張楠楠　楊淑殷　陳柳瑩　尹　珊　王十錦　劉三?！⊥踺磔?3　汪　錚　李　海#

上海交通大學(xué)醫(yī)學(xué)院附屬仁濟(jì)醫(yī)院消化科　上海市消化疾病研究所1(200001) 首都醫(yī)科大學(xué)附屬北京地壇醫(yī)院傳染病研究所2　新發(fā)突發(fā)傳染病研究北京市重點(diǎn)實(shí)驗(yàn)室3

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·論著·

ConA重復(fù)給藥建立ACLF免疫狀態(tài)動(dòng)態(tài)轉(zhuǎn)變的小鼠模型*

張楠楠1楊淑殷1陳柳瑩1尹珊1王十錦1劉三海2王蓓蓓2,3汪錚1李海1#

上海交通大學(xué)醫(yī)學(xué)院附屬仁濟(jì)醫(yī)院消化科上海市消化疾病研究所1(200001) 首都醫(yī)科大學(xué)附屬北京地壇醫(yī)院傳染病研究所2新發(fā)突發(fā)傳染病研究北京市重點(diǎn)實(shí)驗(yàn)室3

背景：慢加急性肝衰竭(ACLF)是我國(guó)常見的肝衰竭類型，目前尚缺乏能有效模擬ACLF免疫狀態(tài)動(dòng)態(tài)轉(zhuǎn)變的動(dòng)物模型。目的：通過刀豆球蛋白A(ConA)重復(fù)給藥，建立模擬ACLF免疫狀態(tài)動(dòng)態(tài)轉(zhuǎn)變的動(dòng)物模型。方法：小鼠隨機(jī)分為對(duì)照組和ConA重復(fù)給藥組，ConA重復(fù)給藥組小鼠給予球后內(nèi)眥靜脈叢注射ConA 15 mg/kg，每隔48 h一次，共5次，對(duì)照組給予等體積0.9%NaCl溶液。CBA法檢測(cè)外周血IL-6、IL-10、IL-12、TNF-α、IFN-γ、MCP-1水平，并測(cè)定IL-10/TNF-α比值；流式細(xì)胞術(shù)檢測(cè)外周血中單核細(xì)胞HLA-DR表達(dá)、CD4+T細(xì)胞數(shù)量及其比例以及PD-1表達(dá)。結(jié)果：隨著給藥次數(shù)增加，ConA重復(fù)給藥組小鼠外周血細(xì)胞因子從促炎細(xì)胞因子為主轉(zhuǎn)變成抗炎細(xì)胞因子為主。與對(duì)照組相比，ConA重復(fù)給藥組外周血中單核細(xì)胞HLA-DR表達(dá)下降(P<0.05)；CD4+T細(xì)胞數(shù)量和比例下降(P<0.05)，PD-1表達(dá)上調(diào)(P<0.05)。結(jié)論：本研究通過ConA重復(fù)刺激成功建立了模擬ACLF免疫狀態(tài)從全身炎癥反應(yīng)綜合征(SIRS)到代償性抗炎反應(yīng)綜合征(CARS)動(dòng)態(tài)轉(zhuǎn)變的動(dòng)物模型。

關(guān)鍵詞慢加急性肝衰竭；伴刀豆球蛋白A；免疫狀態(tài)；細(xì)胞因子類；模型，動(dòng)物

Establishment of Model of Dynamic Change of Immune Status of ACLF Induced by ConA Repeated Administration in MiceZHANGNannan1,YANGShuyin1,CHENLiuying1,YINShan1,WANGShijin1,LIUSanhai2,WANGBeibei2,3,WANGZheng1,LIHai1.1DivisionofGastroenterologyandHepatology,RenJiHospital,SchoolofMedicine,ShanghaiJiaoTongUniversity;ShanghaiInstituteofDigestiveDisease,Shanghai(200001);2InstituteofInfectiousDiseases,BeijingDitanHospital,CapitalMedicalUniversity,Beijing;3BeijingKeyLaboratoryofEmergingInfectiousDiseases,Beijing

