大鼠心脏移植排斥反应时细胞间粘附分了—1的表达及霉酚酯对其的抑制作用

目的 探讨大鼠同种异位心脏移植排斥反应期间移植心组织细胞间粘附分子－1（ICAM－1）的表达霉酚酯酯（MMF）对移植心ICAM－1表达和排斥反应的抑制作用。方法 建立大鼠心脏腹腔移植模型。设Wistar 到Wistar大鼠的同系心脏移植对照组、SD大鼠到Wistar大鼠的同种移植组和同种移植MMF治疗组。采集移植心组织标本行免疫组织化学和组织病理学检查,应用多媒体彩色图文分析系统对移植心组织ICAM－1的表达进行定量检测。结果 对照组移植心组织ICAM－1表达较弱;同种移植组在排斥反应期间移植心组织毛细血管内皮细胞ICAM－1的表达强度和数量明显增加,且伴有大量淋巴细胞浸润;MMF治疗组移植心仅微弱表达ICAM－1,同时少有淋巴细胞浸润;检测移植心组织ICAM－1的表达改变比普通组织病理学检查可以提早2－3d发现排斥反应。结论 ICAM－1的表达水平与排斥反应的发生和发展有关;MMF能显著抑制移植心ICAM－1的表达和淋巴细胞的浸润,明显延长移植物的存活。     

大鼠异体肢体移植术后急性排斥反应阶段血管内皮细胞ICAM-1表达的动态变化

目的 研究大鼠异体肢体移植术后急性排斥反应阶段,移植肢体血管内皮细胞细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)动态变化及环孢素A(cyclosporine A,CsA)抗免疫排斥的作用.方法 建立大鼠肢体移植动物模型,随机分为对照组(Wistar大鼠→Wistar大鼠)、排斥组(SD大鼠→ Wistar大鼠)和CsA治疗组(SD大鼠→Wistar大鼠),术后1、4、7 d获取移植肢体股动脉行病理学观察,采用免疫组化法检测移植肢体血管ICAM-1表达的变化.结果 对照组供体移植肢体股动脉血管内皮细胞仅出现轻微肿胀与ICAM-1表达微弱;排斥组血管内皮细胞肿胀明显,淋巴细胞大量浸润,ICAM-1的表达强度和数量明显增加;CsA治疗组移植肢体血管内皮细胞仅有少量淋巴细胞浸润,ICAM-1表达较弱.结论 大鼠异体肢体移植术后急性排斥反应阶段,血管内皮细胞ICAM-1表达水平与排斥反应的发生和发展有关,CsA可降低移植肢体血管内皮细胞ICAM-1表达,抑制复合组织移植术后急性排斥反应.     

大鼠接受小鼠异种心脏移植后粘附分子ICAM-1的表达

目的观察大鼠接受小鼠异种心脏移植后延迟性排斥反应(DXR)的病理特征,研究异种心脏移植中粘附分子ICAM-1的表达变化,探讨DXR的发生机制。方法建立Lewis大鼠接受BALB/c小鼠异种心脏移植模型。随机将接受异种心脏移植的大鼠分为5组,每组5只,分别于移植后12、24、36、48 h及移植心停跳时切取移植心;另取5只术前健康雄性BALB/c小鼠心脏作为对照。进行病理学检查、免疫组织化学检测及图象分析定量,研究供者移植心组织中ICAM-1的表达,以及应用半定量逆转录-聚合酶链式(RT-PCR)方法检测移植心组织中ICAM-1 mRNA的表达。结果病理学检查显示移植后12 h,移植心组织中即有间质充血、出血以及少量的炎性细胞浸润;随时间的推移病变不断加重,直至排斥反应的终点呈现典型的DXR。术前对照组心脏组织中ICAM-1表达微弱;异种心脏移植后随着时间的推移,心脏组织中ICAM-1的表达不断增强。半定量RT-PCR结果显示,移植心组织中ICAM-1 mRNA亦随时间的推移进行性增高。结论异种心脏移植后,移植心组织中供者型ICAM-1的表达明显增强,与DXR的发生和发展密切相关;供者型ICAM-1表达增强可较早提示DXR的发生。     

