【作者】 张松林；

【导师】 孙宗全；

【作者基本信息】 华中科技大学 ， 心血管外科学， 2010， 博士

【摘要】 第一部分应用Tail-cuff技术改良小鼠颈部异位心脏移植模型目的小鼠颈部心脏移植模型是研究移植缺血再灌注损伤和排斥反应的理想模型,由于显微血管吻合的技术问题,限制了其在医学研究中的广泛应用。在本实验室既往应用套管法(cuff technique)建立该模型的基础上,本研究应用Tail-cuff技术(带尾套管法)改良小鼠颈部异位心脏移植模型。方法昆明小鼠用作预实验,C57BL/6和BALB/c小鼠用作正式试验。应用24G和22G静脉内导管制作成带有尾巴的动脉或静脉套管(Tail-cuff),在准备受体血管时,Tail-cuff通过右颈外静脉或右颈总动脉近心端后,用显微血管夹一起固定其近心端和Tail-cuff管的尾巴,翻转血管并固定后分别与供心的肺动脉主干和升主动脉连接固定。结果应用这种改良技术,预实验手术成功率为72.2%(13/18),总操作时间为56.2±8.9min。正式实验总操作时间为49.6±7.4 min,血管翻转和与供心连接时间分别为3.6±1.4min和4.1±1.1 min,正式实验(12例同系移植,24例同种移植)的成功率为100%(36/36)。结论应用Tail-cuff技术建立小鼠颈部异位心脏移植模型显示出一定的优越性,是一种理想的方法。第二部分诱导受体产生一氧化碳减轻小鼠移植心的冷缺血再灌注损伤目的一氧化碳(carbon monoxide, CO)是血红素加氧酶催化亚铁血红素生成的代谢产物之一,对大鼠肝、肺、肾和小肠的缺血再灌注损伤(ischemia/reperfusion injury, IRI)具有明显的保护作用。本研究在小鼠心脏移植模型的基础上,观察以二氯甲烷(methylene chloride, MC)诱导受体小鼠产生CO对移植心冷IRI的影响,并探讨其可能的机制。方法以Balb/c小鼠作为供、受者,建立颈部异位心脏移植模型。实验共分为4组,分别为MC100mg组(n=10)、MC500mg组(n=12)、橄榄油组(IR组,n=10)和正常对照组(N,n=5)。前3组在麻醉前3 h分别用经橄榄油稀释的MC100mg/kg、MC500mg/kg以及单纯橄榄油0.15ml(IR组)对受者进行灌胃处理,然后行颈部异位心脏移植；N组小鼠仅作麻醉处理,不进行心脏移植。分别于灌胃后0、1、3、6、12和24 h剪尾采血,测定血液中CO的含量[用碳氧血红蛋白(COHb)占总血红蛋白的百分比表示],并于相应时间点取心肌组织,检测心肌组织中CO的含量；各组移植后3h和24 h分别处死半数受者,检测移植后3h和24 h血清中肌钙蛋白I(cTnI)、心肌组织中肿瘤坏死因子α(TNF-α)、白细胞介素10(IL-10)、细胞凋亡与相关基因Bcl-2和Bax mRNA以及核因子κB(NF-κB)的表达,并观察移植心心肌的超微结构。结果与橄榄油灌胃比较,以MC100 mg和MC500 mg灌胃受者血液中COHb浓度与心肌组织中CO含量明显升高,均在灌胃后3 h达到峰值(P<0.01)。与IR组比较,MC100 mg和MC500 mg组受者心脏移植后3h和24 h,能显著降低血清中cTnI水平(P<0.01),并以MC500 mg组降低最为明显;可明显下调促炎症基因TNF-a mRNA水平(P<0.01),而抗炎症基因IL-10 mRNA上调不明显(P>0.05),同时可以显著上调抗凋亡基因Bcl-2 mRNA水平(P<0.01),并抑制促凋亡基因Bax mRNA的转录(P<0.01)；心肌超微结构损伤明显减轻,以MC500mg组最为显著。正常对照组可见少量NF-κB表达,而IR组、MC100mg组和MC500 mg组NF-κB活性均显著增强(P<0.01),但后三组之间在相同时间点的NF-κB活性无明显区别(P>0.05)。结论诱导受体小鼠产生CO明显减轻小鼠移植心冷IRI,其主要机理与CO的抗炎和抗凋亡作用有关,且不受NF-κB信号转导通路的调节。第三部分诱导受体产生一氧化碳经PI3K/Akt信号途径抑制冷缺血再灌注诱导的移植心细胞凋亡目的CO是体内的一种重要信号分子。外源性低浓度的CO具有缓解血管损伤、排斥反应和急性肺损伤等功能,但涉及到的信号机理尚未完全阐明。本研究在小鼠心脏移植的基础上,观察诱导受体产生CO对移植心冷IRI中细胞凋亡的影响,并探讨其可能的信号机制。方法以Balb/c小鼠建立同系移植心冷IRI模型。受体用二氯甲烷(MC,500mg/kg体重)灌胃诱导受体小鼠产生CO(MC组,n=12),或用橄榄油灌胃(IR组,n=12),在MC组的基础上受体在移植心恢复血供前1h腹腔注射PI3K抑制剂LY294002(40mg/kg,LY组,n=10)或二甲亚砜(DMSO组,n=10)；检测移植后3h和24h(每一时间点n=5～6／组)移植心细胞凋亡并计算凋亡指数(AI)、磷酸化Akt(p-Akt)蛋白、Bcl-2与Bax蛋白的表达及Caspase-3蛋白酶活性;设立正常对照组(N组,n=5)。结果受体以MC灌胃后血液中COHb浓度与心肌组织CO含量均在3h达到峰值,分别为(9.82±0.84)%和2.25±0.08 pmol/mg；与IR组比较,MC组明显降低移植心AI[3h:(8.65±2.01)％vs.(19.28±4.94)％,P<0.01；24h:(5.82±2.36)％vs.(10.54±3.66)％,P<0.05].激活Akt蛋白(3h:P<0.01；24h:P<0.05).抑制Caspase-3蛋白酶活性(3h:2.19±0.18 vs.3.31±0.36,P<0.05)、上调Bcl.2/Bax比值(3h：1.97±0.16比0.46±0.07,P<0.01；24h：1.89±0.10比0.51±0.04,P<0.01)；而与MC组比较,LY组明显增加AI[3h:(17.95±4.92)％,P<0.01:24h:(9.75±3.14)%；P<0.01]、抑制Akt蛋白激活(3h：P<0.01；24：P<0.05)、增强Caspase-3蛋白酶活性(3h：3.27±0.24,P<0.05)、下调Bcl.2/Bax比值(3h：0.47±0.06,P<0.01；24h:0.52±0.03,P<0.01),从而逆转其抗凋亡作用；DMSO组与MC组的各个指标无明显统计学意义(P>0.05)。结论诱导受体产生CO能明显抑制冷缺血再灌注诱导的移植心的细胞凋亡,其机理可能与通过P13K/Akt信号途径对凋亡相关蛋白Caspase-3、Bcl-2和Bax的调节有关。