纤维连接蛋白及其肝素结合域多肽对重症感染的作用和免疫机制

发布时间：2018-01-07 06:10

  本文关键词：纤维连接蛋白及其肝素结合域多肽对重症感染的作用和免疫机制　出处：《福建医科大学》2015年博士论文　论文类型：学位论文

  更多相关文章： 重症感染 纤维连接蛋白 脓毒症动物模型 T淋巴细胞 巨噬细胞 细胞因子

【摘要】：目的:1.分析重症急性胰腺炎(SAP)、脓毒症、多器官功能障碍(MODS)等重症感染患者病程多时间点血浆纤维连接蛋白(pFN)、Th1/Th2/Th17细胞因子水平的变化,以期寻找早期诊断和准确判断重症感染患者疾病严重程度和预后的临床指标;2.以半乳糖胺(D-GaL)敏化的内毒素(LPS)诱导的小鼠脓毒症模型,观察人血浆纤维连接蛋白(pFN)和重组人FN羧基端肝素结合域多肽(rhFNHC-36)的保护作用,研究其对脓毒症小鼠的炎症反应和免疫功能尤其对CD4+T淋巴细胞功能的影响,从而探讨pFN、rhFNHC-36对脓毒症动物的保护机制,为重症感染治疗新策略提供实验基础和思路。方法:1.收集34例SAP、20例脓毒症、45例MODS患者患病临床确诊时的血浆样品和15例普通胰腺炎(AP)、46例健康对照者血浆,用流式液相多重蛋白定量技术(CBA)检测血浆Th1/Th2/Th17细胞因子(IL-2、IL-4、IL-6、IL-10、IL-17A、IFN-γ、TNF-α)的浓度;ELISA检测血浆FN、CRP浓度,分析各类重症感染患者血浆FN和细胞因子与AP、健康对照的差别。其中49例不同转归的SAP和MODS患者收集病程多个时间点血浆样品,分析FN和细胞因子的动态变化与病情进展的关系;2.建立稳定的D-GaL敏化LPS诱导的BALB/C小鼠脓毒症模型;3.观察pFN、rhFNHC-36对脓毒症模型小鼠的保护作用及其对炎症反应的影响3.1以pFN、rhFNHC-36多次尾静脉注射给药的治疗方案,观察和记录小鼠的状况和72小时存活率。实验分组:正常对照组,脓毒症PBS对照组(尾静脉注射PBS),脓毒症rhFNHC-36治疗组,脓毒症pFN治疗组;3.2 HE染色观察各组组织器官(肝、脾、肺)的炎症情况。收集各组2h、4h、8h、24h、72h血浆,ELISA检测FN、IL-4浓度,流式液相多重蛋白定量技术(CBA)检测炎症因子IL-1β、IL-6、IL-12p70、IFN-γ、MCP-1、TNF的浓度;3.3分离正常小鼠腹腔巨噬细胞,分别以LPS+rhFNHC-36、LPS+pFN共培养4h和16h,Real-time PCR检测小鼠腹腔巨噬细胞M1相关分子(i NOS、IL-1β、IL-6、IL-12p40、IFN-γ、HMGB1、TNFα)和M2相关分子(Argnase-1、IL-10)m RNA表达水平;4.探讨pFN、rhFNHC-36对脓毒症模型小鼠辅助性T淋巴细胞功能的影响4.1体外实验:免疫磁珠分选脓毒症小鼠和正常小鼠脾脏CD4+T淋巴细胞,以抗CD3/CD28单抗和不同浓度rhFNHC-36或pFN刺激培养,收集12h和72h细胞培养上清,ELISA检测上清Th1和Th2细胞代表性细胞因子IFN-γ、IL-4的浓度;培养72h收集细胞,流式细胞术检测表面分子CD44、CD62L的表达水平;流式细胞术CFSE标记法检测T细胞增殖能力;4.2体内实验:流式细胞术检测LPS腹腔注射6h后各组动物脾脏Th细胞亚群(Th1、Th2、Th17)的胞内细胞因子TNF-α、IFN-γ、IL-4、IL-17A的表达;流式细胞术检测各组动物脾脏CD4+T淋巴细胞表面分子CD25、CD69、CD28、PD-1的表达水平;流式细胞术PE Annexin V和7-AAD标记检测各组动物脾脏T淋巴细胞的凋亡。结果:1.与健康对照(335.6±89.6μg/ml)比较,AP患者血浆FN浓度无明显改变(273.9±116.2μg/ml,P�e0.05)。脓毒症、MODS和SAP患者血浆FN浓度与AP、健康对照比较均显著降低,分别为146.88±53.99μg/ml、139.6±63.66μg/ml、191.7±60.22μg/ml(P0.001)。血浆F N浓度的动态变化可反应MODS、SAP患者的病情,缓解组患者随病程好转血浆FN浓度逐渐升高至241.5±52.14μg/ml,恶化死亡组患者随病情发展血浆FN浓度呈持续性降低至124.7±65.84μg/ml,病程中出现FN浓度低于100μg/m L者,预后极差。2.与健康对照(3.08±2.95μg/ml)比较,AP、SAP、MODS患者血浆CRP浓度均显著升高(P0.01)。AP、SAP和MODS患者血浆CRP浓度分别为352.5±553.2μg/ml、779±1445μg/ml和510±909.2μg/ml,三者之间无显著差异P�e0.05。3.生理情况下血浆IL-6、IL-2、IL-4、IL-10、IL-17A、IFN-γ、TNF-α浓度均低于检测范围(20 pg/ml)。脓毒症、MODS和SAP患者血浆IL-6浓度显著升高(P0.001),但呈非正态分布,个体差异大,分别为460.7±1260.9 pg/ml、992.6±2587 pg/ml、728.4±2125 pg/ml。