细胞间粘附分子-1的反义寡核苷酸在小鼠心脏移植中的作用

目的 探讨细胞间粘附分子-1(ICAM-1)的反义寡核苷酸(ICAM-1-ASO)在小鼠心脏移植中对ICAM-1表达以及在排斥反应中的作用.方法 以BALB/c小鼠为供者,C57小鼠为受者,建立颈部异位心脏移植模型,移植后将受者随机分为3组,每组10只.分别为单纯移植组、对照寡核苷酸组(对照ODN)和ICAM-1-ASO组.各组移植后24 h开始经受者静脉分别注射双蒸水0.3 ml、对照ODN 10 mg/kg和ICAM-1-ASO 10 mg/kg,每天1次,连续5 d.移植后第8天各组随机取5只受者切取移植心,进行病理学检查;采用免疫组织化学方法对移植心组织中ICAM-1的表达进行观察;采用半定量逆转录聚合酶链反应(RT-PCR)对ICAM-1mRNA表达进行定量分析.每组中余下的小鼠继续观察直至移植心停跳,记录各组移植心的存活时间.结果 单纯移植组心脏移植后第8天可观察到明显的排斥反应,移植心心跳减慢、心律不齐、心律紊乱、搏动无力;病理学检查可见大量的淋巴细胞浸润,心肌间质水肿及大片心肌坏死;与移植前对比,内皮细胞和心肌细胞ICAM-1表达明显增强;ICAM-1mRNA表达也明显增加;移植心存活时间平均为(9.8±1.48)d.对照ODN组与单纯移植组差异无统计学意义.而ICAM-1-ASO组与单纯移植组相比,移植心ICAM-1的表达明显减弱,淋巴细胞浸润明显减少,排斥反应程度减轻;移植后第9天心律基本规整,偶有心律不齐,心跳力度较单纯移植组增强;移植心存活时间明显延长至(14.6±1.14)d.结论 ICAM-1-ASO抑制移植心组织中ICAM-1的表达和淋巴细胞的浸润,抑制移植后排斥反应的发生和发展,改善移植物的功能,延长移植物的存活时间,其作用具有序列特异性.     

鼠纤维介素在小鼠心脏移植急性排斥反应中的表达及其意义

目的　探讨小鼠同种心脏移植急性排斥反应期间鼠纤维介素 (mfgl2 )在移植心脏组织中的表达及其与组织病理学改变的关系。方法　采用BALB/c小鼠到C5 7BL/ 6小鼠的颈部异位心脏移植作为同种排斥反应模型 ,以抗mgfl2多克隆抗体干预 ,并设同系小鼠心脏移植对照组。采集移植心组织标本作病理学检查 ,以免疫组织化学方法测定mfgl2在移植心脏组织细胞上的表达 ,并对mgfl2的表达进行半定量分析。结果　同系移植对照组移植心脏组织结构正常 ,未见mfgl2表达 ;同种移植组移植心脏组织出现进行性坏死 ,大量单个核细胞浸润 ,并伴有mfgl2的表达 ,且血管内皮细胞表达mfgl2 ;抗mfgl2抗体干预组移植心脏组织损伤较轻 ,移植物的存活时间延长 (P <0 .0 1) ,巨噬细胞、淋巴细胞的浸润和mfgl2表达量也显著减少。结论　mfgl2表达水平与排斥反应所致移植心脏病理损害程度相关 ;抗mfgl2抗体干预能显著减少移植心脏巨噬细胞、淋巴细胞的浸润 ,明显延长移植心脏存活时间。     