第四部分诱导受体产生一氧化碳减轻同种移植心排斥反应不依赖于Foxp3表达的上调目的CO由于具有强大的干扰体内氧运输的特性而通常被认为是一种有害的废弃物。事实上,低浓度的CO通过调节血管张力、抗炎、抗凋亡等作用缓解移植物缺血再灌注损伤。本研究在小鼠心脏移植的基础上,观察诱导受体小鼠产生CO对同种移植排斥反应和移植心存活时间的影响,并探讨其可能的机制。方法建立小鼠颈部异位心脏移植模型(C57BL/6→Balb/c小鼠)。受体小鼠自移植前1d至移植后第6d(MC1w组,n=11)或至第13d(MC2w组,n=10)以含二氯甲烷(MC)100mg/kg体重灌胃,或以同体积不含MC的橄榄油灌胃(Tx组,n=6)。观测移植心存活时间,并于移植后6d、10d分别检测受体血清TNF-α、IL-10的含量、心脏移植物和受体脾脏TNF-α、IL-10、Foxp3 mRNA及Foxp3蛋白的表达、心脏移植物ICAM-1(细胞间粘附分子-1)及Caspase-3蛋白的表达；观察移植后6d、10d或移植物心跳停止时移植心胶原纤维增生程度及组织病理学变化。结果受体以MC灌胃后血液中COHb浓度在3h达到峰值,为(5.24±0.45)%；受体诱导CO可以明显延长移植心存活时间[Tx:(6.33±0.56)d; MC1w: (12.14±0.87)d, P<0.01vs.Tx; MC2w组：(19.38±0.95)d,P<0.01vs.Tx and MC1w];降低血清TNF-a的含量(MC1w组：P<0.01vs.Tx; MC2w组:P<0.01vs.Tx and P<0.05 vs. MC1w);抑制心脏移植物和受体脾脏TNF-αmRNA的表达(MC1w组：P<0.01vs.Tx; MC2w组：P<0.01vs.Tx and MC1w);抑制心脏移植物ICAM-1 (MC1w组及MC2w组：P<0.01vs.Tx)及Caspase-3的表达(MC1w组：P<0.01vs.Tx; MC2w组：P<0.01vs.Tx and MC1w);减轻移植物内胶原纤维增生及淋巴细胞和单核细胞侵润的程度,以MC2w组最为明显；各组血清IL-10含量、心脏移植物和受体脾脏IL-10mRNA、Foxp3 mRNA及Foxp3蛋白的表达无统计学意义(P>0.05)。结论受体小鼠诱导CO明显减轻同种移植排斥反应并延长移植心的存活时间,其主要机制与可能与CO的抗炎症和抗凋亡功能密切相关,而并不依赖于移植物和受体脾脏内Foxp3表达的上调。更多还原

【Abstract】 PartⅠImprovements of mouse cervical heterotopic heart transplantation model using the tail-cuff techniqueObjective The model of mouse cervical heart transplantation is an ideal medical research tool for study of transplant-induced ischemia/reperfusion injury and immunological rejection. However, technical problems have limited the widespread use of mouse cervical vascularized heart transplantation. The aim of this study is to describe an improved method for performing cervical heterotopic heart transplantation by the tail-cuff technique.Methods The kunming mice were used for preliminary experiment. And for formal experiment, inbred male C57BL/6 mice and Balb/c mice were used for isogeneic transplantation from BALB/c to BALB/c mice and allogeneic transplantation from C57BL/6 to BALB/c mice. The right common carotid artery and the external jugular vein of the recipient were equipped with a tail cuff made from 24G and 22G intravenous catheter, and everted over the cuff, and then connected with the aorta and the pulonary artery of donor heart, respectively. Results For preliminary experiment, the successful rate was 72.2%(13/18) and the the total surgical procedures took 56.2±8.9min. For formal experiment, the total surgical procedures took 49.6±7.4 min, the average time for evert and reconnection of both vessels was 3.6±1.4min and 4.1±1.1 min, and the successful rate was 100%(36/36).Conclusions This improved cuff technique shows its superiority, and can serve as an ideal method for the establishment of mouse cervical heterotopic heart transplantation model.Part IIInduction of carbon monoxide in recipients ameliorates cold ischemia/reperfusion injury in