仅20%的重症感染患者血浆IL-10浓度升高,23%的重症感染患者TNF-α升高,但IL-6、IL-10、TNF-α的动态变化与病情进展无明显关系。IL-2、IL-4、IL-17A、IFN-γ在重症感染患者血浆中也低于检测范围。4.pFN、rhFNHC-36多次尾静脉给药治疗,显著改善D-Gal敏化的LPS诱导的脓毒症模型小鼠72h生存率(48%、38%比0%)。脓毒症小鼠脾脏皮质区明显萎缩,肝细胞发生大面积坏死。pFN、rhFNHC-36治疗均可改善肝、脾组织的病变程度。5.正常小鼠血浆FN浓度为531.8±148.5 pg/ml,脓毒症小鼠血浆FN浓度在LPS腹腔注射后2h即显著减少(284±39.8 pg/ml,P0.05),至24h持续降低至112.25±20.5 pg/ml。pFN、rhFNHC-36治疗均可改善其血浆FN的降低,治疗组24h的血浆FN浓度分别为445.6±36.25 pg/ml、345.5±112.25 pg/ml。6.正常小鼠血浆各炎症因子浓度甚微,脓毒症小鼠血浆MCP-1、IL-6、TNF、IL-10明显升高,均在LPS腹腔注射后2h达到峰值,pFN、rhFNHC-36显著抑制血浆MCP-1、IL-6、TNF的升高,但对血浆IL-10浓度无影响。pFN、rhFNHC-36治疗组较PBS对照组2h血浆浓度分别为MCP-1(5635±449.7 pg/ml、5184±675.9 pg/ml比12649±1126 pg/ml)、IL-6(1981±268 pg/ml、2249±325 pg/ml比7412±444 pg/ml)、TNF(75.63±19.31 pg/ml、84.06±23.52 pg/ml比204.9±43.11pg/ml)。7.pFN、rhFNHC-36显著抑制LPS刺激后4h、16h小鼠腹腔巨噬细胞M1相关分子IL-1β、IL-6、TNF-α、IFN-γ、i NOS、IL-12基因表达,抑制16hHMGB-1基因表达;促进M2相关分子Argnase-1、IL-10的基因表达。8.脓毒症小鼠LPS腹腔注射后6h,脾脏即出现Th1、Th17亚群比例减少和Th2亚群比例增加,正常小鼠和脓毒症小鼠脾脏各群细胞占CD4+T细胞的比例分别为CD4+-TNF-α+(54.9±7.2%比35.4±7.3%)、CD4+-IFN-γ+(6.14±0.89%比1.56±0.75%)、CD4+-IL-17+(1.42±0.22%比0.53±0.17%)、CD4+-IL-4+(1.92±0.18%比3.83±0.5%)。pFN、rhFNHC-36治疗可部分恢复TNF-α+、IFN-γ+、IL-17+Th细胞,减少IL-4+Th细胞比例,两治疗组细胞比例分别为TNF-α+Th(44±3.7%、46±4.36%)、IFN-γ+Th(3.65±0.27%、3.84±0.89%)、IL-17+Th(1.1±0.28%、1.03±0.22%),IL-4+Th(2.83±0.35%、2.43±0.21%)。9.脓毒症小鼠Th细胞CD28表达降低,PD-1表达增强,出现以CD4+T细胞为主的淋巴细胞凋亡。pFN、rhFNHC-36治疗组与PBS对照组比较,CD4+T细胞中CD28阳性细胞增多(71.3±3.25%、74.2±2.69%比53.6±3.25%,P0.05),PD-1阳性细胞减少(15.84±0.52%、15.57±0.62%比20.26±1.61%,P0.05),rhFNHC-36治疗组CD4+T细胞凋亡减少(10.7%比20.2%)。10.分离正常小鼠CD4+T细胞,体外用抗CD3/CD28单抗共刺激72h,上清IL-4浓度为706.9±115.5 pg/ml,IFN-γ浓度为1846.4±301.4 pg/ml。脓毒症小鼠CD4+T细胞体外用抗CD3/CD28单抗共刺激72h,上清IL-4浓度显著高于正常对照(1814.4±132.82 pg/ml,P0.01),IFN-γ浓度明显低于正常小鼠(395.6±139.1pg/ml,P0.01);加入pFN、rhFNHC-36的共刺激培养可剂量依赖性地降低脓毒症小鼠上清IL-4浓度,恢复上清IFN-γ的浓度。0.5 mg/ml pFN和rhFNHC-36作用组72h细胞培养上清IL-4的浓度分别为997.42±137.5 pg/ml、1019.7±125.5 pg/ml,IFN-γ浓度分别为1223.6±190.3 pg/ml、1105.7±220.7 pg/ml。11.脓毒症小鼠淋巴细胞体外用抗CD3/CD28单抗共刺激后的增殖能力比正常小鼠显著降低(39.8±5.06%比66±15.4%,P0.05),CD44+CD62L-效应T细胞比例比正常小鼠显著减少(84.3%比26.2%,P0.01);加入pFN、rhFNHC-36共培养可部分恢复T淋巴细胞的增殖活性(54±14.3%,56.4±15.0%),增加CD44+CD62L-T细胞比例(50.3%、48.2%)。结论1.与AP、健康对照比较,SAP、脓毒症、MODS等重症感染患者血浆FN浓度显著降低。SAP和MODS患者血浆FN浓度的动态变化可反映病情进展,恶化或死亡患者血浆FN浓度呈持续降低趋势,缓解者血浆FN浓度逐渐升高。病程中出现FN浓度低于100μg/ml者,预后极差。FN可作为区分普通炎症和重症感染的诊断指标和反映病情变化的监测指标。