本文關(guān)鍵詞： 高遷移率族蛋白Bl 抗HMGB1抗體 缺血 再灌注損傷 腎 小鼠 HMGB1 抗HMGB1抗體 異種心臟移植 大鼠 小鼠　出處：《華中科技大學(xué)》2010年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分上調(diào)HO-1對(duì)缺血再灌注損傷的保護(hù)機(jī)制與抑制HMGB1釋放有關(guān) [目的]本課題組已經(jīng)發(fā)現(xiàn)Copp誘導(dǎo)HO-1高表達(dá)能減輕大鼠腎臟缺血再灌注損傷,目前實(shí)驗(yàn)為了進(jìn)一步深入研究保護(hù)性基因HO-1在減輕大鼠腎臟缺血再灌注損傷中的作用及其機(jī)制,探討HMGB1在其中是否發(fā)揮炎癥因子效應(yīng)。 [方法]課題前期實(shí)驗(yàn)以Wistar大鼠為對(duì)象,建立大鼠腎臟在體原位缺血再灌注損傷模型,夾閉大鼠左側(cè)腎蒂阻斷血供47分鐘,恢復(fù)血流后切除右腎。實(shí)驗(yàn)分三組：1)缺血對(duì)照組：缺血再灌后無處理；2)CoPP治療組：手術(shù)前48h和24h分別腹腔內(nèi)注射CoPP (2.5mg/kg);3)正常組。左腎恢復(fù)血供即再灌注24h后檢測(cè)血清肌酐(Cr)和尿素氮(BUN)值,并取正常及缺血再灌注腎臟標(biāo)本行HE病理檢查,Western-blot檢測(cè)腎臟組織中HO-1表達(dá),觀察CoPP治療對(duì)腎臟長(zhǎng)時(shí)間缺血(80min)再灌注后大鼠存活的影響。目前實(shí)驗(yàn)以免疫組化檢測(cè)其腎組織內(nèi)HMGB1、TLR4、MPO、TNF-α的表達(dá)情況,以及TUNEL法檢測(cè)細(xì)胞凋亡并計(jì)數(shù)。 [結(jié)果]CoPP治療組與對(duì)照組相比腎功能(Cr和BUN)明顯改善(p0.05),存活實(shí)驗(yàn)發(fā)現(xiàn)CoPP治療組5只大鼠全部存活,而對(duì)照組6只大鼠中,4只分別于觀察第5、5、7、13天死亡,兩組具有明顯差異(p0.05)。CoPP治療組腎組織中HMGB1及TLR4表達(dá)明顯弱于對(duì)照組,且代表單核細(xì)胞標(biāo)志的MPO及細(xì)胞因子TNF-α也同樣弱于對(duì)照組。細(xì)胞凋亡情況通過統(tǒng)計(jì)后對(duì)照組明顯高于HO-1保護(hù)組,并具有統(tǒng)計(jì)學(xué)差異(p0.05)。 [結(jié)論]CoPP可效誘導(dǎo)腎臟HO-1高表達(dá)保護(hù)大鼠腎臟缺血再灌注損傷,其作用機(jī)制可能與抑制HMGB1的釋放有關(guān),進(jìn)一步抑制炎癥反應(yīng),減輕細(xì)胞凋亡,進(jìn)而減少損傷。 第二部分抗HMGB1中和抗體對(duì)小鼠腎臟缺血再灌注損傷的保護(hù)作用 [目的]探討高遷移率簇蛋白B1 (HMGB1)在小鼠腎臟缺血再灌注損傷(IRI)模型中的作用,通過抗體阻斷HMGB1研究對(duì)小鼠腎臟IRI的保護(hù)作用。 [方法]以BALB/C小鼠為實(shí)驗(yàn)對(duì)象,腎缺血前24小時(shí)和前30分鐘實(shí)驗(yàn)組(n=7)分別于腹腔注射兔抗小鼠HMGB1抗體,而對(duì)照組(n=7)以等量生理鹽水替代作為參照。在體阻斷小鼠左腎血流60min,恢復(fù)左腎血流的同時(shí)切除小鼠右腎,恢復(fù)血供再灌注0,3,6或24小時(shí)后,在每組每個(gè)時(shí)間點(diǎn)(n=2)分別取其外周血及左腎。測(cè)定血清肌酐(sCr)和尿素氮(BUN)水平,觀察腎組織學(xué)變化(PAS),免疫組化及Western blot檢測(cè)腎組織中HMGB1的表達(dá),并檢測(cè)抗HMGB1抗體的沉積,免疫組化檢測(cè)腎組織中髓過氧化物酶(MPO)表達(dá),免疫熒光檢測(cè)TNF-a表達(dá),原位末端脫氧核苷酸轉(zhuǎn)移酶標(biāo)記法(TUNEL)檢測(cè)腎組織中細(xì)胞凋亡情況。 [結(jié)果]正常組腎臟內(nèi)HMGB1均表達(dá)于細(xì)胞核內(nèi),而對(duì)照組HMGB1則在核外明顯表達(dá),且在缺血后即有核外表達(dá),隨著再灌注時(shí)間延長(zhǎng)(分別為0,3,6,24小時(shí)),HMGB1表達(dá)量增多且部位分布更廣(細(xì)胞外)。