【作者】 张波；

【导师】 滕木俭；

【作者基本信息】 山东大学 ， 外科学（普外）（专业学位）， 2018， 博士

【摘要】 背景急性胰腺炎(AP)是常见的急性腹部炎性疾病,主要表现为腺泡细胞损伤、氧化应激和胰腺炎症,经常由胆结石疾病或过量饮酒引起,目前仍有较高的发病率和死亡率。大部分急性胰腺炎患者的预后良好,可以完全治愈,但仍有大约20%的患者会有发生器官衰竭的风险,急性胰腺炎患者是否有脏器功能衰竭以及持续时间决定了早期病情的严重程度。2012年美国亚特兰大会议将急性胰腺炎分为轻症、中度重症和重症急性胰腺炎3种。轻症急性胰腺炎患者不合并脏器功能障碍,病死率几乎为零;中度重症急性胰腺炎患者往往伴有一过性器官功能衰竭(持续时间不超过48小时),病死率介于轻症急性胰腺炎与重症胰腺炎之间。病程当中一旦出现持续性脏器功能障碍,很容易将患者划分为轻度或重度胰腺炎,但对改善预后没有帮助,只有在疾病的早期就能够很好的预测急性胰腺炎疾病发展的进程和结果,才能尽早的制定有效的治疗方案以及采取干预措施,避免发生多脏器功能衰竭,从而改善预后。尽管许多单个生物预后标志物和多个评分系统己经被试用,但目前仍然没有一个被广泛接受的。因此,迫切需要寻找新的诊断和治疗靶点来改善急性胰腺炎尤其是重症患者的生存率。RNA干扰技术(RNAi)是最近几年来新产生的一种在基因功能研究和基因治疗上具有广阔的应用前景的生物技术,因为它的实施简单有效,并且研究表明应用微量的siRNA即可达到使其编码的致病基因产物的含量下降90%以上的目的,有的甚至可以达到完全基因敲除的效果,所以逐渐成为了代替基因敲除的有力工具。慢病毒载体是一种基因治疗载体,具有极好的包装能力,可以在不同哺乳动物细胞中维持长期稳定的转基因的表达,并且不出现病理损害现象。慢病毒载体具有广泛的细胞趋性,无论对分裂细胞还是非分裂细胞均具有感染能力,可以容纳片段比较大的外源性基因,是目前常用的RNA干扰技术,它的发展形成了一个可以应用于减少目标基因的表达系统。双特异性磷酸酶-1(DUSP1)又被称为丝裂素活化蛋白激酶磷酸酶-1(MKP1),属于丝裂原活化蛋白激酶(MAPK)磷酸酶家族中的一员,其表达受许多细胞外刺激物诱导,包括生长因子、细胞因子和应激等,对MAPK具有强有力的负监管作用,DUSP1基因被认为是肿瘤抑制因子和癌症相关炎症的调节器,更重要的是DUSP1在抗炎方面也发挥着重要的作用。丝裂原活化蛋白激酶(MAPKs)是所谓的进化守恒的丝氨酸和苏氨酸蛋白酶,它参与到连接细胞表面受体和主要调节细胞核和细胞内靶体的信号转导通路中。MAPKs被视为急性胰腺炎发展的早期阶段的主要信号传导器,在哺乳动物中,有多种MAPK酶负责细胞增殖、凋亡、分化和存活。MAPK磷酸酶(包括DUSP1)可以通过MAPK脱磷酸化或干扰作用于MAPK的效应分子结合,从而抑制信号通路的转导和细胞因子激活。急性胰腺炎病情加重甚至恶化的关键是在急性胰腺炎期间失去对多种炎症介质的控制,从而引发级联反应,导致胰腺组织的大量坏死,并因系统性多器官衰竭而变得更为复杂。就这一点而言,细胞因子的作用一直是关注的焦点。本研究旨在通过慢病毒载体沉默DUSP1从而增强MAPK信号通路的作用,PD98059作为MAPK信号通路的抑制剂抑制MAPK信号通路,观察急性胰腺炎小鼠模型血清及组织中促炎因子的表达以及MAPK通路相关基因蛋白表达情况,探索其对促炎因子释放的调控机制及对多脏器损伤的影响。目的通过慢病毒载体沉默DUSP1基因,观察急性胰腺炎小鼠模型血清及组织中促炎因子的表达以及MAPK通路相关基因蛋白表达情况,探索慢病毒载体沉默DUSP1基因通过MPK通路介导急性胰腺炎小鼠促炎因子释放的调控机制以及对多脏器损伤的影响。方法1.表达siRNADUSP1载体的构建:设计2条siRNA和一条阴性对照序列siRNA1、5’-TAGCGTCAAGACATTTGCTGA-3’,siRNA2、5’-CTGTACTATCCTGTAAATATA-3’,scramble siRNA、5’-GGCAAGCTGACCCTGAAGTTC-3’检测DUSP1的表达情况,筛选沉默效率高的siRNA。2慢病毒包装及滴度测定选择沉默效率高的siRNA,采用脂质体法,分别与慢病毒包装质粒混合物共转染293T细胞,并进行病毒滴度测定。3小鼠急性坏死性胰腺炎模型制备及分组选取105只清洁级健康雄性KM小鼠,制模小鼠在实验前禁食12h,自由饮水。小鼠按4g/kg体质量予腹腔内注射20%L-精氨酸,分两次腹腔内注射,每次注射间隔1h,制备小鼠急性胰腺炎模型。按随机数字表法将小鼠分为7组,每组15只:(1)control组:健康小鼠,注射相同剂量的生理盐水。(2)siRNA组:健康小鼠,腹腔注射0.88mg/kgDUSP1-siRNA慢病毒。(3)AP组:模型小鼠。(4)AP+PD98059组:在AP诱导前0.5h与诱导后1h腹腔注射PD98059 10mg/kg,其余各组采用相同剂量的生理盐水代替PD98059。(5)AP+siRNA组:小鼠造模成功后,予以腹腔注射0.88mg/kgDUSP1-siRNA慢病毒。(6)AP+scramble组:小鼠造模成功后,予以腹腔注射0.88mg/kg scramble-siRNA慢病毒。(7)AP+PD98059+siRNA组:在AP诱导前0.5h与诱导后1h腹腔注射PD9805910mg/kg,造模成功后,予以腹腔注射0.88mg/kgDUSP1-siRNA慢病毒。各组小鼠分别在制模后相应时间点即12小时、24小时、48小时经全麻心脏取血(作为血液标本),于建模48小时后处死小鼠,立即取得胰腺组织、肝脏组织、肺脏组织和肾脏组织。4 Elisa法检测血清中促炎因子及HMGB1和S100A12含量于建模后12小时、24小时、48小时,分别选取各组5只小鼠行心脏取血,严格按照试剂盒要求采用Elisa方法检测各组小鼠血清中促炎因子(TNF-α、IL-1β、IL-6)以及 HMGB1 和 S100A12 含量。5测定急性胰腺炎小鼠血清淀粉酶、脂肪酶水平各组小鼠建模后12小时、24小时、48小时测定血清中淀粉酶、脂肪酶水平,各指标的检测严格按照试剂包的要求进行操作。6 HE染色将小鼠的胰腺、肝脏、肾脏和肺组织用4%甲醛固定,石蜡包埋切片,HE法进行染色,用光学显微镜观察各脏器组织病理学改变,并采取照片。7 RT-qPCR根据病毒核酸提取的说明书,通过试剂盒一步法提取组织标本中的总RNA,并且测定总RNA的质量和浓度。采用两步法进行提取RNA的逆转录。采用TaqMan探针法进行RT-qPCR检测,检测各脏器组织中促炎因子(TNF-α、IL-1β和 IL-6)、DUSP1、HMGB1 和 S100A12 的 mRNA 表达量。8 Western提取各组胰腺、肝脏、肾脏和肺组织样本,清除血液和其他组织,用超声波粉碎。采用蛋白免疫印迹法检测各组织中DUSP1及MAPK通路相关基因蛋白的表达量。结果1.设计的siRNA2序列具有较高的沉默效率,采用pSIH-DUSP1-siRNA2包装的慢病毒对293T细胞的感染率超过90%。2.与对照组相比,siRNA组中血清淀粉酶、脂肪酶水平以及血清和组织中的促炎因子、HMGB1和S100A12都有升高,但都无显著差异;而在其他5组中各项指标的表达显著增加。3.与AP组相比,AP + siRNA组组织中DUSP1表达下降,而血清中各项指标的表达明显升高,并且组织中TNF-α、IL-1β、IL-6、HMGB1 和 S100A12mRNA的表达以及MAPK信号通路中相关基因蛋白p-ERK、p-JNK和p-p38的表达也明显升高,胰腺组织的病理改变加重;AP + PD98059组血清中各项指标的表达下降,并且组织中TNF-α、IL-1β、IL-6、HMGB1 和 S100A12mRNA的表达以及MAPK信号通路中相关基因蛋白p-ERK、p-JNK和p-p38的表达也下降,而胰腺组织的水肿程度减轻;AP+scramble组和AP+PD98059+siRNA组无显著性差异。