天然免疫分子HMGB1在肾脏缺血再灌注损伤和异种移植排斥反应中的作用研究

发布时间：2018-03-01 10:20

本文关键词： 高迁移率族蛋白Bl 抗HMGB1抗体缺血再灌注损伤肾小鼠 HMGB1 抗HMGB1抗体异种心脏移植大鼠小鼠　出处：《华中科技大学》2010年博士论文　论文类型：学位论文

【摘要】：第一部分上调HO-1对缺血再灌注损伤的保护机制与抑制HMGB1释放有关 [目的]本课题组已经发现Copp诱导HO-1高表达能减轻大鼠肾脏缺血再灌注损伤,目前实验为了进一步深入研究保护性基因HO-1在减轻大鼠肾脏缺血再灌注损伤中的作用及其机制,探讨HMGB1在其中是否发挥炎症因子效应。 [方法]课题前期实验以Wistar大鼠为对象,建立大鼠肾脏在体原位缺血再灌注损伤模型,夹闭大鼠左侧肾蒂阻断血供47分钟,恢复血流后切除右肾。实验分三组：1)缺血对照组：缺血再灌后无处理；2)CoPP治疗组：手术前48h和24h分别腹腔内注射CoPP (2.5mg/kg);3)正常组。左肾恢复血供即再灌注24h后检测血清肌酐(Cr)和尿素氮(BUN)值,并取正常及缺血再灌注肾脏标本行HE病理检查,Western-blot检测肾脏组织中HO-1表达,观察CoPP治疗对肾脏长时间缺血(80min)再灌注后大鼠存活的影响。目前实验以免疫组化检测其肾组织内HMGB1、TLR4、MPO、TNF-α的表达情况,以及TUNEL法检测细胞凋亡并计数。 [结果]CoPP治疗组与对照组相比肾功能(Cr和BUN)明显改善(p0.05),存活实验发现CoPP治疗组5只大鼠全部存活,而对照组6只大鼠中,4只分别于观察第5、5、7、13天死亡,两组具有明显差异(p0.05)。CoPP治疗组肾组织中HMGB1及TLR4表达明显弱于对照组,且代表单核细胞标志的MPO及细胞因子TNF-α也同样弱于对照组。细胞凋亡情况通过统计后对照组明显高于HO-1保护组,并具有统计学差异(p0.05)。 [结论]CoPP可效诱导肾脏HO-1高表达保护大鼠肾脏缺血再灌注损伤,其作用机制可能与抑制HMGB1的释放有关,进一步抑制炎症反应,减轻细胞凋亡,进而减少损伤。第二部分抗HMGB1中和抗体对小鼠肾脏缺血再灌注损伤的保护作用 [目的]探讨高迁移率簇蛋白B1 (HMGB1)在小鼠肾脏缺血再灌注损伤(IRI)模型中的作用,通过抗体阻断HMGB1研究对小鼠肾脏IRI的保护作用。 [方法]以BALB/C小鼠为实验对象,肾缺血前24小时和前30分钟实验组(n=7)分别于腹腔注射兔抗小鼠HMGB1抗体,而对照组(n=7)以等量生理盐水替代作为参照。在体阻断小鼠左肾血流60min,恢复左肾血流的同时切除小鼠右肾,恢复血供再灌注0,3,6或24小时后,在每组每个时间点(n=2)分别取其外周血及左肾。测定血清肌酐(sCr)和尿素氮(BUN)水平,观察肾组织学变化(PAS),免疫组化及Western blot检测肾组织中HMGB1的表达,并检测抗HMGB1抗体的沉积,免疫组化检测肾组织中髓过氧化物酶(MPO)表达,免疫荧光检测TNF-a表达,原位末端脱氧核苷酸转移酶标记法(TUNEL)检测肾组织中细胞凋亡情况。 [结果]正常组肾脏内HMGB1均表达于细胞核内,而对照组HMGB1则在核外明显表达,且在缺血后即有核外表达,随着再灌注时间延长(分别为0,3,6,24小时),HMGB1表达量增多且部位分布更广(细胞外)。对照组肾脏缺血再灌注24小时后血清Cr及BUN分别为(82.46±30.13)μmol/L和(46.46±9.61)μmol/L显著高于实验组的(32.24±22.51)μmol/L和(25.81±11.41)μmol/L(p0.05)。正常肾及对照组肾组织中未检测到HMGB1抗体沉积,使用抗HMGB1抗体的实验组可见明显HMGB1抗体沉积。PAS显示对照组肾小管细胞大片坏死,管型形成,并有大量出血,与之相比,抗HMGB1治疗组肾组织病变程度明显减轻。对照组肾组织中MPO表达明显强于使用HMGB1的实验组,同时对照组细胞凋亡较实验组显著增多,凋亡细胞数计数具有统计学差异(p0.05)：对照组肾组织中细胞因子TNF-α也明显强于实验组。 [结论]HMGB1在肾脏IRI中发挥重要作用,而缺血前使用抗HMGB1抗体可明显减轻小鼠IRI,这种保护作用可能通过中和炎性因子HMGB1来抑制肾脏IRI中的炎症反应,并减少炎症导致的凋亡来实现。第三部分抗HMGB1中和抗体延长大鼠到小鼠异种心脏移植存活的实验研究 [目的]探讨HMGB1对新生大鼠心脏移植到小鼠异种移植心的作用,及其通过抗HMGB1抗体阻断HMGB1能否延长异种移植心存活,并阐明其机制。 [方法]以1周龄SD大鼠为供心供者,BALB/C小鼠为移植心受者,实施颈部心异位心脏移植,抗体治疗组(n=10)于心脏移植前1天起隔天腹腔注射兔抗小鼠HMGB1抗体,而对照组(n=7)不做任何处理。术后观察移植心存活时间,移植心停止搏动即为发生排斥时留取心脏及血清,治疗组另有3例行在第6天留取标本(与对照组进行同期对组),进行病理学检测(HE),免疫组化观察组织中MPO, HMGB1, TLT4的表达及抗体IgG、IgM的沉积,以及补体调节蛋白CD55和CD59,免疫荧光检测JG12表达,TUNEL法检测心脏组织内细胞凋亡情况,流式细胞仪分析循环中抗体IgG、IgM水平。 [结果]对照组心脏存活5-6天,抗体治疗组为7-11天,明显长于对照组(p0.01)。组织病理上发现对照组排斥心脏呈急性血管性排斥反应特征,而治疗组同期的病变明显轻于前者；同时通过细胞计数显示抗HMGB1抗体治疗组较对照组MPO阳性的浸润细胞显著减少,TUNEL显示细胞凋亡或坏死显著减轻,统计具有显著性差异(p0.05),而血管内皮完整性的特异性标志JG12则显著强于对照组；另外组化显示抗体治疗组心脏组织内HMGB1及其受体TLR4表达均明显弱于对照组。流式检测以及免疫组化均显示对照组心脏组织内和循环中的异种抗体IgG显著增加,而同期的抗体治疗组IgG的产生和沉积显著减弱。此外,免疫组化显示对照组心脏组织中CD55和CD59明显弱于正常心脏及实验组。 [结论]本研究首次证明,拮抗HMGB1可明显延长大鼠到小鼠异种移植心脏的存活,其可能的机制是通过中和炎性细胞释放的HMGB1,从而抑制一系列炎症反应,阻遏异种抗体的产生并增强补体调节蛋白表达。
[Abstract]:Part 1 up-regulation the protective mechanism of HO-1 on ischemia-reperfusion injury and the inhibition of HMGB1 release
