【作者】 张旭；

【导师】 秦涛；

【作者基本信息】 郑州大学 ， 普通外科学， 2022， 博士

【摘要】 目的:探讨P-选择素糖蛋白配体-1（p-selectin glycoprotein ligand-1,PSGL-1）在重症急性胰腺炎（severe acute pancreatitis,SAP）中的作用及机制。方法:收集2018年1月至2019年1月就诊于郑州大学人民医院（河南省人民医院）的SAP患者外周血血液标本,并收集同时期健康人对照组外周血血液标本。利用流式细胞术检测外周血单核/巨噬细胞和中性粒细胞的数目,检测这两种细胞表面PSGL-1的表达情况。利用PSGL-1敲除基因小鼠(PSGL-1^-/-)构建SAP小鼠模型,应用淀粉酶试剂盒检测小鼠血清淀粉酶水平,通过组织病理学评分评价胰腺病理损伤情况。应用酶联免疫吸附实验（enzyme linked immunosorbent assay,ELISA）试剂盒检测小鼠外周血白细胞介素-6（interleukin,IL-6）和白细胞介素-1β（interleukin,IL-1β）的表达水平。应用蛋白质免疫印迹法（western blot,WB）检测胰腺组织中IL-6和IL-1β的表达情况。采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（Td T-mediated d UTP nick-end labeling,TUNEL）检测小鼠胰腺腺泡细胞的细胞凋亡水平。利用流式细胞术检测小鼠外周血单核/巨噬细胞和中性粒细胞的数目,并检测小鼠外周血单核/巨噬细胞和中性粒细胞PSGL-1的荧光强度,进而反映PSGL-1的表达情况。应用免疫组织化学染色（immunohistochemistry,IHC）的方法检测胰腺组织中单核/巨噬细胞和中性粒细胞的浸润情况。应用雨蛙肽（caerulein,Cae）诱导大鼠胰腺外分泌细胞系（AR42J）,构建急性胰腺炎（acute pancreatitis,AP）细胞模型,利用外周血单核/巨噬细胞-内皮细胞共培养系统检测内皮粘附情况。构建PSGL-1小干扰RNA（small interfering RNA,si RNA）,转染小鼠单核巨噬细胞白血病细胞系（RAW264.7）,采用实时荧光定量PCR（real-time fluorescence quantitative PCR,q PCR）检测PSGL-1的信使RNA（messenger RNA,m RNA）表达水平,采用WB检测PSGL-1、精氨酸酶-1（arginase-1,ARG1）、一氧化氮合成酶（inducible nitric oxide synthase,i NOS）蛋白表达情况。利用转录组测序技术检测敲低PSGL-1后下游基因的变化情况,并通过基因本体（gene ontology,GO）分析、京都基因和基因组百科全书（kyoto encyclopedia of genes and genomes,KEGG）分析和基因集富集分析（gene set enrichment analysis,GSEA）,进一步分析下游信号通路的变化。结果:1、SAP组患者外周血中单核/巨噬细胞和中性粒细胞数目明显高于健康人对照组,并且其表面PSGL-1表达明显高于健康人对照组。2、构建小鼠SAP模型,SAP小鼠模型外周血单核/巨噬细胞和中性粒细胞上PSGL-1表达明显高于对照组小鼠。3、构建小鼠SAP模型,PSGL-1^-/-小鼠血清中淀粉酶水平、IL-6表达、IL1β表达以及胰腺组织病理损伤的程度显著低于对照组野生型小鼠。4、构建小鼠SAP模型,PSGL-1^-/-小鼠胰腺组织中单核/巨噬细胞和中性粒细胞浸润数目明显低于对照组野生型小鼠。5、构建小鼠SAP模型,PSGL-1^-/-小鼠外周血单核/巨噬细胞与内皮细胞粘附数量明显少于对照组野生型小鼠。6、体外实验表明,敲低PSGL-1表达可以抑制单核/巨噬细胞向M1型分化,并促进单核/巨噬细胞向M2型分化。7、转录组测序、GO分析、KEGG分析、ESGA分析结果表明,敲低PSGL-1后可能通过激活Wnt信号通路诱导单核/巨噬细胞向M2型分化。结论:PSGL-1通过介导单核/巨噬细胞和中性粒细胞与内皮细胞粘附,促进单核/巨噬细胞和中性粒细胞胰腺组织浸润,并可能通过调节Wnt信号通路调控单核/巨噬细胞M1/M2极化方向,在SAP早期促进炎症反应的发生。更多还原