對(duì)照組腎臟缺血再灌注24小時(shí)后血清Cr及BUN分別為(82.46±30.13)μmol/L和(46.46±9.61)μmol/L顯著高于實(shí)驗(yàn)組的(32.24±22.51)μmol/L和(25.81±11.41)μmol/L(p0.05)。正常腎及對(duì)照組腎組織中未檢測(cè)到HMGB1抗體沉積,使用抗HMGB1抗體的實(shí)驗(yàn)組可見明顯HMGB1抗體沉積。PAS顯示對(duì)照組腎小管細(xì)胞大片壞死,管型形成,并有大量出血,與之相比,抗HMGB1治療組腎組織病變程度明顯減輕。對(duì)照組腎組織中MPO表達(dá)明顯強(qiáng)于使用HMGB1的實(shí)驗(yàn)組,同時(shí)對(duì)照組細(xì)胞凋亡較實(shí)驗(yàn)組顯著增多,凋亡細(xì)胞數(shù)計(jì)數(shù)具有統(tǒng)計(jì)學(xué)差異(p0.05)：對(duì)照組腎組織中細(xì)胞因子TNF-α也明顯強(qiáng)于實(shí)驗(yàn)組。 [結(jié)論]HMGB1在腎臟IRI中發(fā)揮重要作用,而缺血前使用抗HMGB1抗體可明顯減輕小鼠IRI,這種保護(hù)作用可能通過中和炎性因子HMGB1來抑制腎臟IRI中的炎癥反應(yīng),并減少炎癥導(dǎo)致的凋亡來實(shí)現(xiàn)。 第三部分抗HMGB1中和抗體延長(zhǎng)大鼠到小鼠異種心臟移植存活的實(shí)驗(yàn)研究 [目的]探討HMGB1對(duì)新生大鼠心臟移植到小鼠異種移植心的作用,及其通過抗HMGB1抗體阻斷HMGB1能否延長(zhǎng)異種移植心存活,并闡明其機(jī)制。 [方法]以1周齡SD大鼠為供心供者,BALB/C小鼠為移植心受者,實(shí)施頸部心異位心臟移植,抗體治療組(n=10)于心臟移植前1天起隔天腹腔注射兔抗小鼠HMGB1抗體,而對(duì)照組(n=7)不做任何處理。術(shù)后觀察移植心存活時(shí)間,移植心停止搏動(dòng)即為發(fā)生排斥時(shí)留取心臟及血清,治療組另有3例行在第6天留取標(biāo)本(與對(duì)照組進(jìn)行同期對(duì)組),進(jìn)行病理學(xué)檢測(cè)(HE),免疫組化觀察組織中MPO, HMGB1, TLT4的表達(dá)及抗體IgG、IgM的沉積,以及補(bǔ)體調(diào)節(jié)蛋白CD55和CD59,免疫熒光檢測(cè)JG12表達(dá),TUNEL法檢測(cè)心臟組織內(nèi)細(xì)胞凋亡情況,流式細(xì)胞儀分析循環(huán)中抗體IgG、IgM水平。 [結(jié)果]對(duì)照組心臟存活5-6天,抗體治療組為7-11天,明顯長(zhǎng)于對(duì)照組(p0.01)。組織病理上發(fā)現(xiàn)對(duì)照組排斥心臟呈急性血管性排斥反應(yīng)特征,而治療組同期的病變明顯輕于前者；同時(shí)通過細(xì)胞計(jì)數(shù)顯示抗HMGB1抗體治療組較對(duì)照組MPO陽性的浸潤(rùn)細(xì)胞顯著減少,TUNEL顯示細(xì)胞凋亡或壞死顯著減輕,統(tǒng)計(jì)具有顯著性差異(p0.05),而血管內(nèi)皮完整性的特異性標(biāo)志JG12則顯著強(qiáng)于對(duì)照組；另外組化顯示抗體治療組心臟組織內(nèi)HMGB1及其受體TLR4表達(dá)均明顯弱于對(duì)照組。流式檢測(cè)以及免疫組化均顯示對(duì)照組心臟組織內(nèi)和循環(huán)中的異種抗體IgG顯著增加,而同期的抗體治療組IgG的產(chǎn)生和沉積顯著減弱。此外,免疫組化顯示對(duì)照組心臟組織中CD55和CD59明顯弱于正常心臟及實(shí)驗(yàn)組。 [結(jié)論]本研究首次證明,拮抗HMGB1可明顯延長(zhǎng)大鼠到小鼠異種移植心臟的存活,其可能的機(jī)制是通過中和炎性細(xì)胞釋放的HMGB1,從而抑制一系列炎癥反應(yīng),阻遏異種抗體的產(chǎn)生并增強(qiáng)補(bǔ)體調(diào)節(jié)蛋白表達(dá)。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 許永華;李長(zhǎng)賢;陳杰;姚愛華;李相成;;高遷移率族蛋白A1(HMGA1)在肝細(xì)胞癌中的表達(dá)及臨床意義[J];南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版);2010年03期