2.AP、SAP、脓毒症、MODS患者血浆CRP浓度均显著升高,各感染组间无差异,CRP不能区分普通炎症和重症感染。SAP、脓毒症、MODS等重症感染患者血浆IL-6浓度升高,但个体差异大,IL-6的浓度和变化提示机体炎症反应情况,在与病情转归的研究中,FN优于IL-6。仅部分感染患者血浆IL-10、TNF-α浓度升高。3.pFN和rhFNHC-36多肽对D-Gal敏化LPS诱导的脓毒症小鼠均具有保护作用。pFN和rhFNHC-36减轻脓毒症小鼠肝脾组织坏死,控制血浆FN浓度降低和MCP-1、IL-6、TNF等炎症因子水平升高。pFN和rhFNHC-36抑制LPS诱导的小鼠腹腔巨噬细胞向M1亚群极化,促进其向M2亚群分化。4.pFN和rhFNHC-36促进脓毒症小鼠Th细胞向Th1、Th17亚群分化,抑制其向Th2亚群偏移;促进Th细胞CD28分子表达,抑制PD-1分子表达,减少Th细胞凋亡。rhFNHC-36促进脓毒症小鼠Th细胞对抗CD3/CD28抗体体外刺激的反应性,促进其增殖、分化和分泌IFN-γ。
[Abstract]:Objective: analysis of 1. patients with severe acute pancreatitis (SAP), sepsis, multiple organ dysfunction (MODS) and other severe infections in patients with disease time of plasma fibronectin (pFN), changes of Th1/Th2/Th17 cytokine levels, in order to find the early diagnosis and accurate judgment of disease severity and prognosis of severe infection in 2. clinical indicators; D-galactosamine (D-GaL) sensitized by lipopolysaccharide (LPS) induced mouse model of sepsis, observation of human plasma fibronectin (pFN) and recombinant human FN C-terminal heparin binding domain polypeptide (rhFNHC-36) and the protective effect on septic mice inflammatory reaction and immune function in particular influence on the function of CD4+T lymphocyte thus, the effects of pFN, rhFNHC-36 on the protective mechanism of sepsis animal, for severe infection of new therapeutic strategies to provide experimental basis and ideas. Methods: 1. of 34 cases of SAP, 20 cases of sepsis, 45 cases of MODS patients The clinical diagnosis of the disease of plasma samples and 15 cases of common acute pancreatitis (AP) and 46 healthy controls by plasma flow liquid multiple protein quantification (CBA) detection of plasma Th1/Th2/Th17 cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN- y, TNF- a) the concentration of plasma FN; ELISA the concentration of CRP, analysis of patients with plasma FN and cytokines and AP of severe infection, healthy controls. The difference between the 49 cases of SAP patients with different prognosis and MODS course of collecting multiple time points in plasma samples, the dynamic changes of FN and cytokine analysis and condition; 2. to establish a stable D-GaL sensitization induced by LPS the BALB/C mouse model of sepsis; 3. to