本文關(guān)鍵詞：纖維連接蛋白及其肝素結(jié)合域多肽對(duì)重癥感染的作用和免疫機(jī)制　出處：《福建醫(yī)科大學(xué)》2015年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 重癥感染 纖維連接蛋白 膿毒癥動(dòng)物模型 T淋巴細(xì)胞 巨噬細(xì)胞 細(xì)胞因子

【摘要】：目的:1.分析重癥急性胰腺炎(SAP)、膿毒癥、多器官功能障礙(MODS)等重癥感染患者病程多時(shí)間點(diǎn)血漿纖維連接蛋白(pFN)、Th1/Th2/Th17細(xì)胞因子水平的變化,以期尋找早期診斷和準(zhǔn)確判斷重癥感染患者疾病嚴(yán)重程度和預(yù)后的臨床指標(biāo);2.以半乳糖胺(D-GaL)敏化的內(nèi)毒素(LPS)誘導(dǎo)的小鼠膿毒癥模型,觀察人血漿纖維連接蛋白(pFN)和重組人FN羧基端肝素結(jié)合域多肽(rhFNHC-36)的保護(hù)作用,研究其對(duì)膿毒癥小鼠的炎癥反應(yīng)和免疫功能尤其對(duì)CD4+T淋巴細(xì)胞功能的影響,從而探討pFN、rhFNHC-36對(duì)膿毒癥動(dòng)物的保護(hù)機(jī)制,為重癥感染治療新策略提供實(shí)驗(yàn)基礎(chǔ)和思路。方法:1.收集34例SAP、20例膿毒癥、45例MODS患者患病臨床確診時(shí)的血漿樣品和15例普通胰腺炎(AP)、46例健康對(duì)照者血漿,用流式液相多重蛋白定量技術(shù)(CBA)檢測(cè)血漿Th1/Th2/Th17細(xì)胞因子(IL-2、IL-4、IL-6、IL-10、IL-17A、IFN-γ、TNF-α)的濃度;ELISA檢測(cè)血漿FN、CRP濃度,分析各類(lèi)重癥感染患者血漿FN和細(xì)胞因子與AP、健康對(duì)照的差別。其中49例不同轉(zhuǎn)歸的SAP和MODS患者收集病程多個(gè)時(shí)間點(diǎn)血漿樣品,分析FN和細(xì)胞因子的動(dòng)態(tài)變化與病情進(jìn)展的關(guān)系;2.建立穩(wěn)定的D-GaL敏化LPS誘導(dǎo)的BALB/C小鼠膿毒癥模型;3.觀察pFN、rhFNHC-36對(duì)膿毒癥模型小鼠的保護(hù)作用及其對(duì)炎癥反應(yīng)的影響3.1以pFN、rhFNHC-36多次尾靜脈注射給藥的治療方案,觀察和記錄小鼠的狀況和72小時(shí)存活率。實(shí)驗(yàn)分組:正常對(duì)照組,膿毒癥PBS對(duì)照組(尾靜脈注射PBS),膿毒癥rhFNHC-36治療組,膿毒癥pFN治療組;3.2 HE染色觀察各組組織器官(肝、脾、肺)的炎癥情況。收集各組2h、4h、8h、24h、72h血漿,ELISA檢測(cè)FN、IL-4濃度,流式液相多重蛋白定量技術(shù)(CBA)檢測(cè)炎癥因子IL-1β、IL-6、IL-12p70、IFN-γ、MCP-1、TNF的濃度;3.3分離正常小鼠腹腔巨噬細(xì)胞,分別以LPS+rhFNHC-36、LPS+pFN共培養(yǎng)4h和16h,Real-time PCR檢測(cè)小鼠腹腔巨噬細(xì)胞M1相關(guān)分子(i NOS、IL-1β、IL-6、IL-12p40、IFN-γ、HMGB1、TNFα)和M2相關(guān)分子(Argnase-1、IL-10)m RNA表達(dá)水平;4.探討pFN、rhFNHC-36對(duì)膿毒癥模型小鼠輔助性T淋巴細(xì)胞功能的影響4.1體外實(shí)驗(yàn):免疫磁珠分選膿毒癥小鼠和正常小鼠脾臟CD4+T淋巴細(xì)胞,以抗CD3/CD28單抗和不同濃度rhFNHC-36或pFN刺激培養(yǎng),收集12h和72h細(xì)胞培養(yǎng)上清,ELISA檢測(cè)上清Th1和Th2細(xì)胞代表性細(xì)胞因子IFN-γ、IL-4的濃度;培養(yǎng)72h收集細(xì)胞,流式細(xì)胞術(shù)檢測(cè)表面分子CD44、CD62L的表達(dá)水平;流式細(xì)胞術(shù)CFSE標(biāo)記法檢測(cè)T細(xì)胞增殖能力;4.2體內(nèi)實(shí)驗(yàn):流式細(xì)胞術(shù)檢測(cè)LPS腹腔注射6h后各組動(dòng)物脾臟Th細(xì)胞亞群(Th1、Th2、Th17)的胞內(nèi)細(xì)胞因子TNF-α、IFN-γ、IL-4、IL-17A的表達(dá);流式細(xì)胞術(shù)檢測(cè)各組動(dòng)物脾臟CD4+T淋巴細(xì)胞表面分子CD25、CD69、CD28、PD-1的表達(dá)水平;流式細(xì)胞術(shù)PE Annexin V和7-AAD標(biāo)記檢測(cè)各組動(dòng)物脾臟T淋巴細(xì)胞的凋亡。