a murine heart transplantation modelObjective Carbon monoxide (CO), a product of heme degradation by heme oxygenase, induces cytoprotection against ischemia/reperfusion (I/R) injury in a variety of organs such as the liver, lung, kidney, and small intestine. The aim of this study is to examine whether induction of carbon monoxide (CO) with methylene chloride (MC) in recipients would protect heart grafts against cold I/R injury associated with transplantation and explore the possible mechanisam in a murine heart transplantation model..Methods Inbred male Balb/c mice were used as donors and recipients to establish transplant-induced cold I/R injury model with 2 hours cold preservation. Recipients were treated with either methylene chloride (MC) (100mg/kg or 500mg/kg, per os)(Group MC100mg, n=10; Group MC 500mg, n=12) or olive oil(0.15ml, per os, Group IR, n=10) 3h prior to anesthesia. Age-matched normal mice served as controls(Group N, n=5). The serum COHb and the CO content of myocardial tissue were measured at Oh, 1h,3h,6h,12h, 24h after oral MC administration. Half of Recipients mice were killed at 3h and 24h after transplantation for serum or cardiac graft samples(n=5-6 per time point). Recipients mice were killed at 3h and 24h after transplantation for serum or cardiac graft samples. The serum cTnI levels, the mRNA levels of TNF-α、IL-10、Bcl-2、Bax, the protein of NF-κB and the ultrastructure of myocardium were measured, respectively.Results The serum COHb and tissue CO increased significantly and peaked within 3h in Group MC100mg and Group MC 500mg(P<0.01 vs Olive oil). Compared with Group IR, The serum cTnI levels in Group MC100mg and Group 500mg were significantly decreased(P<0.01), especially in Group 500mg. Induction of CO in recipients in Group MC100mg and Group MC 500mg significantly inhibited the pro-inflammatory gene expression of TNF-a mRNA and the pro-apoptotic gene expression of Bax mRNA(P<0.01), and increased the anti-apoptotic gene expression of Bcl-2 mRNA(P<0.01), but did not increase the anti-inflammatory gene expression of IL-10 mRNA(P>0.05) in cardiac grafts. Compared with Group N, the myocardial NF-κB activation increased significantly in Group Olive, Group MC100mg and Group MC500mg(P<0.01), but did not differ between the later three groups at the same point(P>0.05).The myocardial ultrastructure was also improved significantly in Group MC100mg and Group MC500mg, especially in Group MC500mg.Conclusions Induction of CO in recipients with MC suppresses pro-inflammatory and pro-apoptotic gene expression and efficiently ameliorates transplant-induced heart cold I/R injury. The possible mechanism does not seem to be associated with down-regulation of the NF-κB signaling pathway. Part IIIInduction of carbon monoxide