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1 章保新;五加苷元對(duì)D-GaIN/LPS誘導(dǎo)的暴發(fā)型肝衰竭的保護(hù)作用及其機(jī)制初步研究[D];中南大學(xué);2011年

2 董月青;高遷移率族蛋白B1誘導(dǎo)腦膜瘤發(fā)生及侵襲生長(zhǎng)的機(jī)制研究[D];中國人民解放軍軍醫(yī)進(jìn)修學(xué)院;2008年

3 龔權(quán);HMGB1參與實(shí)驗(yàn)性急性肝、肺損傷的作用及其機(jī)制[D];華中科技大學(xué);2009年

4 周蓉蓉;肝衰竭時(shí)肝細(xì)胞高遷移率族蛋白1的移位及釋放研究[D];中南大學(xué);2009年

5 姜飚;HMGB1在胃癌中表達(dá)及siRNA對(duì)胃癌細(xì)胞活性影響[D];天津醫(yī)科大學(xué);2013年

相關(guān)碩士學(xué)位論文 前5條

1 許永華;高遷移率族蛋白A1（HMGA1）在肝細(xì)胞癌中的表達(dá)及臨床意義[D];南京醫(yī)科大學(xué);2010年

2 石苗茜;HMGB1對(duì)心肌成纖維細(xì)胞增殖和Ⅰ、Ⅲ型膠原蛋白分泌的影響及意義[D];第四軍醫(yī)大學(xué);2011年

3 杜曉琴;HMGB1在宮頸癌組織、細(xì)胞中的表達(dá)及其與RAGE關(guān)系研究[D];天津醫(yī)科大學(xué);2008年

4 章雪蓮;HMGB1mRNA和蛋白在宮頸鱗癌組織中的表達(dá)及臨床意義研究[D];中南大學(xué);2008年

5 謝叢良;HMGB1在頭頸鱗癌中的表達(dá)及其與臨床預(yù)后的關(guān)系[D];中南大學(xué);2010年

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