【Abstract】 Objectives:To investigate the role and mechanism of P-selectin glycoprotein ligand-1（PSGL-1）in the occurrence and development of severe acute pancreatitis（SAP）.Methods:We collected the peripheral blood samples from patients who is diagnosed as SAP from January 2018 to January 2019 in the people’s Hospital of Zhengzhou University.We collected the peripheral blood samples from healthy people during the same time.We used flow cytometry to detect the number of monocytes/macrophages and neutrophils in the two groups.The expression of PSGL-1 on the surface of these cells was detected by same method,too.The mouse model of severe acute pancreatitis was established in PSGL-1 knockout mice(PSGL-1^-/-),and the amylase level and pancreatic pathological injury were detected.IL-6 and IL-1βin peripheral blood of mice were detected by ELISA Expression level of IL-6 and IL-1βin pancreatic tissue were detected by western blot（WB）.We used TUNEL assay to detect the apoptosis of pancreatic acinar cells in AP mouse model.The number of monocytes/macrophages and neutrophils from peripheral blood of AP mouse model was detceted by flow cytometry.The expression of PSGL-1 on the surface of monocytes/macrophages and neutrophils in AP mouse model was detected by the same method.The infiltration of monocytes/macrophages and neutrophils in pancreatic tissue was detected by immunohistochemical staining.We use Cae to stimulate AR42J cell line to construct AP model in vitro.For detecting the ability of adhesion with monocytes/macrophages and endothelial cell,we created a co-culture system.Si RNA was constructed and transfected into RAW264.7 cells,the expression of PSGL-1 m RNA was detected by q PCR,and the expression of PSGL-1,ARG1 and i NOS protein was detected by Western Blot.Using transcriptome sequencing technology to detect the changes of downstream genes after knockdown of PSGL-1,and further analyzes the changes of downstream signal pathways through GO analysis,KEGG analysis and GSEA analysis.Results:1.Compared with the healthy group,the SAP group has higher quantitative monocytes/macrophages and neutrophils number significantly.The SAP group has higher expressive PSGL-1 on the surface of monocytes/macrophages and neutrophils compared with the healthy group.2.The SAP mouse model group has higher expressive PSGL-1 on the surface of monocytes/macrophages and neutrophils than control group.3.Serum amylase level,IL-6 and IL-1βexpression,pathological injury of pancreatic tissue in PSGL-1^-/-mice with severe pancreatitis were significantly lower than that of wild-type mice.4.The infiltration of monocytes/macrophages and neutrophils in the pancreas of PSGL-1^-/-mice with severe pancreatitis was significantly lower than that of wild-type mice.5.The adhesion between peripheral blood monocytes/macrophages and endothelial cells in PSGL-1^-/-mice was significantly lower than that in control wild-type mice.6.Knockdown of PSGL-1 expression can inhibit the differentiation of monocytes/macrophages into M1 type and promote the differentiation of monocytes/macrophages into M2 type7.Transcriptome sequencing,GO analysis,KEGG analysis and GSEA analysis showed that PSGL-1 may induce monocytes/macrophages to differentiate into M2type by regulating Wnt signaling pathway.Conclusion:PSGL-1 promotes the infiltration of monocytes/macrophages and neutrophils into pancreatic tissue by mediating the adhesion of monocytes/macrophages and neutrophils to endothelial cells,and may regulate the differentiation of monocyte macrophages into M2 type by activating Wnt signaling pathway.更多还原