observe the pFN protective effect of rhFNHC-36 on sepsis model mice and its effect on the inflammatory reaction in 3.1 pFN, rhFNHC-36 multiple dosing intravenous injection, were observed and recorded the condition and the survival rate for 72 hours. Experimental groups: normal control group, sepsis PBS group (intravenous injection PBS), sepsis rhFNHC-36 group, sepsis pFN group; observed 3.2 HE staining of tissue and organ (liver, spleen, lung inflammation) was collected. 2h, 4h, 8h, 24h, 72h in plasma ELISA, detection of FN, IL-4 concentration, flow liquid multiple protein quantification (CBA) detection of inflammatory cytokine of IL-1, IL-6, IL-12p70, MCP-1, IFN- gamma, TNF concentration; 3.3 isolated from normal mouse peritoneal macrophages, respectively by LPS+rhFNHC-36, 4H and 16h were co cultured with LPS+pFN, Real-time PCR detection of mouse peritoneal macrophages M1 molecular (I NOS, IL-1 IL-6, IL-12p40, IFN-, beta, gamma, HMGB1, TNF alpha) and M2 related molecules (Argnase-1, IL-10) m RNA expression level of pFN; 4., the effect of rhFNHC-36 on sepsis model mice helper T lymphocyte cell 4.1 in vitro: spleen C immunomagnetic separation of sepsis mice and normal mice D4+T lymphocytes, with anti CD3/CD28 monoclonal antibody and different concentrations of rhFNHC-36 or pFN stimulated 12h and collected the culture supernatant of 72h cells, Th2 cells and ELISA was determined by Th1 representative cytokines IFN-, IL-4 concentration; cultured 72h cells were collected and detected the surface molecules of CD44 by flow cytometry, the expression level of CD62L T; cell proliferation the ability of flow cytometry CFSE labeling; 4.2 in vivo experiment: flow cytometry after intraperitoneal injection of LPS 6h animal spleen Th cell subsets (Th1, Th2, Th17) intracellular cytokines TNF-, IFN- gamma, IL-4, IL-17A expression was detected; animal spleen CD4+T lymphocyte surface molecules CD25 flow cytometry, CD69, CD28, PD-1 expression levels of apoptosis; flow cytometry PE Annexin V and 7-AAD markers to detect animal splenic T lymphocytes. Results: 1. healthy controls (335.6 + 89.6 g/ml) compared with AP plasma FN concentration 鏄庢樉鏀瑰彉(273.9卤116.2渭g/ml,P?0.05).鑴撴瘨鐥，

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