【作者】 张惠；

【导师】 龚非力；

【作者基本信息】 华中科技大学 ， 免疫学， 2009， 博士

【摘要】 病毒性肝炎、药物性肝损害、自身免疫性肝炎、肝硬化及肝癌等肝病已成为世界范围内威胁人群健康的主要疾病之一。已有研究表明，肝炎病毒并非直接导致细胞病理性改变，而主要是通过诱导宿主产生针对病毒感染肝细胞的免疫应答，继而损伤肝脏细胞。炎症是不同类型急性和慢性肝病普遍存在的病理现象，它与进行性肝脏损伤和肝组织纤维化有关。现已发现，固有免疫系统的各类细胞通过模式识别受体(patternrecognition receptors，PRR)识别病原微生物，从而被活化，参与启动和维持肝脏炎症过程。这些固有免疫细胞中，树突状细胞可通过处理、加工和提呈抗原而引起适应性T细胞应答；活化的kupffer细胞、募集到肝脏的巨噬细胞及其他炎症细胞可产生大量细胞因子、趋化因子，引起炎症反应反复发生和肝细胞损伤。TLR(Toll-like receptor)是一类重要的PRR，因其与果蝇Toll蛋白家族在结构上具有同源性而得名，TLR通过识别不同病原微生物的PAMP(pathogen associatedmolecular partem)在抗感染固有免疫中发挥重要作用。其中，表达于细胞内TLR9通过识别细菌和病毒DNA中的非甲基化CpG基序或人工合成的脱氧寡核糖核酸(CpG-containing oligodeoxynucleotides，CpG ODNs)序列而被活化，继而促进B细胞增殖、分化，促进DC成熟，上调共刺激分子表达，并诱导Th1细胞应答。目前已知有3种CpG ODN，即D-型(A-class)、K-型(B-class)及C-型，三者结构中均含非甲基化CpG二核苷酸序列，且因结构各异而具有不同生物学功能。CpG ODNs已被用于治疗某些感染性疾病、肿瘤及变态反应性疾病等。本课题研究目标为：建立Con A诱导的小鼠肝损伤模型，观察不同给药时间、不同剂量CpG ODN对肝损伤的影响；用CpG ODN 100μg预处理小鼠3h，初步探讨CpGODN对ConA诱导肝损伤的保护作用及其机制。一、CpG ODN对ConA诱导肝损伤的影响(一)实验分组PBS正常对照组(PBS组)PBS预处理组(PBS／ConA组)CpG ODN预处理组(CpG ODN／ConA组)(二)CpG ODN预处理对ConA诱导肝损伤的影响CpG ODN(100μg／只小鼠)或PBS由尾静脉注射，3h后尾静脉注射Con A(15μg／g体重)，12h后采集血清和肝脏组织，进行血清转氨酶和肝脏组织学检测。1．血清转氨酶检测：PBS预处理组，Con A静脉注射导致ALT与AST水平急剧升高。CpG ODN预处理可使Con A注射后ALT与AST水平显著降低。CpG ODN单独作用未引起转氨酶升高。2．组织学检测：PBS预处理组肝脏大面积坏死，CpG ODN预处理组肝组织未见明显炎性病灶和坏死区域。(三)CpG ODN对Con A诱导肝脏损伤的保护作用具有时间和剂量依赖性1．Con A注射前后不同时间点经尾静脉注射CpG ODN(100μg／小鼠)；2．Con A注射前3h，尾静脉注射不同剂量CpG ODN；ConA．注射后12h采集血清。3．血清转氨酶检测：(1)CpG ODN预处理1 2h和3h对Con A所致肝损伤均具有保护作用；Con A注射后2h给予同等剂量CpG ODN，未见有保护作用。(2)静脉注射CpG ODN 50μg／小鼠和100μg／小鼠均可对Con A所致肝损伤产生保护作用。(四)CpG ODN预处理对小鼠存活时间的影响静脉注射CpG ODN 100μg／小鼠，3h后尾静脉注射致死剂量Con A(25μg／g体重)，观察小鼠存活时间。小鼠存活率分析：CpG ODN预处理可明显延长小鼠存活时间，与PBS预处理组相比有显著差异(五)CpG预处理对肝脏SOD、MDA、MPO表达的影响检测肝脏组织超氧化物歧化酶(SOD)、丙二醛(MDA)及髓过氧化物酶(MPO)：与PBS预处理组(PBS／ConA组)相比，CpG ODN预处理组(CpG ODN／ConA组)SOD水平在Con A注射后12h和24h明显升高，而MDA在6h和24h水平显著下降。检测肝组织MPO含量可间接反映中性粒细胞浸润情况，CpG ODN预处理组(CpGODN／ConA组)MPO水平明显低于PBS预处理组。上述结果显示，CpG ODN对Con A所致肝损伤具有保护作用，该效应具有时间和剂量依赖性，以CpG ODN(100μg／小鼠)预处理小鼠3h保护作用最显著，可明显提高小鼠存活率。二、CpG ODN预处理对Con A所致肝损伤保护作用的机制(一)实验分组PBS正常对照组(PBS组)PBS预处理组(PBS／ConA组)CpG ODN预处理组(CpG ODN／ConA组)尾静脉注射CpG ODN(100μg／只小鼠)或PBS，3小时后尾静脉注射Con A(15μg／g体重)，于ConA注射后不同时间点收集肝组织和血清。(二)CpG ODN预处理对Con A所致肝细胞凋亡的影响1．TUNEL检测：PBS预处理组(PBS／ConA组)注射ConA 12h后，肝细胞出现大范围凋亡，而CpG ODN预处理组(CpG ODN／ConA组)肝细胞凋亡明显减轻。2．RT-PCR检测bcl-2和bax mRNA表达水平：与PBS预处理组(PBS／ConA组)相比，CpG ODN预处理组(CpG ODN／Con A组)抗凋亡基因bcl-2 mRNA表达水平明显增加，而促凋亡基因bax mRNA表达水平下降。(三)CpG ODN预处理抑制Con A所致炎症反应1．ELISA检测血清细胞因子水平：与PBS预处理组(PBS／ConA组)相比，CpGODN预处理组(CpG ODN／ConA组)血清IFN-γ水平在6h和12h明显降低。PBS预处理组(PBS／Con A组)动物血清中TNF-α水平在Con A注射后2h达到最高，而CpG ODN预处理组(CpG ODN／Con A组)TNF-α水平显著下降；Con A注射后12h后，PBS预处理组(PBS／ConA组)和CpG ODN预处理组(CpG ODN／ConA组)血清中均未检出TNF-α。2．RT-PCR检测肝脏IFN-γ和TNF-αmRNA水平：CpG ODN预处理组(CpGODN／Con A组)肝组织IFN-γ和TNF-αmRNA表达水平比PBS预处理组(PBS／Con A组)明显降低。