血小板源性生长因子A在大鼠移植心脏血管病变及纤维化中的作用

目的 探讨血小板源性生长因子A(PDGF-A)在移植心脏血管病变及心肌纤维化中的作用.方法 选择近交系健康雄性Wistar大鼠及SD大鼠,建立大鼠异位心脏移植模型.实验分为四组,每组8只.正常对照组:取正常Wistar大鼠的心脏作为空白对照.无排斥组:心脏移植的供、受者均为Wistar大鼠,移植后第60天取移植心脏.急性排斥组和慢性排斥组:心脏移植的供者均为Wistar大鼠,受者均为SD大鼠;急性排斥组术后未行免疫抑制治疗,术后第5天取移植心脏;慢性排斥组术后给予环孢素A 10 mg·kg-1·d-1,皮下注射,移植后第60天取移植心脏.采用免疫组织化学染色法检测移植心脏的巨噬细胞浸润(CD68阳性细胞数)情况;逆转录聚合酶链反应(RT-PCR)分析PDGF-A mRNA的表达水平.结果 正常对照组和无排斥组未见巨噬细胞浸润;急性排斥组巨噬细胞浸润主要见于心肌及冠状血管周围;慢性排斥组巨噬细胞浸润见于心肌及血管周围,在心肌坏死纤维化较严重的区域,巨噬细胞浸润尤为明显;正常对照组、无排斥组、急性排斥组和慢性排斥组移植心组织中PDGF-A mRNA的相对表达量分别为:0.26±0.06、0.31±0.04、0.88±0.12和0.94±0.11,慢性排斥组和急性排斥组中PDGF-A mRNA的相对表达量明显高于正常对照组和无排斥组(P＜0.01).结论 巨噬细胞浸润及血小板源性生长因子表达水平与移植心脏血管病变及心肌纤维化有关.     

大鼠异种心脏移植中P选择素和细胞间粘附分子-1表达的意义

目的探讨大鼠异种心脏移植中P选择素和细胞间粘附分子-1(ICAM-1)表达的意义。方法建立金黄地鼠到SD大鼠的异种异位(腹部)心脏移植模型,然后将模型大鼠随机分为4组:对照组术后不采取任何治疗措施;环孢素A组(CsA组)于手术当天起每天腹腔注射CsA10mg/kg;脾切除组于手术当天切除脾脏;CsA联合脾切除组于手术当天切除脾脏,每天腹腔注射CsA10mg/kg。观察每组移植心脏的存活时间,定时和发生排斥反应时切取移植心组织,进行病理学检查,并以免疫组化方法观察移植物中P选择素和ICAM-1的表达。结果CsA联合脾切除组移植心脏存活时间为(34.2±8.98)d,较对照组、CsA组和脾切除组明显延长(P<0.05)。CsA联合脾切除组术后1~5d移植物中未见明显病理改变;7、14d时移植心脏组织结构清晰,未见血栓及出血;30d时移植心脏组织结构基本正常,排斥反应级别为Ⅰ~Ⅱ;其它三组的病理改变明显,对照组于术后1~2d、脾切除组及CsA组于术后4~7d发生延迟性排斥反应。CsA联合脾切除组术后各观察时点移植心脏组织中均未见P选择素和ICAM-1表达,其它三组的P选择素和ICAM-1均呈阳性。结论异种移植发生延迟性排斥反应时,P选择素和ICAM-1均有表达,可以作为判断异种移植免疫抑制治疗效果的指标之一。     

泡状棘球蚴感染对大鼠免疫学状态及移植心存活的影响

目的 研究肝泡状棘球蚴感染对大鼠免疫学状态及移植心存活的影响。方法 建立SD大鼠到Wistar大鼠的颈部异位心脏移植模型。对照组以未接种泡状棘球蚴的Wistar大鼠为受者；实验组以感染泡状棘球蚴的Wistar大鼠为受者。术后观察移植心的存活时间、组织病理学变化、心肌组织内T淋巴细胞和嗜酸粒细胞的浸润情况，测定血清内白细胞介素4（IL-4）和γ干扰素（IFN-γ）水平。结果实验组移植心的存活时间与对照组相比，差异有统计学意义（P〈0．05），组织病理学分级及心肌组织中CD4^＋T淋巴细胞数与对照组相比，差异无统计学意义，而CD8^＋T淋巴细胞数、嗜酸粒细胞数与对照组相比，差异均有统计学意义。发生排斥反应时，实验组血清内IL-4水平高于对照组，而IFN-γ水平低于对照组。结论 泡状棘球蚴感染造成TH1／TH2向TH2类细胞因子偏移，有利于移植物的存活，嗜酸粒细胞浸润可能是移植心发生排斥反应的原因之一。     

