【作者】 苏兆亮；

【导师】 许化溪；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2011， 博士

【摘要】 高迁移率族蛋白B1 (HMGB1)为HMG蛋白家族成员之一,广泛表达于多种组织细胞,具有调节基因转录、稳固胞核结构以及释放后具有炎症介质功能的核蛋白,也是一种重要的损伤相关的分子识别模式(Damage Associated Molecular Patterns, DAMPs)。HMGB1结构高度保守,由215个氨基酸组成,包括A、B-box两个构象高度类似的DNA绑定区域、受体晚期糖基化终末产物结合区(Receptor for advanced glycation end products, RAGE)和具有负调控作用的羧基端尾部。机械损伤或坏死的细胞被动释放HMGB1,而活化的巨噬细胞(Mφ)、树突状细胞(DC)则主动分泌HMGB1,释放或分泌的HMGB1通过与其配体RAGE、TLR2、4、7等结合既发挥着对免疫应答的调节作用,也参与肿瘤、缺血再灌注损伤、自身免疫性疾病的发生、发展。自身免疫性心肌炎是Th17细胞介导的一种自身免疫性疾病,继发于病毒性心肌炎(VMC)；病毒的感染导致心肌细胞破坏、心肌球蛋白释放,进而引起自身免疫性心肌炎。自身免疫性心肌炎是扩张型心肌病、心衰的最主要因素。迄今为止,自身免疫性心肌炎的致病机制尚不十分清楚,然而Th17细胞介导的进行性心肌损伤是造成自身免疫性心肌炎的主要原因。实验性自身免疫性心肌炎(EAM)是研究这一疾病的有效模型,可由柯萨奇病毒或心肌球蛋白α链(MyHC-α)诱导产生。研究显示EAM时血浆HMGB1水平增高、循环Th17细胞增加,心肌组织中有大量中性粒细胞、Mφ和Th17细胞浸润。HMGB1和Th17细胞均在EAM时呈现高水平,那么两者的关系如何?HMGB1是否通过调控Th17细胞参与自身免疫性心肌炎的致病?在环境压力改变时心肌细胞是否会主动分泌HMGB1,分泌的HMGB1是否参与心肌炎症的致病?有待于研究证实。目的(1)已有研究发现释放或分泌到胞外的HMGB1与抗原提呈细胞(APC)表面的RAGE或TLR4、2、7等模式识别受体(PRR)结合,通过MyD88依赖或MyD88非依赖途径,调节Th1、Treg等CD4+T细胞的分化、增殖。前期预试验发现EAM时血清HMGB1水平增加,Th17细胞浸润增加。HMGB1水平增加与Th17细胞的浸润有无关系,其对新发现的Th17细胞有无调控作用等,正是本研究所瞄准的科学问题。通过项目的实施,力争阐明HMGB1对Th17细胞的调节作用及其在实验性自身免疫性心肌炎发生中的作用。(2) HMGB1是一重要的危险警报分子,长期以来人们一直认为HMGB1主要由活化的巨噬细胞、树突状细胞主动分泌或者是由坏死或凋亡的细胞被动释放。在心脏组织外界环境压力改变时作为心脏主要组成成分的心肌细胞是否会主动分泌HMGB1?分泌的HMGB1对心脏的炎症或心肌功能有何影响?因此,通过本研究的实施阐明心肌细胞能否主动分泌HMGB1,其分泌HMGB1所依赖的分子机制及其在心肌炎症损伤过程中的作用。(3) HMGB1作为一重要的炎症介质,参与免疫应答的调控及肿瘤、败血症、缺血/再灌注损伤、自身免疫性疾病等许多炎症疾病的致病,中和或抑制HMGB1可以缓解炎症性疾病的症状。因此,本课题制备HMGB1的单克隆抗体并观察其在心肌炎中的治疗作用,为其今后的人源化及其临床推广做准备。方法(1) HMGB1 B-box重组蛋白及其单克隆抗体的制备HMGB1 B-box的质粒由第三军医大学何凤田教授馈赠,重组质粒转化E. coli DH5a,然后37℃培养5-7h,待OD值(A600)为1～1.5时加入IPTG,诱导6h后收集细菌。超声裂解法提取蛋白,层析柱进行纯化。纯化后的蛋白经多粘菌素B柱,抑制细菌内毒素活性,-20℃保存待用。用15μg纯化的B box蛋白与福氏完全佐剂混合并完全乳化后,经皮下多点注射免疫BALB/c小鼠。隔2周同法进行第2次免疫,一周后,经尾静脉加强免疫数次。细胞融合、亚克隆及mAb腹水的制备均按实验室常规方法进行。(2)小鼠心肌炎模型的诱导及其病理分析MyHC-α614-629与弗氏完全佐剂(CFA)1：1混合,分别在第0和7天,进行小鼠免疫,对照组用CFA和PBS进行免疫。3周后,眼球采血处死小鼠,取出心脏10%的福尔马林固定。ELISA检测血清HMGB1、TNFα、IL-1β、IFN-γ、IL-17; RT-qPCR分析心肌组织HMGB1以及脾脏中IFN-γ、IL-17、T-bet、RORγt的表达。HE染色分析心肌病理损伤程度、免疫荧光观察Th17细胞在心肌组织中的浸润情况。(3) HMGB1对Th17细胞的调控作用及抗HMGB1 B box单克隆抗体在EAM发生中的干预作用阳性选择法分离CD4+T细胞,在anti-CD3、anti-CD28存在的体系下加入HMGB1, FACS观察HMGB1对其分化、增殖的影响。并将制备的抗HMGB1 B box单克隆抗体腹腔注射入EAM小鼠病理分析心肌损伤程度、免疫荧光观察Th17细胞浸润情况。(4)LPS对心肌细胞主动分泌HMGB1的影响及其分子机制用160U/ml的胶原酶Ⅱ37℃消化1h,分离新生小鼠心肌细胞反复吹打后收集细胞,接种到细胞培养板中,静置贴壁1h,收集未贴壁的细胞转置于另一新的细胞培养板,在含有10%FCS的M 199培养基中37℃培养备用。LPS刺激后观察其凋亡情况及其HMGB1主动分泌水平。体内通过LPS诱导的内毒素模型观察心肌细胞HMGB1的主动分泌功能。免疫荧光、western-blot及TLR4-/-、PI3Kγ-/-小鼠阐明心肌细胞主动分泌HMGB1的分子机制。(5)心肌细胞主动分泌的HMGB1对心肌功能的影响通过WT、PI3Kγ-/- C57BL/6小鼠模型诱导与干预,采用Millar tip transducer catheter (SPR-893,1.4 Fr.)仪器评估小鼠心脏功能并通过HE染色分析心肌的损伤及其炎症损伤程度。结果(1) HMGB1 B-box质粒经测序正确后,转化E. coli DH5α后,表达HMGB1 Bbox蛋白,SDS-PAGE鉴定电泳位置正确；通过杂交瘤技术制备抗HMGB1 B box的单克隆抗体,筛选到两株能够稳定分泌抗HMGB1 B box的单克隆抗体细胞株(1D2F4E3和2D4E3A2),两株细胞株分泌的抗体均为IgGα,κ型,效价达到106以上,与E. coli DH5α基因组蛋白质无交叉反应。