Correspondence to: LI Hai, Email: haili_17@126.com

慢加急性肝衰竭(ACLF)是臨床常見的肝衰竭類型，晚期常合并多臟器功能衰竭，死亡率可高達(dá)30%～50%[1]。東方ACLF的病因以乙型肝炎感染為主，西方則以酒精性肝炎為基礎(chǔ)。ACLF的急性誘因包括感染、過度飲酒、外科手術(shù)、肝臟毒性藥物等[2-4]。盡管東西方ACLF的病因和急性誘因不一，但引起機(jī)體免疫應(yīng)答和免疫演變的過程大致相同，主要表現(xiàn)為急性肝損傷誘因?qū)е碌臋C(jī)體早期免疫亢進(jìn)即全身炎癥反應(yīng)綜合征(SIRS)，逐漸演變?yōu)橐钥寡准?xì)胞因子白細(xì)胞介素(IL)-10為主的代償性抗炎反應(yīng)綜合征(CARS)[4-8]。

2014年世界胃腸病組織工作小組提出了ACLF的“優(yōu)先和次優(yōu)先”研究主題，其中發(fā)展有效的動(dòng)物實(shí)驗(yàn)?zāi)Ｐ鸵陨钊胙芯緼CLF病理生理機(jī)制和研制可能的治療藥物作為三大優(yōu)先主題之一，因此建立模擬ACLF動(dòng)態(tài)免疫演變的動(dòng)物模型是深入闡明該疾病的關(guān)鍵切入點(diǎn)。本研究通過給予小鼠刀豆球蛋白A(ConA)重復(fù)刺激，旨在建立反復(fù)急性肝損傷誘導(dǎo)的ACLF免疫狀態(tài)動(dòng)態(tài)演變的動(dòng)物模型。

材料與方法

一、實(shí)驗(yàn)動(dòng)物、主要試劑和儀器

1. 實(shí)驗(yàn)動(dòng)物：SPF級(jí)6～8周齡C57BL/6雄性小鼠20只，體質(zhì)量20～23 g，購于中國(guó)醫(yī)學(xué)科學(xué)院醫(yī)學(xué)實(shí)驗(yàn)動(dòng)物研究所。給予小鼠清潔飲食，晝夜規(guī)律光照。

2. 主要試劑和儀器：ConA購于美國(guó)Sigma公司；低溫多用途離心機(jī)購于德國(guó)Thermo公司；小鼠CBA試劑盒、抗小鼠CD45-FITC抗體、抗小鼠NK1.1-PE、抗小鼠CD3-PerCP抗體、抗小鼠CD4-APC抗體、抗小鼠CD11b-FITC抗體、抗小鼠Gr-1 PE抗體、抗小鼠MHC-Ⅱ-PerCP抗體、抗小鼠CD48-APC、抗小鼠PD-1-FITC抗體、絕對(duì)細(xì)胞計(jì)數(shù)管、流式細(xì)胞儀(BD FACS Calibur)購于美國(guó)BD公司。

二、實(shí)驗(yàn)方法

1. 模型制備：小鼠隨機(jī)分為對(duì)照組和ConA重復(fù)給藥組，每組各10只。ConA重復(fù)給藥組小鼠給予球后內(nèi)眥靜脈叢注射ConA 15 mg/kg，每隔48 h一次，共5次，建立ConA重復(fù)給藥模型。對(duì)照組給予等體積0.9% NaCl溶液。

2. 細(xì)胞因子的檢測(cè)：每次給予ConA或0.9% NaCl溶液后3 h眼球取血，2 000 r/min離心 20 min，分離血清。根據(jù)CBA試劑盒說明書，通過流式細(xì)胞儀檢測(cè)IL-6、IL-10、IL-12、TNF-α、IFN-γ、單核細(xì)胞趨化蛋白(MCP)-1水平。

3. 單核細(xì)胞HLA-DR表達(dá)、CD4+T細(xì)胞數(shù)量和程序性死亡受體(PD)-1表達(dá)的檢測(cè)：ConA末次刺激后48 h取小鼠外周血，制成單細(xì)胞懸液，應(yīng)用流式細(xì)胞儀檢測(cè)單核細(xì)胞HLA-DR表達(dá)、CD4+T細(xì)胞數(shù)量和PD-1表達(dá)。