血管内皮生长因子在大鼠心脏移植急性排斥期的表达

目的　观察血管内皮生长因子(VEGF)在大鼠心脏移植急性排斥期中的表达及其和排斥的关系。方法　大鼠分对照组,CSA组,每组12只。分别静脉给予生理盐水及CSA干预,采用颈部心脏异位移植术式建立移植模型。常规监测排斥反应发生情况。每组5只用于观察移植物存活时间,7只用于动态切取标本。应用逆转录 聚合酶链反应(RT PCR)检测移植物局部VEGF的表达水平。结果　CSA组移植心存活时间(2 0 .4±5 .1)d显著长于对照组(8.6±1.5 )d ,CSA组VEGF的表达强度弱于对照组,其时间变化趋势和高峰时间较对照组推迟并和移植心存活延长时间有相关性。病理观察显示移植心局部的炎性细胞和淋巴细胞浸润和VEGF表达有关。结论　VEGF高表达促进排斥反应的发生缩短移植心存活时间,提示VEGF的表达和移植心的炎性浸润以及急性排斥有密切关系     

大鼠供心转染CTLA4-Ig基因抑制心脏移植后排斥反应

目的研究大鼠供心转染CTLA4-Ig基因抑制心脏移植术后排斥反应的可行性。方法以BN大鼠为供者,Lewis大鼠为受者,建立心脏移植模型。将实验分为2组。对照组:供心获取过程中不给予任何干预处理;实验组:在供心获取的过程中,以质粒载体携带CTLA4-Ig(pUF1-CTLA4-Ig)经过冠状动脉灌注供心。采用逆转录聚合酶链反应(RT-PCR)检测供心组织中CTLA4-IgmRNA的表达,观察移植心存活时间;酶联免疫法(ELISA)检测血清γ-干扰素(IFN-γ)和白细胞介素-4(IL-4)水平;观察移植心的组织学改变;用免疫组织化学SABC法检测移植心组织中CD4 和CD8 T细胞的浸润。结果实验组移植心存活时间较对照组明显延长[(11.70±1.24)dvs(5.62±0.74)d,P<0.05]。移植后5d,实验组移植心组织中可见CTLA4-IgmRNA的表达;对照组病理学检查可见心肌间质出现弥漫性的炎性细胞浸润,伴有局部的心肌坏死,组织间质水肿,实验组仅有局灶性血管外周及心肌间质内炎性细胞浸润,未见坏死;对照组移植心组织中浸润的CD4 和CD8 T细胞数量明显高于实验组(P<0.01);实验组血清IFN-γ水平明显低于对照组(P<0.01),但血清IL-4水平差异无统计学意义(P>0.05)。结论经冠状动脉灌注转染CTLA4-Ig基因,可以抑制心脏移植后排斥反应。     

肝组织ICAM-1及LFA-1表达对急性排斥反应的诊断价值

目的 探讨大鼠移植肝组织内细胞间粘附分子-1（ICAM-1）和淋巴细胞功能相关抗原-1（LFA-1）和淋巴细胞功能相关抗原-1（LFA-1）与急性排斥反应的关系。方法 将SD大鼠、Listar大鼠随机分为正常对照组、同种同基因移植组、同种异基因移植组。分别对正常肝组织及移植后2，4，7d肝组织进行活检，用单克隆抗体免疫组化染色，然后进行图像半定量分析，以检测正常及移植肝组织中ICAM-1和LFA-1的表达水平。结果 同种异基因移植组大鼠肝组织ICAM-1和LFA-1分子表达水平显著高于同种同基因移植组及正常对照组，且其升高与移植术后的时间呈正相关。结论 移植肝组织中ICAM-1和LFA-1表达的检测对肝移植术后急性排斥反应的诊断具有参考价值。     

Increased expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in rat cardiac allografts