(2) MyHC-α614-629诱导小鼠EAM,病理分析发现心肌组织边缘区有大量的淋巴细胞浸润,免疫荧光进一步确认浸润的细胞为Th17细胞,血清ELISA发现IL-17水平显著增高(667.09±120.44 pg/ml vs.68±31.11 pg/ml,p<0.05), HMGB1水平在14天、21天分别为：3717.48±210.93pg/ml、5030.17±1102.08pg/ml,而对照组为2000±1193.39,p<0.05；对小鼠脾脏Th17细胞相关的转录因子和细胞因子定量检测也发现RORyt和IL-17表达水平增高,而IFN-γ和T-bet没有明显变化。(3)用制备的抗HMGB1 B box的单克隆抗体中和HMGB1。21天后处死小鼠,发现心肌损伤程度明显减轻；且免疫荧光和ELISA检测也发现Th17细胞的浸润减轻,血清IL-17明显降低。体外HMGB1能够有效地增加Th17细胞的比例；HMGB1中和前后EAM小鼠血清TGF-β、IL-6、IL-1β检测结果分别为：1063.58±233.22pg/ml与250.64±152.74 pg/ml；482.81±87.04pg/ml与100.80±73.77pg/ml；8.90±1.84pg/ml与6.47±0.81pg/ml.(4)小鼠腹腔注射LPS,12～24h后,血浆HMGB1水平明显增加(200±67.3ng/ml),HMGB1的增加滞后于TNF-α(4h) (575±25.5pg/ml)；免疫荧光分析发现,在对照组小鼠心肌组织中HMGB1的表达主要集中于细胞核,而在LPS刺激后HMGB1在细胞核、浆中均表达增加。体外用不同浓度的LPS刺激新生鼠心肌细胞,发现用10μg/ml LPS刺激心肌细胞并不影响心肌细胞活性,但会导致心肌细胞主动分泌HMGB1。LPS刺激心肌细胞后在5～15min时PI3Kγ磷酸化程度增强,相同的现象在TLR4-/-的小鼠分离的心肌细胞中并没有被观察到。而且也发现PI3Kγ抑制剂(AS605240),能够有效地抑制心肌细胞主动分泌HMGB1:同样现象也在PI3Kγ-/-的心肌细胞中被观察到。(5)免疫组化分析发现,在WT小鼠内毒素模型的心肌组织HMGB1表达水平增加,而心肌组织并没有炎症和病理改变,小鼠注射LPS 24h后心肌收缩功能发生障碍,且这种障碍能够通过注射HMGB1拮抗剂A-box或者是抑制剂GZA缓解。小鼠腹腔注射HMGB1蛋白,1h后检测循环中HMGB1的水平并评估心肌功能,发现直接注射HMGB1并没有影响心肌功能，直到血清HMGB1增加后心脏收缩发生障碍。结论(1)成功地筛选到了两株分泌抗HMGB1 B box的单克隆抗体细胞株,两株细胞株分泌的抗体均为IgG2a,κ型,效价达到106以上,有良好的特异性。(2)利用MyHCα614-629成功的诱导了小鼠EAM,而且证实在MyHCα614-629诱导的心肌炎中以Th17细胞浸润为主,并伴随有HMGB1江苏大学博士学位论文：HMGB1在心肌炎中的致病机制及调控作用研究分泌水平的增高；利用自制的抗HMGB1 B box的单克隆抗体中和循环中的HMGB1能够有效减轻心肌病理损伤,降低Th17细胞浸润。这提示心肌炎时由于心肌细胞损伤、免疫细胞浸润而释放的HMGB1可能对Thl7细胞具有调控作用；HMGB1有望成为自身免疫性心肌炎潜在治疗靶位。(3)在MyHCα614-629诱导的EAM模型中中和HMGB1能够减少Th17细胞浸润,这提示HMGB1的分泌与Th17细胞的浸润存在着相关性。因此,体外研究了HMGB1对Th17细胞的调控作用,发现HMGB1可能通过调节Th17细胞的增殖、分化、极化,导致了EAM小鼠心肌组织Th17细胞浸润增多,这也是首次提出HMGB1对CD4+Th17细胞具有调控作用。(4)用LPS刺激分离的新生鼠心肌细胞发现心肌细胞会主动分泌HMGB1,这一结论在体内败血症/内毒素血症模型中也得以证实,这是继垂体细胞、肝细胞和上皮细胞后第四个能主动分泌HMGB1的非免疫细胞。在此基础之上,我们进一步探索了心肌细胞主动分泌HMGB1所依赖的分子机制,发现心肌细胞主动分泌HMGB1的机制与免疫细胞主动分泌HMGB1机制一样均是PI3Kγ依赖的。(5)我们用LPS在C57BL/6小鼠诱导内毒素血症模型24小时后分析发现,在短期内由于外界环境压力刺激心肌细胞主动释放的HMGB1没有导致心肌的炎症,但是会导致心肌收缩功能障碍。这一结论与以往关于HMGB1是一晚期炎症介质的报道相一致。更多还原

【Abstract】 High mobility group box 1 (HMGB1), a non-histone nuclear protein, has been functionally characterized as an alarmin or damage associated molecular protein (DAMP). It is constitutively expressed in quiescent cells and stored preformed in the nucleus. HMGB1 is the most evolutionarily conserved protein in eukaryotes. HMGB1 was merely a structural protein that resided in the nucleus where it functions to stabilize DNA structure and modulate transcriptional activity. The major structural features of HMGB1 are its two DNA-binding domains, termed the A and B boxes, and a C-terminal domain. HMGB1 was actively secreted by activated macrophages and dendritic cells or passively secreted by necrosis or apotosis cells. HMGB1 widely involved in the inflammatory and cancer, sepesis, autoimmune disease.Autoimmune myocarditis, caused by viral myocarditis, was mediated by Th17 cells. Because of virus infected cardiomyocytes, myosin was released and led to autoimmune myocarditis. Autoimmune myocarditis also was a main factor of