三、統(tǒng)計(jì)學(xué)分析

結(jié)果

一、促炎/抑炎細(xì)胞因子的動(dòng)態(tài)變化過程

第1次ConA刺激后，促炎細(xì)胞因子TNF-α(P<0.000 1)水平、IL-12(P=0.015 2)、IFN-γ(P=0.001 8)、MCP-1(P<0.000 1)、IL-6(P=0.000 2)較刺激前顯著升高，抗炎細(xì)胞因子IL-10水平顯著升高(P=0.002 3)；第5次ConA刺激后，TNF-α、IL-12、IFN-γ、MCP-1、IL-6水平均由峰值明顯降低，而IL-10繼續(xù)維持高水平(圖1A)。給予ConA刺激后，IL-10/TNF-α比值持續(xù)升高(圖1B)。說明隨著ConA刺激次數(shù)增加，小鼠的主導(dǎo)細(xì)胞因子由第1次ConA刺激后以促炎細(xì)胞因子為主導(dǎo)的SIRS轉(zhuǎn)變?yōu)榈?次ConA刺激后以抑炎細(xì)胞因子IL-10為主導(dǎo)的CARS階段。

二、HLA-DR表達(dá)

ConA第5次給藥后，外周血單核細(xì)胞HLA-DR表達(dá)較對(duì)照組明顯下降(平均熒光強(qiáng)度：3.56對(duì)11.76)，差異有統(tǒng)計(jì)學(xué)意義(P<0.000 1)(圖2、圖3)。說明ConA重復(fù)刺激后機(jī)體處于先天性免疫系統(tǒng)受損狀態(tài)。

三、外周血CD4+T細(xì)胞數(shù)量和PD-1表達(dá)

與對(duì)照組相比，ConA第5次刺激后，小鼠外周血CD4+T細(xì)胞數(shù)量明顯下降[(1 417±1 386)/mL對(duì)(1 986±323)/mL，P=0.350 5]，CD4+T細(xì)胞比例亦明顯下降(33.69%對(duì)59.97%，P<0.000 1)(圖3)。說 明ConA重復(fù)刺激后小鼠適應(yīng)性免疫系統(tǒng)受損。

A：外周血細(xì)胞因子水平；B：IL-10/TNF-α比值

圖2　流式細(xì)胞術(shù)檢測(cè)外周血HLA-DR和PD-1表達(dá)

圖3　外周血HLA-DR表達(dá)、CD4+T細(xì)胞比例、數(shù)量和PD-1表達(dá)

與對(duì)照組相比，ConA重復(fù)給藥組小鼠的外周血CD4+T細(xì)胞表面PD-1表達(dá)明顯上調(diào)(平均熒光強(qiáng)度：6.90對(duì)4.79，P<0.000 1)(圖2、圖3)，表明具有正向免疫調(diào)節(jié)功能的CD4+T細(xì)胞受損。

討論

已有研究表明免疫狀態(tài)的相互轉(zhuǎn)變?cè)贏CLF發(fā)病和轉(zhuǎn)歸中起有重要作用[4-8]。ACLF的免疫狀態(tài)可分為SIRS和隨后的CARS[8]。外周血細(xì)胞因子是反映機(jī)體免疫狀態(tài)的重要指標(biāo)，SIRS主要表現(xiàn)為外周血促炎細(xì)胞因子(TNF-α等)明顯升高且占主導(dǎo)地位的細(xì)胞因子風(fēng)暴；CARS主要表現(xiàn)為促炎細(xì)胞因子明顯降低而抗炎細(xì)胞因子(IL-10等)分泌增加并占主導(dǎo)地位[9-11]。

本研究中，ConA首次給藥后，小鼠外周血促炎細(xì)胞因子TNF-α、IFN-γ、MCP-1、IL-6急劇升高，隨著給藥次數(shù)增加，促炎細(xì)胞因子水平逐漸下降，末次給藥時(shí)基本處于正常范圍；但I(xiàn)L-12水平變化不明顯。抗炎細(xì)胞因子IL-10在首次給藥輕微升高，第2次給藥后繼續(xù)升高并達(dá)到峰值，隨這給藥次數(shù)增加，IL-10仍維持較高水平。細(xì)胞因子的變化說明首次給藥后小鼠處于免疫亢進(jìn)狀態(tài)(SIRS)，即促炎細(xì)胞因子急劇升高，而抗炎細(xì)胞因子處于較低水平；第3次ConA刺激后，促炎細(xì)胞因子水平逐漸下降而抗炎細(xì)胞因子維持較高水平，小鼠呈現(xiàn)免疫耐受狀態(tài)(CARS)。