(四)CpG ODN预处理对肝脏组织NF-κB活性的影响1．EMSA检测NF-κB的活性：与PBS预处理组(PBS／ConA组)相比，CpG ODN预处理组ConA注射2h后可显著抑制NF-κB活性。2．Western blot检测IκB降解：CpG ODN预处理可明显抑制IκB降解。(五)CpG ODN预处理对肝脏和脾脏淋巴细胞的影响流式细胞术检测肝脏和脾脏淋巴细胞亚群：与PBS预处理组(PBS／ConA组)相比，CpG ODN预处理组(CpG ODN／ConA组)肝脏和脾脏T细胞(CD3~+)、NKT细胞(CD3~+NK1．1~+)、NK细胞(CD3~+NK1．1~-)比例无明显变化，但CpG预处理可抑制肝脏中三类细胞活化(CD69~+)。以上结果表明：CpG ODN预处理可明显抑制肝脏细胞凋亡，减弱Con A所致炎症反应，抑制淋巴细胞活化。三、CpG ODN预处理对巨噬细胞的影响小鼠巨噬细胞系RAW264．7体外培养；实验分组：CpG ODN单独作用组(5μg／ml)Con A单独作用组(10μg／ml)CpG ODN预处理加Con A作用组[CpG ODN(5μg／ml)+Con A(10μg／ml)]于不同时间点收集上清和细胞1．ELISA检测：与ConA单独作用组相比，CpG ODN预处理组上清中TNF-α水平明显降低。2．RT-PCR检测：与CpG ODN单独作用组和Con A单独作用组相比，CpG ODN预处理组IRAK1 mRNA表达水平明显降低，而IRAK-M mRNA表达水平无明显变化。上述结果提示：CpG ODN预处理可使巨噬细胞对Con A刺激产生耐受，其机制可能与IRAK1表达水平下降相关。结论1．CpG ODN预处理对Con A所致肝损伤具有保护作用；2．CpG ODN预处理对ConA所致肝损伤保护作用的机制可能为：抑制淋巴细胞活化；诱导巨噬细胞低反应性；减弱ConA所致炎症反应；抑制肝细胞凋亡。更多还原

【Abstract】 Viral hepatitis,drug-induced hepatic injury,autoimmune hepatitis,hepatic cirrhosisand other liver diseases are major threats to human health worldwide.It has been shownthat hepatitis virus itself can not induce liver injury,which mostly mediated by the immuneresponse to the hepatocyte infected with virus.It has been shown that the inflammatory response play an key role in pathogenesis ofdifferent types of acute and chronic liver diseases,which contributes to liver damage,fibrosis and dysfunction.There are approximately 10¹⁰lymphocytes in human liver,including lymphocyte subpopulations of the innate systems such as NKT and NK cells,andadaptive immune systems,for example T and B cells.For its large population of innateimmune cells,the liver is considered to be an organ with innate immunity features.Emerging evidence suggests that liver play critical roles in first line of host immunedefense against invading microorganisms and modulation of liver injury and repair.Cells of the innate immune system recognized microorganisms by pattern recognitionreceptors（PRR）and then were activated to produce various proinflammatory cytokine andother mediator,which lead to initiate and maintain hepatic inflammation and hepatocytedamage.Innate immune cells,particularly dendritic cells,have a pivotal role in processingpathogens and initiating adaptive immune responses.Moreover,activated Kupffer cells,macrophages which were recruited to liver from blood and other inflammatory cells releasecytokines and chemokines that contribute to liver injury and impaired liver regeneration.Toll-like receptors（TLRs）are the best understood family of PRRs and are highlyevolutionarily conserved.There are 13 members in the TLRs family.TLRs recognize related PAMPs and trigger the downstream signaling pathway and production ofproinflammatory cytokines and