dilated cardiomyopathy and heart failure. Th17 cells involved in the pathogenesis of myocarditis. Experimental autoimmune myocarditis (EAM) is a mouse model of postinfectious myocarditis, characterized by inflammatory infiltration of the myocardium and cardiomyocytes necrosis. EAM can be induced by inoculation with the coxsackie B virus or cardiac myosin. And we also found that HMGB1 level was increased in the plasma and Th17 cells infiltrated the heart. Therefore, we aimed to clarify whether Thl7 cells are involved in EAM pathogenesis; whether HMGB1 contributes to the progression of EAM; whether HMGB1 expression is associated with Th17 cell activity in EAM. And whether cardiomyocytes could actively secrete HMGB1. Objective(1) HMGB1 released into the extracellular milieu, binds to the receptor for advanced glycation endproducts (RAGE) or toll like receptor 2/4/7 (TLR2/4/7), and has restorative effects on CD4+ T helper cell regulation. And we also found that HMGB1 level was increased in the plasma and Thl7 cells infiltrated the heart. Therefore, we aimed to clarify the HMGB1 effects on Th17 cells and the pathogenesis on EAM.(2) HMGB1 has been functionally characterized as an alarmins, was actively secreted by activated macrophages and dendritic cells or passively secreted by necrosis or apotosis cells. Whether cardiomyocytes could actively secrete HMGB1 on the milieu stress. Therefore, we want to clarity the mechanism of cardiomyocytes actively secreted HMGB1 and affect on heart.(3) HMGB1 has recently been shown to also be associated with inflammatory diseases. Therefore whether inhibition of HMGB1 could ameliorate the degree of myocardial injury in this experimental model. And we produced the mAb of HMGB1 B box.Methods(1) HMGB1 B-box expression and producing of monoclonal antibodies The plasmid of HMGB1 B-box provided by Prof FengTian He from the third military medical university. The plasmid was transformed DH5a, cultivating 5～7h, keeping OD value (A600) for 1～1.5, then IPTG added. After 6h, bacteria were collected. The protein was extracted and purified. The polymyxin B column was used to inhibit endotoxin activity.15μg protein with complete freund’adjuvant was immunized BALB/c mice by i.p. every two weeks. After one week of second immunization, immunizaed again by vein. Cell fusion, cloning and mAb producing by conventional method. (2) The myocarditis induction and pathological analysisMyHCα614-629 with complete freund’ adjuvant (CFA) immunized BALB/c mouse on day 0 and day 7. And control group immunized by PBS with CFA. After three weeks, the serum was collected and the heart was fixed in 10% formalin. Serum HMGB1, TNFα, IL-1β, IFN-γand IL-17 were detected by ELISA. The HMGB1, IFN-γ, IL-17, T-bet, RORγT mRNA were analyzed by RT-qPCR. HE staining were used to analysis myocardial pathology, Th17 cells infiltrated the myocardial tissue by immunofluorescence.