediates the occurrence of APALI, and can be used as an early predictor of the severity of acute pancreatitis.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R657.5

【相似文献】