[Objective] this research group has found that high expression can reduce renal ischemia reperfusion injury in rats induced by HO-1 Copp, the current experiment in order to further study the protective gene in HO-1 could reduce the effect and mechanism of reperfusion injury in rat renal ischemia, to explore whether HMGB1 in which the effect of inflammatory factors.
[Methods] previous experiments using Wistar rats as the object, the establishment of rat kidney in vivo model of ischemia reperfusion injury, clamping the left renal pedicle of rats to block the blood supply for 47 minutes, blood flow is restored after resection of the right kidney. The experiment was divided into three groups: 1) ischemia group: ischemia reperfusion after treatment; 2) CoPP group: preoperative 48h and 24h were treated with intraperitoneal injection of CoPP (2.5mg/kg); 3) normal group. The left renal blood supply is restored after reperfusion for 24h detection of serum creatinine (Cr) and urea nitrogen (BUN) value, and normal and ischemia reperfusion kidney samples HE expression pathological examination. HO-1 of renal tissue in Western-blot detection, observation of CoPP therapy on renal ischemia long time (80min) affected the survival rats after reperfusion. The experiment with immunohistochemical staining in renal tissue of HMGB1, TLR4, MPO, expression of TNF- alpha and TUNEL assay, cell apoptosis and death count.
[results]CoPP treatment group compared with the control group, renal function (Cr and BUN) significantly improved survival (P0.05), we found that CoPP treatment group 5 rats survived, and 6 rats in the control group, 4 rats respectively to observe the death 5,5,7,13 days, the two groups have obvious difference (P0.05).CoPP treatment group HMGB1 and TLR4 expression in renal tissue was significantly weaker than the control group, and monocyte markers of MPO and cytokine TNF- alpha is also weaker than the control group. The cell apoptosis by statistical control group was significantly higher than HO-1 group, and the difference was statistically significant (P0.05).
[conclusion]CoPP can effectively induce the high expression of HO-1 in kidney to protect renal ischemia-reperfusion injury in rats, and its mechanism may be related to inhibiting the release of HMGB1, further inhibiting inflammatory reaction, reducing cell apoptosis and reducing injury.
The protective effect of anti HMGB1 neutralizing antibody on renal ischemia reperfusion injury in mice in second part
[Objective] to explore the role of high mobility cluster protein B1 (HMGB1) in renal ischemia-reperfusion injury (IRI) model in mice, and to block the protective effect of HMGB1 on renal IRI in mice.