in recipient mice inhibits cold ischemia/reperfusion-induced apoptosis of cardiac grafts via PI3K/Akt signal pathwayObjective Carbon monoxide (CO) is emerging as an important gaseous molecule. And exogenous low-dose CO protects against vascular injury, transplant rejection and acute lung injury. However, little is known regarding the precise signaling mechanisms of CO. The aim of this study is to observe whether induction of carbon monoxide in recipients could inhibit cold ischemia/reperfusion(I/R)-induced apoptosis of cardiac grafts and to explore the possible anti-apoptotic signaling mechanisms in a murine heart transplantation model.Methods Inbred Balb/c mice were used as donors and recipients to establish transplant-induced cold I/R injury model. Recipients were treated with either methylene chloride (500mg/kg, per os, Group MC, n=12) or olive oil(Group IR, n=12) 3h prior to anesthesia, or treated with MC plus either LY294002 of PI3K inhibitor (40mg/kg, i.p, Group LY, n=10) or DMSO(Group DMSO, n=10) lh prior to reperfusion of cardiac grafts. Recipients mice were killed at 3h and 24h after transplantation for cardiac graft samples(n=5-6 for each time point). The apoptosis index(AI), the protein of phosphorylated, the activities of Caspase-3, the protein of Bcl-2 and Bax were measured, respectively. Age-matched normal mice served as controls(Group N).Results Following MC application serum COHb[(9.82±0.84)%]and tissue CO (2.25±0.08 pmol/mg) peaked within 3h in recipients. Compared with Group IR, induction of CO in recipients decreased significantly the level of AI [3h:(8.65±2.01)%vs. (19.28±4.94)%, P<0.01; 24h:(5.82±2.36)%vs.(10.54±3.66)%, P<0.05], increased the Akt phosphorylation (3h:P<0.01; 24h:P<0.05), inhibited the activities of Caspase-3(3h:2.19±0.18 vs.3.31±0.36, P<0.05)and up-regulated the ratios of Bcl-2/Bax(3h:1.97±0.16 vs.0.46±0.07, P<0.01; 24h:1.89±0.10 vs.0.51±0.04, P<0.01) in cardiac grafts. However, compared with Group MC, recipients pretreated with LY294002 could reverse the anti-apoptotic effects of MC by increasing the level of AI[3h:(17.95±4.92)%, P<0.01; 24h:(9.75±3.14)%, P<0.01], inhibiting the Akt phosphorylation(3h:P<0.01; 24:P<0.05), activating Caspase-3(3h:3.27±0.24, P<0.05) and down-regulating the ratio of Bcl-2/Bax (3h:0.47±0.06, P<0.01; 24h:0.52±0.03, P<0.01). And the DMSO had no impact on the anti-apoptotic effects of MC(P>0.05).Conclusion Induction of carbon monoxide in recipient mice inhibits cold I/R-induced apoptosis of cardiac grafts by regulating caspase-3, Bcl-2 and Bax proteins via the activation of PI3K/Akt signal pathway. Part IVAmelioration of cardiac allograft rejection by induction of carbon monoxide with methylene chloride in recipients is independent of up-regulation of Foxp3 expression