4.在各组急性胰腺炎小鼠模型中,血清中HMGB1和S100A12的表达水平与TNF-α、IL-1β和IL-6的表达水平呈正相关。5.与AP组相比,AP + siRNA组胰腺、肝脏、肾脏和肺组织的病理改变明显加重,而AP + PD98059组胰腺、肝脏、肾脏和肺组织的病理改变减轻。结论1.采用pSIH-DUSP1-siRNA2包装的慢病毒可以有效的抑制细胞中DUSP1的表达。2.在急性胰腺炎小鼠模型中DUSP1基因沉默可以促进促炎因子的释放,其作用机制可能是通过对MAPK信号通路中的ERK、JNK和p38的磷酸化而发挥其活性作用,增强了 MAPK信号通路的作用,从而促进促炎细胞因子释放。3.血清中HMGB1和S100A12的表达水平与TNF-α、IL-1β和IL-6的表达水平呈正相关,提示作为MAPK信号通路的上游因子的HMGB1和S100A12与TNF-α、IL-1β和IL-6之间存在正反馈关系,共同促进炎症进程的发展。4.TNF-α、IL-1β、IL-6、HMGB1和S100A12的过度表达可以加重急性胰腺炎小鼠模型胰腺、肝脏、肾脏和肺组织的病理改变,从而更容易发生多脏器功能衰竭,加重急性胰腺炎的病情。5.早期检测DUSP1的表达水平,可以提前预测发生多脏器功能衰竭的几率,通过对DUSP1和MAPK信号通路的调节可以改变急性胰腺炎小鼠各脏器损伤的程度,为治疗提供新的靶点。更多还原

【Abstract】 BackgroundAcute pancreatitis(AP)is an acute inflammatory disease mainly manifested as acinar cell injury,oxidative stress,and pancreatic inflammation,frequently caused by gallstone disease or excess alcohol ingestion,and is a main cause of morbidity and mortality worldwide.Most patients suffering from AP are in a mild form but about 20%of patients develop a complicated life-threatening disease with a risk of organ failures.The severity of early acute pancreatitis is mainly determined by the presence or absence of organ failure and the duration of organ failure.According to the classification criteria of acute pancreatitis in 2012 Atlanta conference,acute pancreatitis was divided into three types:mild,moderate and severe acute pancreatitis.Patients with mild acute pancreatitis have neither organ dysfunction nor local and systemic complications,and generally have a good prognosis with almost zero case fatality.Patients with moderate severe acute pancreatitis are often accompanied by transient organ failure(which can recover spontaneously within 48 hours),or with local or systemic complications without persistent organ failure.Patients with moderate severe acute pancreatitis have a higher mortality than those with mild acute pancreatitis,but much lower than those with severe acute pancreatitis.Sustained organ dysfunction exists in patients with severe acute pancreatitis(lasting for more than 48 hours),and the mortality rate of severe pancreatitis is 36%to 50%,which is higher if combined with infection.Once appearing sustained organ dysfunction,it is easy to dividing the patients with AP into mild or severe acute pancreatitis,but there is no help on how to improve the prognosis.To improve the prognosis,the progression and results of the disease of acute pancreatitis should be well predicted in the early stages of the disease,so that more effective treatments and intervention measures may be made as early as possible to avoid the occurrence of multiple organ function failure.A technique called RNA interference(RNAi)is a kind of new biological technology in recent years,because it can be simply and effectively implemented.More studies have shown that trace of siRNA application can make the coding content dropped more than 90%of the disease-causing gene product,some even can achieve the result of gene knock out completely.As a powerful tool replacing the gene knockout,RNAi has a broader application prospect in gene function research and gene therapy.Lentivirus vector,a gene therapy vector based on human immunodeficiency virus(HIV),is a commonly used RNA interference technique