[method] using BALB/C mice as experimental subjects, renal ischemia 24 hours before and 30 minutes before the experimental group (n=7) intraperitoneal injection of anti mouse HMGB1 antibody in rabbits respectively, while the control group (n=7) with normal saline as a reference. In the block 60min in left renal blood flow, renal blood flow and recovery of left resection of the right kidney of mice, restore blood supply or 0,3,6 reperfusion after 24 hours in each time point (n=2) were collected from the peripheral blood and left kidney. Determination of serum creatinine (sCr) and urea nitrogen (BUN), to observe the pathological change of kidney tissue (PAS), the expression of HMGB1 in renal tissue by immunohistochemistry Western and blot, and the detection of anti HMGB1 antibody deposition, immunohistochemical detection of renal tissue myeloperoxidase (MPO) expression and expression of TNF-a by immunofluorescence, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) of renal cell apoptosis detection.
[results] the normal group in the kidney of HMGB1 was expressed in the nucleus, while the control group, HMGB1 was expressed in the nucleus, and in ischemia after a nuclear expression, with the prolongation of reperfusion (0,3,6,24 hours), the expression of HMGB1 increased and the area more widely distributed (extracellular) control group kidney. After 24 hours of ischemia reperfusion, serum Cr and BUN were (82.46 + 30.13) mol/L and (46.46 + 9.61) mol/L was significantly higher than that of the experimental group (32.24 + 22.51) mol/L and (25.81 + 11.41) mol/L (P0.05). The normal kidney and renal tissue in the control group did not detect the HMGB1 antibody the experimental group showed obvious deposition, deposition of.PAS HMGB1 antibody using anti HMGB1 antibody showed that the control group of renal tubular cell necrosis, tube formation, and a large amount of bleeding, compared with anti HMGB1 degree of pathological changes in group therapy. Control group significantly reduced renal tissue MPO expression was significantly stronger than the real HMGB1 At the same time, the cell apoptosis in the control group was significantly higher than that in the experimental group, and the number of apoptotic cells was statistically different (P0.05): the cytokine TNF- alpha in the renal tissue of the control group was also significantly stronger than that of the experimental group.
[conclusion]HMGB1 plays an important role in renal IRI, and anti HMGB1 antibody before ischemia can significantly reduce IRI in mice. This protective effect may be mediated by neutralizing inflammatory factor HMGB1 to inhibit inflammatory reaction in IRI and reduce inflammation induced apoptosis.
The third part of anti HMGB1 neutralizing antibody prolongs the survival of rat xenotransplantation in mice
[Objective] to explore the effect of HMGB1 on cardiac transplantation of neonatal rats to xenograft heart in mice, and whether blocking HMGB1 by anti HMGB1 antibody can prolong the survival of xenograft heart, and elucidate its mechanism.
[Methods] to 1 week old SD rats as donor heart donors and BALB/C mice as recipients of heart transplantation, implementation of cervical heart heterotopic heart transplantation, antibody treatment group (n=10) in heart transplantation in 1 days before the day after intraperitoneal injection of Rabbit anti mouse HMGB1 antibody, and the control group (n=7) without any treatment. Postoperative survival time, the transplanted heart stopped beating for rejection were collected from the heart and serum, the treatment group and 3 cases in sixth days were collected (compared to group) and the control group, pathological detection (HE), immunohistochemical observation of tissue MPO, HMGB1 expression and IgG TLT4 IgM antibody, and the deposition of complement regulatory protein CD55 and CD59, the expression of JG12 by immunofluorescence, cell apoptosis detection of cardiac tissue by TUNEL method, analysis of circulating antibody IgG, flow cytometry and IgM level.
[results] the hearts in control group survived for 5-6 days, the antibody treatment group was 7-11 days, significantly longer than the control group (P0.01). The pathological control group was found on the rejection of cardiac acute vascular rejection characteristics, while the treatment group were significantly lighter than the former disease; at the same time by cell counting showed anti HMGB1 antibody treatment group significantly compared with the control group decreased MPO positive cell infiltration, TUNEL cell apoptosis or necrosis significantly reduced, with statistical significant difference (P0.05), and the integrity of endothelium specific marker JG12 was significantly stronger than the control group; the other group showed antibody treatment group in heart tissue HMGB1 and its receptor TLR4 expression was weak in the control group. Flow cytometry and immunohistochemistry showed that the control group IgG antibody in heart tissue and in the circulation increased significantly, and the production and deposition of IgG in the treatment group over the same period the antibody significantly weakened. In addition, Immunohistochemistry showed that CD55 and CD59 in the heart tissues of the control group were significantly weaker than those in the normal heart and the experimental group.
[Conclusion] this study demonstrated for the first time, antagonist HMGB1 could significantly prolong the survival of heart xenotransplantation in mice to rats, the possible mechanism is the release of inflammatory cells by neutralizing HMGB1, thereby inhibiting a series of inflammatory reaction, xenogeneic antibodies and enhanced repression of complement regulatory protein expression.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

【共引文献】