ConA是一種植物來源的凝集素，對(duì)肝臟細(xì)胞具有特異性毒性作用[12-13]。ConA單次給藥能引起肝臟大面積壞死，外周血促炎細(xì)胞因子(TNF-α、IFN-γ)顯著升高，機(jī)體處于免疫亢進(jìn)狀態(tài)(SIRS)[14-15]，同時(shí)單核細(xì)胞HLA-DR表達(dá)升高[16-17]。

CARS主要表現(xiàn)為促炎細(xì)胞因子(TNF-α等)明顯降低而抗炎細(xì)胞因子(IL-10等)分泌增加并占主導(dǎo)地位。據(jù)文獻(xiàn)報(bào)道，ACLF患者的免疫狀態(tài)以CARS狀態(tài)為主，持續(xù)CARS狀態(tài)是導(dǎo)致ACLF患者死亡的主要原因[4,18-19]。ACLF的免疫抑制主要表現(xiàn)為先天性和適應(yīng)性免疫系統(tǒng)功能抑制[8,20-24]，因此本研究選擇HLA-DR反映先天性免疫系統(tǒng)在免疫抑制時(shí)單核細(xì)胞呈遞抗原的能力，同時(shí)選擇CD4+T細(xì)胞反映免疫抑制時(shí)適應(yīng)性免疫系統(tǒng)功能的變化。文獻(xiàn)[25-27]報(bào)道，PD-1(CD279)是一種重要的免疫抑制分子，免疫抑制時(shí)CD4+T細(xì)胞通過PD-1介導(dǎo)其凋亡，誘導(dǎo)外周免疫耐受。本研究結(jié)果表明ConA 5次刺激小鼠后，HLA-DR表達(dá)明顯降低，外周血CD4+T細(xì)胞數(shù)量和比例均明顯降低，PD-1表達(dá)明顯升高。說明ConA 5次刺激后機(jī)體免疫系統(tǒng)處于抑制狀態(tài)，即發(fā)生了先天性和適應(yīng)性免疫系統(tǒng)的功能障礙。

綜上所述，本研究通過連續(xù)ConA重復(fù)刺激成功建立了ACLF免疫狀態(tài)動(dòng)態(tài)演變的動(dòng)物模型，對(duì)探索ACLF的病理生理過程、篩選有效的治療藥物以及研究新的有效治療方法等具有重要意義。

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(2015-12-14收稿；2016-01-26修回)

DOI:10.3969/j.issn.1008-7125.2016.06.002

Background: Acute-on-chronic liver failure (ACLF) is a commonly seen liver failure in China, and lacking an animal model that can effectively simulate the dynamic change of immune status of ACLF. Aims: To establish an animal model that can simulate dynamic change of immune status of ACLF by repeated administration of concanavalin A (ConA). Methods: Mice were randomly divided into normal control group and ConA repeated administration group. Mice in ConA repeated administration group were injected with ConA 15 mg/kg through retrobulbar angular vein every 48 hours for 5 times, and mice in control group were injected with same volume of 0.9% NaCl solution. Serum levels of IL-6, IL-10, IL-12, TNF-α, IFN-γ, MCP-1 in peripheral blood were assessed by CBA assay, and the ratio of IL-10/TNF-α was calculated. The expression of HLA-DR, number and proportion of CD4+T cells and the expression of PD-1 of monocytes in peripheral blood were detected by flow cytometry. Results: Peripheral blood cytokines changed from predominated proinflammatory cytokines into predominated anti-inflammatory cytokines with the increasing in time of administration in ConA repeated administration group. Compared with control group, HLA-DR expression of monocytes in peripheral blood was significantly decreased (P<0.05), number and proportion of CD4+T cells were significantly decreased (P<0.05), and PD-1 expression was significantly increased (P<0.05) in ConA repeated administration group. Conclusions: An animal model of ACLF immune status from systemic inflammatory response syndrome (SIRS) to compensatory anti-inflammatory response syndrome (CARS) induced by repeated ConA stimulation is successfully established.

Key wordsAcute-On-Chronic Liver Failure;Concanavalin A;Immune Status;Cytokines;Models, Animal

*本課題為國(guó)家自然基金項(xiàng)目(30971333、81170421、81470869)

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