currently,which can maintain long-term stability in different mammalian cells,and infect both mitotic and non-mitotic cells.Lentiviral vectors have excellent packaging ability,extensive cellular tropism,and have developed a system that can be used to reduce the expression of target genes.Compared with other retroviruses,lentivirus has significant advantages such as being able to infect non-dividing cells,accommodate large fragments of exogenous genes,and express for a long time without pathologic damage.Dual-specificity phosphatase-1(DUSP1),also known as mitogen-activated protein kinase(MAPK)phosphatase 1,is a member of the MAPK phosphatase family and a potent negative regulator of MAPK activity.Its expression is induced by many extracellular stimuli,including growth factors,cytokines and stress.The DUSP1 gene is considered a tumor suppressor and a regulator of cancer-associated inflammation.DUSP1 is also important in anti-inflammation effects.MAPKs are considered evolutionarily well-conserved serine and threonine protein enzymes,which are involved in signal transduction pathways linking cell surface receptors with main regulative nuclear and intracellular targets.MAPKs are considered to be main signal transducers in the early stage of the development of AP.In mammals,there are several MAPK enzymes responsible for cell proliferation,apoptosis,differentiation and survival.MAPK phosphatases,including DUSP1,inhibit signal transduction and cytokine activation via MAPK dephosphorylation or interference with effector molecules binding to MAPKs.The key to the exacerbation or even deterioration of acute pancreatitis is the loss of control over multiple inflammatory mediators during acute pancreatitis,leading to a cascade of reactions that leads to massive necrosis of pancreatic tissue and a complicated condition because of systemic multi-organ failure.In this regard,the role of cytokines has been the focus of attention.This study aims to observe the proinflammatory factor expression and MAPK pathways related gene protein expression in serum and tissues in mice model of acute pancreatitis,and to explore the proinflammatory factor release control mechanism and the effects on multiple organ injury by lentivirus vector silencing DUSP1 to enhance the role of MAPK signal pathway and PD98059(MAPK signal pathway inhibitors)inhibiting the MAPK signaling pathways.ObjectiveThis study aims to observe the proinflammatory factor expression and MAPK pathways related gene protein expression in serum and tissues in mice model of acute pancreatitis,and to explore the proinflammatory factor release control mechanism and the effects on multiple organ injury by lentivirus vector silencing DUSP1 to enhance the role of MAPK signal pathway.Methods1.Construction and detection of the psiRNA-DUSP1 vectorTwo DUSP1-siRNAs and one negative control(NC)sequence were designed according to the mouse DUSP1 gene sequence in the gene bank as follows:siRNA1:5’-TAG CGT CAA GAC ATT TGC TGA-3’;siRNA2:5’-CTG TAC TAT CCT GTA AAT ATA-3’;and negative:5’-AAC TGG ACT TCC AGA AGA ACA-3’.The expression of DU SP1 was detected and siRNA with high silencing efficiency was screened2.Lentivirus packaging and titer determination.The pSIH-DUSP1-siRNA and pSIH-NC with high silencing efficiency were selected