結(jié)果:1.與健康對(duì)照(335.6±89.6μg/ml)比較,AP患者血漿FN濃度無(wú)明顯改變(273.9±116.2μg/ml,P㧐0.05)。膿毒癥、MODS和SAP患者血漿FN濃度與AP、健康對(duì)照比較均顯著降低,分別為146.88±53.99μg/ml、139.6±63.66μg/ml、191.7±60.22μg/ml(P0.001)。血漿F N濃度的動(dòng)態(tài)變化可反應(yīng)MODS、SAP患者的病情,緩解組患者隨病程好轉(zhuǎn)血漿FN濃度逐漸升高至241.5±52.14μg/ml,惡化死亡組患者隨病情發(fā)展血漿FN濃度呈持續(xù)性降低至124.7±65.84μg/ml,病程中出現(xiàn)FN濃度低于100μg/m L者,預(yù)后極差。2.與健康對(duì)照(3.08±2.95μg/ml)比較,AP、SAP、MODS患者血漿CRP濃度均顯著升高(P0.01)。AP、SAP和MODS患者血漿CRP濃度分別為352.5±553.2μg/ml、779±1445μg/ml和510±909.2μg/ml,三者之間無(wú)顯著差異P㧐0.05。3.生理情況下血漿IL-6、IL-2、IL-4、IL-10、IL-17A、IFN-γ、TNF-α濃度均低于檢測(cè)范圍(20 pg/ml)。膿毒癥、MODS和SAP患者血漿IL-6濃度顯著升高(P0.001),但呈非正態(tài)分布,個(gè)體差異大,分別為460.7±1260.9 pg/ml、992.6±2587 pg/ml、728.4±2125 pg/ml。僅20%的重癥感染患者血漿IL-10濃度升高,23%的重癥感染患者TNF-α升高,但I(xiàn)L-6、IL-10、TNF-α的動(dòng)態(tài)變化與病情進(jìn)展無(wú)明顯關(guān)系。IL-2、IL-4、IL-17A、IFN-γ在重癥感染患者血漿中也低于檢測(cè)范圍。4.pFN、rhFNHC-36多次尾靜脈給藥治療,顯著改善D-Gal敏化的LPS誘導(dǎo)的膿毒癥模型小鼠72h生存率(48%、38%比0%)。膿毒癥小鼠脾臟皮質(zhì)區(qū)明顯萎縮,肝細(xì)胞發(fā)生大面積壞死。pFN、rhFNHC-36治療均可改善肝、脾組織的病變程度。5.正常小鼠血漿FN濃度為531.8±148.5 pg/ml,膿毒癥小鼠血漿FN濃度在LPS腹腔注射后2h即顯著減少(284±39.8 pg/ml,P0.05),至24h持續(xù)降低至112.25±20.5 pg/ml。pFN、rhFNHC-36治療均可改善其血漿FN的降低,治療組24h的血漿FN濃度分別為445.6±36.25 pg/ml、345.5±112.25 pg/ml。6.正常小鼠血漿各炎癥因子濃度甚微,膿毒癥小鼠血漿MCP-1、IL-6、TNF、IL-10明顯升高,均在LPS腹腔注射后2h達(dá)到峰值,pFN、rhFNHC-36顯著抑制血漿MCP-1、IL-6、TNF的升高,但對(duì)血漿IL-10濃度無(wú)影響。pFN、rhFNHC-36治療組較PBS對(duì)照組2h血漿濃度分別為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顯著抑制LPS刺激后4h、16h小鼠腹腔巨噬細(xì)胞M1相關(guān)分子IL-1β、IL-6、TNF-α、IFN-γ、i NOS、IL-12基因表達(dá),抑制16hHMGB-1基因表達(dá);促進(jìn)M2相關(guān)分子Argnase-1、IL-10的基因表達(dá)。8.膿毒癥小鼠LPS腹腔注射后6h,脾臟即出現(xiàn)Th1、Th17亞群比例減少和Th2亞群比例增加,正常小鼠和膿毒癥小鼠脾臟各群細(xì)胞占CD4+T細(xì)胞的比例分別為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治療可部分恢復(fù)TNF-α+、IFN-γ+、IL-17+Th細(xì)胞,減少I(mǎi)L-4+Th細(xì)胞比例,兩治療組細(xì)胞比例分別為T(mén)NF-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.