This study was designed to investigate the role of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) during chronic cardiac allograft rejection. Wistar rats were used as donors, and SD rats as recipients heterotopic cardiac transplants. Recipients pretreated with inoculation of donor splenocytes (SPC) followed by cyclophosphamide (CP) were divided into 4 groups: (A) untreated group (n = 18) without immunosuppression; (B) SPC plus CP-treated group (n = 18) that were euthanized at 15-120 days posttransplantation; (C) CsA-treated group (n = 18) euthanized at 2-3 months posttransplantation; and (D) tolerance group (n = 18) treated with SPC plus CP and monitored for at least 1 year posttransplantation. Cardiac allografts were harvested at various times for immunohistochemical studies performed to evaluate the expression of ICAM-1 and VCAM-1. Pretreatment of animals with SPC and CP induced long-term cardiac allograft survival. Immunohistochemical staining demonstrated a low level of ICAM-1 and VCAM-1 expression in cardiac allograft muscle and coronary arteries among Groups B and D. In contrast, the expressions of ICAM-1 and VCAM-1 in cardiac allografts of Groups A and C were significantly higher than those in Groups B and D. Our results suggested that the expression of ICAM-1 and VCAM-1 plays an important role during the development of chronic cardiac allograft rejection.     

FK778, a novel immunosuppressive agent, reduces early adhesion molecule up-regulation and prolongs cardiac allograft survival.

The adhesion molecules, P-selectin, ICAM-1, and VCAM-1 are important mediators of T-cell adhesion and T-cell co-stimulation. We investigated the effect of the malononitrilamide FK778 on cardiac allograft survival, acute allograft rejection, and adhesion molecule up-regulation in a heterotopic, cardiac transplantation model. Rats received low- or high-dose FK778 or no treatment. Grafts were harvested on the fifth postoperative day for histologic examinations. To assess allograft survival, recipients were treated for a maximum of 10 days and grafts were harvested after cessation of the contractile activity. FK778 low dose showed a mild but significant decrease in mononuclear infiltration but failed to markedly reduce histologic rejection, adhesion molecule up-regulation, or to prolong allograft survival. However, high-dose FK778 treatment significantly reduced early up-regulation of P-selectin, ICAM-1, and VCAM-1, abolished infiltration, reduced histologic rejection and resulted in prolonged cardiac allograft survival. Therefore, FK778 is a novel, highly desirable immunosuppressive drug for transplantation medicine.     

3-Deazaadenosine prevents leukocyte invasion by suppression of adhesion molecule expression during acute cardiac allograft rejection: involvement of apoptotic cell death.

BACKGROUND: In the initial phase after cardiac transplantation, mononuclear cells infiltrate the graft, initiating a relevant impulse for rejection. 3-Deazaadenosine (c3Ado), an analog of adenosine, has proven anti-inflammatory properties both in vitro and in vivo. We hypothesized that c3Ado can serve as a therapeutic tool to reduce cellular infiltration in cardiac allograft transplantation. METHODS: Using the Wistar-Furth-to-Lewis rat cardiac allograft model, animals were treated with 5 mg c3Ado subcutaneously twice per day. Allografts of untreated animals served as controls. Grafts were harvested on Days 1, 3 and 6 after transplantation for further examination (n = 4 per group and timepoint). RESULTS: Immunohistochemical examination of c3Ado-treated grafts revealed up to 80% reduction of infiltrating major histocompatability complex (MHC) II-positive cells and T-cell-receptor-positive cells (R73) as well as ED1-positive monocytes and macrophages at Days 3 and 6 after transplantation. Adhesion molecule (ICAM-1 and VCAM-1) expression at Days 1 and 3 was almost completely abolished in c3Ado-treated grafts. However, c3Ado treatment did not prevent apoptotic cell death (TUNEL assay, DNA laddering) at Day 6, nor did it prolong allograft survival. As in controls, grafts were rejected at Day 7. CONCLUSION: c3Ado significantly reduces graft infiltration by preventing leukocyte invasion, most likely through suppression of adhesion molecule expression. Although graft survival was not prolonged, treatment with c3Ado may still serve as a strategy to protect hearts from early damage after transplantation. Further studies will show whether peri-operative use of c3Ado can bridge the critical phase after transplantation when standard immunosuppression is not yet completely efficacious.     