to co-transfect 293T cells with a packaging plasmid mixture respectively using the lipofection method,and the virus titer was measured.3.Preparation and grouping of the AP mouse model.A total of 105 male KM mice in healthy condition were used.Prior to the experiment,the mice used for the AP model were fasted for 12 h for gastrointestinal decompression,however,they all had free access to water.The mice were intraperitoneally injected twice with a 1 h interval with 20%L-arginine at 4 g/kg to establish the AP mouse model.Using a Table of random numbers,the mice were divided into seven groups with 15 mice in each group:Control group(mice injected with the same dose of normal saline);siRNA group(mice injected with 0.88 mg/kg DUSP1-siRNA lentivirus);AP group(AP mouse model);AP+PD98059 group[0.5 h pre-induction and 1 h post-induction,the mice were intraperitoneally injected with PD98059 at 10 mg/kg;AP+siRNA group(following successful model establishment,lentiviruses containing 0.88 mg/kg DUSP1-siRNA were injected into mice intraperitoneally);AP+scramble group(following successful model establishment,0.88 mg/kg scramble-siRNA lentiviral vectors were injected intraperitoneally);AP+PD98059+siRNA group(0.5 h pre-induction and 1 h post-induction,mice were intraperitoneally injected with PD98059 at 10 mg/kg,and following successful model establishment,lentiviruses containing 0.88 mg/kg DUSP1-siRNA were injected into mice intraperitoneally).The mice in each group received cardiac blood sampling(as blood specimen)at 12,24 and 48 h post-modeling,respectively.After 48 h,the mice were sacrificed and pancreatic tissue,liver tissue,lung tissue and kidney tissue were immediately obtained.4.Elisa was used to measure the proinflammatory factors,HMGB1 and S100A12 in serum.At 12,24 and48 h post-model establishment,five mice in each group were selected for cardiac blood sampling.Elisa was used to measure the proinflammatory factors,HMGB1 and S100A12 in serum,Strictly following the kit requirements.5.Determination of serum amylase and lipase levels.The levels of amylase and lipase in serum were determined at 12,24 and 48 h post-model establishment in each group.The test of each index was performed strictly in accordance with the requirements of the reagent package6.Hematoxylin and eosin(HE)staining.The pancreatic,liver,kidney and lung tissues of mice in each group were removed and fixed with 4%formaldehyde,and immersed and embedded in paraffin.The paraffin-embedded pancreatic tissues were cut into sections,Using the morphological image analysis system,images of the HE-stained sections of pancreatic,liver,kidney and lung tissues in each group were captured.7.Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)analysis.Based on the manufacturer’s protocol of the TRIzol kit,total RNA from the tissue specimens was extracted via the TRIzol one-step method.The quality and concentration of total RNA were identified and determined.The reverse transcription of the extracted RNA was performed in two steps according to the protocol of the kits.The RT-qPCR was performed using the TaqMan probe method according to the kit protocol to measure the mRNA expressions of pro-inflammatory factors(TNF-a,IL-1β and IL-6),DUSP1,HMGB1,and S100A12 in various organs.8.Western blot analysis.Pancreatic,liver,kidney