in miceObjective Carbon monoxide (CO) is commonly viewed as a poison in high concentrations due to its ability to interfere with oxygen delivery. However, exogenous low-dose carbon monoxide inhibits transplant-induced ischemia/reperfusion injury via its vasodilation, anti-inflammatory and anti-apoptotic effects in various models. The aim of this study is to investigate the protective effects of induction of CO in recipients on amelioration of allograft rejection and prolongation of allograft survival and the possible mechanisms in a mouse heart transplantation model.Methods Inbred male C57BL/6 mice were used as donors and inbred male Balb/c mice were used as recipients to establish allogenic heart transplantation model. Recipients were treated with either methylene chloride (100mg/kg, per os, Group MC) or olive oil(Group Tx, n=6) day 1 prior to transplantation to day 6 posttransplantation (Group MC1w, n=11) or to day 13 posttransplantation (Group MC2w, n=10). The survival of cardiac grafts was recorded. Recipients mice were killed at day 6 and day 10 or the time of non-heartbeating after transplantation for serum, cardiac graft and spleen samples(n=5-6 for each time point). The serum TNF-a and IL-10, the TNF-a mRNA and IL-10 mRNA, the Foxp3 mRNA and Foxp3 protein in cardiac grafts and spleen were measured, respectively. The intercellular adhesion molecule-1(ICAM-1) and Caspase-3 protein in cardiac grafts, the proliferation of collagen fibers and histopathologic changes of cardiac grafts were also recorded. Results Following MC application serum COHb peaked within 3h in recipients[(5.24±0.45)%]. Induction of CO in recipients prolonged significantly the cardiac graft survival(median survival time, MST) [Tx:(6.33±0.56)d; MC1w: (12.14±0.87)d, P<0.01vs.Tx; MC2w:(19.38±0.95)d, P<0.01vs.Tx and MC1w], down-regulated the levels of serum TNF-a(MC1w:P<0.01vs.Tx; MC2w: P<0.01vs.Tx and P<0.05 vs. MC1w)and TNF-a mRNA (MC1w:P<0.01vs.Tx; MC2w: P<0.01vs.Tx and MC1w)of cardiac grafts and spleen in recipient mice, inhibited the protein expression of ICAM-1(MC1w and MC2w: P<0.01vs.Tx) and Caspase-3(MC1w:P<0.01vs.Tx; MC2w:P<0.01vs.Tx and MC1w) of cardiac grafts, and inhibited, especially in Group MC2w, the proliferation of collagen fibers and lymphocyte and monocyte infiltration in cardiac grafts. The levels of serum IL-10, the IL-10 mRNA, the Foxp3 mRNA and the Foxp3 protein in cardiac grafts and spleen of recipients did not differ between the groups(P>0.05).Conclusions Induction of carbon monoxide in recipients could relieve cardiac allograft rejection and prolong cardiac allograft survival via its anti-inflammation and anti-apoptotic effects, not via up-regulation of Foxp3 mRNA and Foxp3 protein in cardiac grafts and spleen in recipient mice.更多还原