大鼠移植心脏的细胞凋亡及其与急性排斥反应的关系

目的 观察移植心脏的细胞凋亡现象及其与急性排斥反应的关系。方法 建立大鼠异位心脏移植模型,用ＨＥ梁色和原位末端标记（ＴＵＮＥＬ）技术检测移植心脏切片,进行排斥反应的病理分级,计算凋亡指数（ＡＩ）。结果 发生凋亡的细胞主要是心肌细胞,在各级排斥反应中均可见凋亡细胞存在,且ＡＩ与急性排斥发生的分级成正相关;各级的ＡＩ与０级（无排斥反应）比较,差异均有显著性。结论 细胞凋亡与移植心脏急性排斥反应的严重程     

Antisense oligodeoxynucleotides prevent acute cardiac allograft rejection via a novel, nontoxic, highly efficient transfection method.

BACKGROUND: We hypothesized that ex vivo donor allograft transfection with antisense oligodeoxynucleotide (AS ODN) would inhibit the expression of intercellular adhesion molecule (ICAM)-1, an important mediator of T-cell adhesion and costimulation, and therefore suppress acute cardiac rejection. METHODS: Hearts were transfected ex vivo with AS, reverse AS ODN, or saline by applying 3 atm pressure for 45 min at 4 degrees C. Grafts were then transplanted into allogenic recipients +/- treatment with leukocyte function-associated antigen (LFA)-1 monoclonal antibody (mAb) (1.5 mg/kg intravenously), cyclosporine (2.5 mg/ kg/day p.o.), or rapamycin (0.025 mg/kg/day intraperitoneally). Reperfusion injury was assessed in grafts harvested at early time points using the myeloperoxidase, %wet weight, and %contraction band necrosis assays; transfection efficiency was assessed using fluorescent microscopy; and efficacy of ICAM-1 blockade was assessed using immunohistochemistry. Other grafts were followed until rejection with donor/third-party skin grafting, adoptive transfer, and interleukin 2 infusion studies in selected recipients. RESULTS: Transfection was highly efficient (fluorescein isothiocyanate-ODN in 48+/-5% of total myocardial nuclei), nontoxic, and reduced the ICAM-1-positive area to 53+/-14% versus having no effect on MHC class I expression (n=4). The incidence of survival >60 days after AS ODN + LFA-1 monoclonal antibody was 75%, significantly higher than other regimens. CONCLUSION: AS ODN hyperbaric transfection proved highly efficient, effective at ICAM-1 blockade, and induced cardiac allograft tolerance when combined with LFA-1 monoclonal antibody. This highly targeted alteration of allograft immunogenicity may have an important role in future immunosuppressive strategies.     

HMG-CoA reductase inhibitor cerivastatin prolonged rat cardiac allograft survival by blocking intercellular signals.

BACKGROUND: The development of atherosclerotic cardiovascular complications caused by hyperlipidemia is a common and serious problem for long-term survivors of organ transplantation. However, adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and lymphocyte function-associated antigen (LFA)-1 are involved in allograft rejection, possibly by providing costimulatory signals. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor cerivastatin has been shown to suppress ICAM-1 expression in acute inflammatory responses. METHODS: In this study, we evaluated the immunosuppressive effects of cerivastatin in rat cardiac allografts. The hearts of Fischer rats were transplanted heterotopically into Lewis rats. Cerivastatin (2 mg/kg) was administrated intraperitoneally to recipients for 7 consecutive days from the day before transplantation. RESULTS: Graft survival in the cerivastatin-treated group (n = 8) was significantly longer than in controls (n = 10) (24.6 +/- 2.2 days vs 10.2 +/- 1.3 days, p < 0.05). Mixed lymphocyte reaction (MLR) showed that on Day 8 after grafting, the proliferative response of alloreactive T cells against F344 alloantigen in cerivastatin-treated rats was significantly more suppressed than in Lewis rats. The Interleukin-2 concentration of supernatant in MLR cultures in the cerivastatin-treated group was lower than in the control group. Immunohistochemical analysis showed that the percentage of CD4-positive cells to infiltrating mononuclear cells was less prominent in the cerivastatin-treated group (9.8% +/- 2.2%) than in the control group (20.9% +/- 3.2%). CONCLUSIONS: The HMG-CoA reductase inhibitor cerivastatin effectively suppressed acute graft rejection, possibly by blocking intercellular signals via ICAM/LFA-1, and cerivastatin may be a candidate for treating patients with hyperlipidemia who undergo organ transplantation.