and lung tissue samples were extracted and shattered on ice using ultrasound,following removal of blood and other tissues.Proteins related to the DUSP1 and MAPK pathways were detected by western blotting.Results1.The siRNA2 sequence was designed with high silencing efficiency,and the infection rate of lentivirus packaged by pSIH-DUSP1-siRNA2 to 293T cells was over 90%.2.Compared with the control group,the serum levels of amylase and lipase of mice in the siRNA group were increased as well as pro-inflammatory factors,HMGB1 and S100A12 in serum and tissues,but there was no significant difference.In the other 5 groups,the expression of each index increased significantly.3.Compared with the AP group,the expression of DUSP1 in the AP + siRNA group was decreased,while the expression of various indicators in the serum was significantly increased,and the expression of TNF-α,IL-1β,IL-6,HMGB1 and S100A12 mRNA in the tissue,as well as the related gene proteins p-ERK、p-JNK and p-p38 in the MAPK signaling pathway was also significantly increased,and the pathological changes in the pancreatic tissue were aggravated.In the AP + PD98059 group,the expression of various indexes in serum decreased,and the expression of TNF-a,IL-1β IL-6,HMGB1 and S100A12 mRNA in tissues as well as the related gene proteins p-ERK、p-JNK and p-p38 in MAPK signaling pathway also decreased,while the degree of edema in pancreatic tissues decreased.There was no significant difference between AP+scramble group and AP+PD98059+siRNA group.4.In the mice with acute pancreatitis in each group,the expression levels of HMGB1 and S100A12 in serum were positively correlated with the expression levels of TNF-α,IL-1βand IL-6.5.Compared with the AP group,the pathological changes of pancreas,liver,kidney and lung tissues in the AP + siRNA group were significantly aggravated,while the pathological changes in the AP + PD98059 group were reduced.Conclusion1.The expression of DUSP1 in the cells was effectively inhibited by lentivirus packed with pSIH-DUSP1-siRNA2.2.In the mice model with acute pancreatitis,silencing DUSP1 gene can promote the release of pro-inflammatory cytokines.Its mechanism may be that it plays its active role through phosphoiylation of ERK,JNK and p38 in MAPK signaling pathway,enhancing the effect of MAPK signaling pathway,thus promoting the release of pro-inflammatory cytokines.3.The expression levels of HMGB1 and S100A12 in serum are positively correlated with the expression levels of TNF-α,IL-1β and IL-6,suggesting that there is a positive feedback relationship between the pro-inflammatory cytokines(TNF-α,IL-1β and IL-6)and HMGBI and S100A12,the upstream factors of MAPK signaling pathway,jointly promoting the development of inflammatory processes.4.Overexpression of TNF-α,IL-1β,IL-6,HMGB1 and S100A12 can aggravate the pathological changes of pancreas,liver,kidney and lung tissues in the mice model with acute pancreatitis,making multiple organ failure easier and aggravating the condition of acute pancreatitis.5.Early detection of DUSP1 expression can predict the probability of multiple organ failure in advance.By regulating the DUSP1 and MAPK signaling pathways,the degree of organ damage in acute pancreatitis mice can be changed,providing a new target for treatment.更多还原