chemokines.It has been well knon that TLR9 canrecognize not only the unmethylated CpG motifs from bacteria/virus but also syntheticoligodeoxynucleotides（ODNs）that contain CpG motifs.There are three types of CpG ODN:D-type（A-class）,K-type（B-class）and C-class,all of which possess unmethylated CpG dinucleotides.CpG-containingoligodeoxynucleotides（CpG ODNs）activate the innate/adaptive immune system throughbinding to TLR9 which is expressed in many immunological cells such as B cells,macrophages and plasmacytoid dendritic cells（pDCs）.The TLR9 is then activated toinduce Thl-based immune responses.To our current understanding,the potentialtherapeutic uses of these CpG ODNs have been focused on infectious disease,cancer andallergy therapy.A recent study suggested that CpG ODN have a protective effect on the CpGODN/D-GalN-induced hepatic injury.However.the effects of CpG ODN on ConA-induced hepatitis are less known and need to be further investigated.In the presentstudy,we demonstrated that CpG ODN pretreatment can protect the mice from ConA-induced liver injury.Furthermore,we explored the possible mechanisms underlying thisprotective effect.1.The effect of CpG ODN on Con A-induced hepatitisTo determine the effect of CpG ODN on Con A-induced hepatitis,CpG ODN（100μgper mouse）was administered to mice through the caudal vein three hours before Con A（15μg/g body weight）injection.Serum aminotransferase levels were determined twelvehours after Con A injection.Serum ALT levels were slightly elevated in mice treated withCpG ODN alone.As expected,Con A administration significantly increased the serum levelof aminotransferase.However,the Con A induced ALT was markedly decreased in micepretreated with CpG ODN,but not in mice pretreated with non-CpG ODN.H&E stainingshowed that Con A injection caused massive necrosis in the liver,which was nearlyabolished by CpG ODN pretreatment.The protective effect of CpG ODN on ConA-induced liver injury was dose-dependent.Furthermore,CpG ODN pretreatment beforeCon A challenged（-12hCpG/Con A or -3hCpG/Con A）protected the mice from hepatitis. Delaying ODN administration until 2 h after Con A challenged（2hCpG/Con A）did notprovide any protection.Next,we determined whether CpG ODN pretreatment protectedmice from lethal dose of Con A（25μg/g body weight）.CpG ODN pretreatmentdramatically increased mice survival.2.The possible mechanisms underlying this protective effect.Mice were treated with PBS or CpG（100μg/mouse）,and three hours later,Con A wasinjected（15μg/g）.Liver and serum samples were collected at indicated time pionts afterCon A injection.