(3) HMGB1 promotes Th17 cell expansion and HMGB1 blockade decreases Th17 cell infiltration and ameliorates myocardial injuryCD4+ T-cells were isolated by magnetic activated cell sorting (MACS) using CD4 antibodies according to the manufacturer’s instructions. Isolated T-cells were plated onto a 96-precoated-well plate with anti-CD3 mAb and anti-CD28 mAb. Cultures were supplemented with 1μg/ml HMGB1, and also included control group and unassociated group. For HMGB1 blockade, neutralizing monoclonal antibody against HMGB1 (100μg/mouse i.p.) was administered every other day according to our lab protocol.(4) LPS-induced HMGB1 production by viable cardiomyocytesHearts were harvested, minced and digested with CollagenaseⅡ(160 U/ml). After a washing step, the cells were suspended in M199 with 10% FCS. The myocytes were enriched by a preplating approach (to remove contaminating cells) before being seeded into cell culture plates. Endotoxemia was induced by i.p. injection of 0.5 ml of normal saline (NS) containing LPS (10 mg/kg) to 3-month-old mice. This dosing regimen is in accord with previous studies assessing LPS-induced cardiac function. Control mice received saline. As an in vitro correlate of endotoxemia, the myocytes were exposed to 10μg/ml LPS, a dosing regimen previously to address LPS-induced signaling in isolated cardiomyocytes. And the signaling pathway was analyzed by immunofluorescence, western-blot and TLR4-/-, PI3Kγ-/-(5) LPS decreases myocardial contractility in vivo via a mechanism that involves HMGB1Endotoxemia was induced and intervented on WT, PI3Kγ-/- C57BL/6 mouse. Millar tip transducer catheter (Model SPR-893,1.4 Fr.) was advanced into the left ventricle via the right carotid artery. Pressure-volume loops were generated by occlusion of the inferior caval vein using a PowerLab system connected to theMillar catheter. Left ventricular end systolic elastance (Ees) was considered to be the best index of myocardial contractile function and was determined from pressure-volume loop data.Results(1) The plasmid was sequenced again. HMGB1 B box proteins were expressed, the molecular weight was identified by SDS-PAGE. Two cell lines were isolated namely 1D2F4E3 and 2D4E3A2. And the mAbs were IgG2a,κtype, valence was 106.(2) Myocarditis model was induced by MyHCα614-629. Monocyte, neutrophil, and lymphocyte infiltration in the myocardium was observed. Th17 cell infiltration was identified by immunofluorescence. Serum IL-17 levels by ELISA were significantly increased compared with controls (667.09±120.44 pg/ml vs.68±31.11 pg/ml, p<0.05). However, serum IFN-y levels were unchanged. In the mice spleens, RORyt and IL-17 mRNA expression were significantly increased; T-bet and IFN-γdid not show any change in mRNA expression.