膿毒癥小鼠Th細(xì)胞CD28表達(dá)降低,PD-1表達(dá)增強(qiáng),出現(xiàn)以CD4+T細(xì)胞為主的淋巴細(xì)胞凋亡。pFN、rhFNHC-36治療組與PBS對(duì)照組比較,CD4+T細(xì)胞中CD28陽(yáng)性細(xì)胞增多(71.3±3.25%、74.2±2.69%比53.6±3.25%,P0.05),PD-1陽(yáng)性細(xì)胞減少(15.84±0.52%、15.57±0.62%比20.26±1.61%,P0.05),rhFNHC-36治療組CD4+T細(xì)胞凋亡減少(10.7%比20.2%)。10.分離正常小鼠CD4+T細(xì)胞,體外用抗CD3/CD28單抗共刺激72h,上清IL-4濃度為706.9±115.5 pg/ml,IFN-γ濃度為1846.4±301.4 pg/ml。膿毒癥小鼠CD4+T細(xì)胞體外用抗CD3/CD28單抗共刺激72h,上清IL-4濃度顯著高于正常對(duì)照(1814.4±132.82 pg/ml,P0.01),IFN-γ濃度明顯低于正常小鼠(395.6±139.1pg/ml,P0.01);加入pFN、rhFNHC-36的共刺激培養(yǎng)可劑量依賴(lài)性地降低膿毒癥小鼠上清IL-4濃度,恢復(fù)上清IFN-γ的濃度。0.5 mg/ml pFN和rhFNHC-36作用組72h細(xì)胞培養(yǎng)上清IL-4的濃度分別為997.42±137.5 pg/ml、1019.7±125.5 pg/ml,IFN-γ濃度分別為1223.6±190.3 pg/ml、1105.7±220.7 pg/ml。11.膿毒癥小鼠淋巴細(xì)胞體外用抗CD3/CD28單抗共刺激后的增殖能力比正常小鼠顯著降低(39.8±5.06%比66±15.4%,P0.05),CD44+CD62L-效應(yīng)T細(xì)胞比例比正常小鼠顯著減少(84.3%比26.2%,P0.01);加入pFN、rhFNHC-36共培養(yǎng)可部分恢復(fù)T淋巴細(xì)胞的增殖活性(54±14.3%,56.4±15.0%),增加CD44+CD62L-T細(xì)胞比例(50.3%、48.2%)。結(jié)論1.與AP、健康對(duì)照比較,SAP、膿毒癥、MODS等重癥感染患者血漿FN濃度顯著降低。SAP和MODS患者血漿FN濃度的動(dòng)態(tài)變化可反映病情進(jìn)展,惡化或死亡患者血漿FN濃度呈持續(xù)降低趨勢(shì),緩解者血漿FN濃度逐漸升高。病程中出現(xiàn)FN濃度低于100μg/ml者,預(yù)后極差。FN可作為區(qū)分普通炎癥和重癥感染的診斷指標(biāo)和反映病情變化的監(jiān)測(cè)指標(biāo)。2.AP、SAP、膿毒癥、MODS患者血漿CRP濃度均顯著升高,各感染組間無(wú)差異,CRP不能區(qū)分普通炎癥和重癥感染。SAP、膿毒癥、MODS等重癥感染患者血漿IL-6濃度升高,但個(gè)體差異大,IL-6的濃度和變化提示機(jī)體炎癥反應(yīng)情況,在與病情轉(zhuǎn)歸的研究中,FN優(yōu)于IL-6。僅部分感染患者血漿IL-10、TNF-α濃度升高。3.pFN和rhFNHC-36多肽對(duì)D-Gal敏化LPS誘導(dǎo)的膿毒癥小鼠均具有保護(hù)作用。pFN和rhFNHC-36減輕膿毒癥小鼠肝脾組織壞死,控制血漿FN濃度降低和MCP-1、IL-6、TNF等炎癥因子水平升高。pFN和rhFNHC-36抑制LPS誘導(dǎo)的小鼠腹腔巨噬細(xì)胞向M1亞群極化,促進(jìn)其向M2亞群分化。4.pFN和rhFNHC-36促進(jìn)膿毒癥小鼠Th細(xì)胞向Th1、Th17亞群分化,抑制其向Th2亞群偏移;促進(jìn)Th細(xì)胞CD28分子表達(dá),抑制PD-1分子表達(dá),減少Th細(xì)胞凋亡。rhFNHC-36促進(jìn)膿毒癥小鼠Th細(xì)胞對(duì)抗CD3/CD28抗體體外刺激的反應(yīng)性,促進(jìn)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鹵116.2渭g/ml,P?0.05).鑴?chuàng)姣掔棧?

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