（1）CpG ODN pretreatment prevents hepatocytes apoptosis in Con A-induced hepatitisThe extent of hepatocytes apoptosis was determined by TUNEL assay,massivehepatocytes apoptosis were detected in the livers of mice treated with Con A.CpG ODNpretreatment markedly prevented the apoptosis induced by Con A.Furthermore,weexamined the mRNA expression of antiapoptotic protein Bcl-2 and proapoptotic proteinBax in the livers.Con A upregulated the expression of Bax.On the contrary,CpG ODNpretreatment downregulated the mRNA expression level of Bax and upregulated the mRNAexpression level of Bcl-2.（2）CpG ODN pretreatment inhibits the release of cytokines in Con A- treated miceThe level of IFN-γand TNF-αin serum were measured by ELISA.CpG ODNpretreatment significantly suppressed the elevation of serum IFN-γlevels at 6 h,12 h afterCon A administration.Furthermore,CpG ODN pretreatment inhibited the secretion ofTNF-αat early stage,especially two hours after Con A injection.Of note,TNF-a wasundetectable in serum at 12 h after Con A administration.Furthermore,we analyzed theexpression of proinflammatory cytokines in livers by RT-PCR.CpG ODN pretreatmentdownregulated the levels of both IFN-γand TNF-αin the livers.（3）The effect of CpG ODN pretreatment on NF-κB DNA binding activity.Nuclear and cytoplasmic extracts of liver tissues were subjected to EMSA and westernblot respectively.CpG ODN pretreatment significantly inhibited the activation of NF-κBand the phosphorylation of IκBα.（4）The effect of CpG ODN on subset of lymphocyte in liver/spleenThe effect of CpG ODN on subset of lymphocyte in liver/spleen were measured byFACS.CpG ODN pretreatment failed to prevent Con A-induced recruitment of these cells into the livers.But CpG ODN pretreatment significantly suppressed the activation of T cells（CD3⁺）,NK cells（CD3^-NK1.1⁺）.and NKT cells（CD3⁺NK1.1⁺）.3.The effect of CpG ODN on macrophage cellsRAW 264.7 cells were stimulated with CpG ODN（5μg/ml）for three hours,and thenrinsed with PBS for three times.The cells were then cultured with Con A（10μg/ml）.Thesupernatants and cells were collected at indicated time points after Con A added.TheTNF-αlevel in supernatants was determined with ELISA.The mRNA expression ofIRAK-1 and IRAK-M were measured by RT-PCR.CpG ODN pretreatment markedlyinhibited the production of TNF-αreleased by RAW 264.7 cells in response to Con A.Meanwhile,CpG ODN pretreatment downregulated the levels of IRAK-1,but had no effecton the IRAK-M expression.ConclusionCpG ODN pretreatment can protect mice from Con A-induced hepatitis throughsuppressing the activation of intra-hepatic leukocvtes,preventing the expression ofproinflammatory mediators,inhibiting the apoptosis of hepatocytes.Our study suggests thatCpG ODN may have therapeutic benefits to protect liver from virus infection.更多还原