(3) HMGB1 blockade resulted in a significant reduction of cardiac myocytes as well as a significant decrease in monocyte, neutrophil, and lymphocyte infiltration of the myocardium on HE. Th17 cells infiltration significantly decrease compared to the untreated group. Serum IL-17 was also significantly decrease (212.11±67.30 pg/ml) compared to the untreated group (617.82±146.00 pg/ml, p<0.01). HMGB1 blockade did not affect IFN-γsecretion.(4) HMGB1 was noted in the circulation at 12-24h after LPS administration; much later than the increase in circulating TNF-α; In control mice, HMGB1 was localized predominantly to the nuclei of the myocytes, whereas the LPS-induced increase in myocardial expression of HMGB1 is apparent in both the cytoplasm and nuclei. Challenge of cardiomyocyte monolayers with 10μg/ml LPS did not affect myocyte viability. Challenge of cardiomyocytes with LPS, resulted in a transient increase in phosphorylation of PI3Kγwithin cardiomyocytes, peaking at 5-15min after the challenge. LPS administration to mice decreased myocardial contractility(5) Injection of HMGB1 did not affect myocardial contractility until circulating evels of HMGB1 were increased; at which point decreases in ardiac contractility. The LPS-induced increase in myocardial HMGB1 protein levels and decrease in myocardial contractility.Conclusions(1) Two cell lines were isolated namely 1D2F4E3 and 2D4E3A2. And the mAbs were IgG 2a,κtype, valence was 106.(2) EAM was induced using MyHC-α614-629 peptides in wild-type male BALB/c mice and found that cardiac myocyte necrosis was accompanied by lymphocyte infiltration into the myocardium. These were confirmed to be Th17 cells by immunofluorescence. Similarly, HMGB1 was increased in both cardiac tissue and serum; HMGB1 blockade resulted in a significant reduction in cardiac myocyte necrosis and a significant decrease in Th17 cell myocardial infiltration. Serum IL-17 was also noticeably decreased after treatment. Furthermore, gross severity scores decreased.(3) HMGB1 secreted by injured cardiac myocytes, fibroblasts, and activated macrophages and monocytes was associated with Th17 cell polarization, differentiation, and proliferation in the EAM model.(4) HMGB1 should be extended to include active release of HMGB1 by " stressed," but still viable, cardiomyocytes. the current study provide the first demonstration that viable cardiac myocytes can produce the HMGB1, when challenged by LPS in vivo and in vitro. In addition, some of the components of the cardiomyocyte signaling pathway have been identified and sequentially positioned (i.e., LPS activates a TLR4/PI3Ky pathway to induce HMGB1 secretion).(5) LPS can induce HMGB1 secretion by viable cardiac myocytes through a TLR4/PI3Ky signaling pathway, and HMGB1 plays a role in the LPS-induced myocardial contractile dysfunction. The results of the current study also have broader implications.更多还原