腹主动脉移植后小鼠外周血内皮祖细胞数量的动态变化及意义

目的 观察腹主动脉移植后小鼠移植物动脉硬化的病变过程及外周血内皮祖细胞(EPC)数量的动态变化,探讨EPC与动脉内膜损伤和修复的相互关系.方法 以C57BL/6小鼠和Balb/c小鼠为供、受者,建立腹主动脉原位移植后移植性模型.术后3 d、2周、4周、6周,观察移植动脉病理改变,并采用计算机图像分析系统分析移植血管内膜增生情况.使用流式细胞仪监测术后外周血中EPC数量的变化.结果 术后3 d,移植动脉内皮细胞损伤,并伴有明显的炎症细胞浸润.术后2周,即可观察到移植动脉新生内膜形成,存在急性排斥反应;术后4周、6周内膜厚度逐渐增生,移植动脉管腔明显狭窄.术后早期外周血中EPC的数量增多,3 d时达到高峰,此后迅速减少,术后14和28 d时,显著低于术前水平(P＜0.05).结论 通过同种小鼠腹主动脉原位移植可成功复制出移植物动脉硬化的病变特点;外周血中EPC的数量与移植动脉内膜损伤的修复密切相关,可能成为移植物动脉硬化的发病指标和干预靶点.     

腹主动脉移植后小鼠外周血内皮祖细胞数量的动态变化及意义

目的 观察腹主动脉移植后小鼠移植物动脉硬化的病变过程及外周血内皮祖细胞(EPC)数量的动态变化,探讨EPC与动脉内膜损伤和修复的相互关系.方法 以C57BL/6小鼠和Balb/c小鼠为供、受者,建立腹主动脉原位移植后移植性模型.术后3 d、2周、4周、6周,观察移植动脉病理改变,并采用计算机图像分析系统分析移植血管内膜增生情况.使用流式细胞仪监测术后外周血中EPC数量的变化.结果 术后3 d,移植动脉内皮细胞损伤,并伴有明显的炎症细胞浸润.术后2周,即可观察到移植动脉新生内膜形成,存在急性排斥反应;术后4周、6周内膜厚度逐渐增生,移植动脉管腔明显狭窄.术后早期外周血中EPC的数量增多,3 d时达到高峰,此后迅速减少,术后14和28 d时,显著低于术前水平(P＜0.05).结论 通过同种小鼠腹主动脉原位移植可成功复制出移植物动脉硬化的病变特点;外周血中EPC的数量与移植动脉内膜损伤的修复密切相关,可能成为移植物动脉硬化的发病指标和干预靶点.     

腹主动脉移植后小鼠外周血内皮祖细胞数量的动态变化及意义

目的 观察腹主动脉移植后小鼠移植物动脉硬化的病变过程及外周血内皮祖细胞(EPC)数量的动态变化,探讨EPC与动脉内膜损伤和修复的相互关系.方法 以C57BL/6小鼠和Balb/c小鼠为供、受者,建立腹主动脉原位移植后移植性模型.术后3 d、2周、4周、6周,观察移植动脉病理改变,并采用计算机图像分析系统分析移植血管内膜增生情况.使用流式细胞仪监测术后外周血中EPC数量的变化.结果 术后3 d,移植动脉内皮细胞损伤,并伴有明显的炎症细胞浸润.术后2周,即可观察到移植动脉新生内膜形成,存在急性排斥反应;术后4周、6周内膜厚度逐渐增生,移植动脉管腔明显狭窄.术后早期外周血中EPC的数量增多,3 d时达到高峰,此后迅速减少,术后14和28 d时,显著低于术前水平(P＜0.05).结论 通过同种小鼠腹主动脉原位移植可成功复制出移植物动脉硬化的病变特点;外周血中EPC的数量与移植动脉内膜损伤的修复密切相关,可能成为移植物动脉硬化的发病指标和干预靶点.

Abstract：

Objective To investigate changes in the number of endothelial progenitor cells (EPC) from peripheral blood and pathological feature in the development of transplant arteriosclerosis in mouse abdominal aortic allografts, and discuss their correlations. Methods A segment of abdominal aorta was transplanted orthotopically from C57BL/6 to Balb/c mice. The grafts were harvested at 3rd day, 2nd week, 4th week and 6th week after the operation and studied by light and electronic microscopy. Regional changes in the lumen and intima were measured with computer imaging analysis system. EPC from peripheral blood were quantified by flow cytometry. Results Endothelium injury and inflammatory cells infiltration were seen in the aortic allografts at 3rd day after transplantation.Neointimal lesions and acute rejection were observed as early as 2nd week after surgery. The lumen of allografts was significantly narrowed due to neointima hyperplasia and had progressed at 4th and 6th week postoperatively. The number of circulation EPC was increased from 1 st day after operation and reached the peak at 3rd day. Thereafter the number of EPC was decreased rapidly and significantly less at 14th and 28th day postoperation than that pre-operation. Conclusion Abdominal aortic transplantation from C57BL/6 to Balb/c mice presents typical pathological feature of transplant arteriosclerosis. The number of EPC from peripheral blood is related to the process of injured endothelial repair and neointima formation of aortic grafts. EPC count may be considered a novel biological marker and therapeutic intervention for transplant arteriosclerosis.     

紫杉醇对大鼠移植动脉内膜增生及硬化的抑制作用

目的观察紫杉醇对同种大鼠移植动脉的影响,探讨紫杉醇对移植物动脉硬化的抑制作用及机制。方法以W istar大鼠为受者,SD大鼠为供者,按供鼠、受鼠不同分为3组,每组8对:同系对照组W istar大鼠接受W istar大鼠的胸腹主动脉移植;同种对照组W istar大鼠接受SD大鼠的胸腹主动脉移植;同种实验组W istar大鼠接受SD大鼠的胸腹主动脉移植,术后1~14 d腹腔注射紫杉醇2 mg.kg-1.d-1。两个对照组每日腹腔注射相同体积的生理盐水。术后30 d,取出移植动脉,进行病理学观察,测出管腔面积和内膜截面积,计算再狭窄率,用免疫组织化学方法测定增殖细胞核抗原(PCNA)的表达。结果同种实验组移植动脉内膜厚度、内膜PCNA表达情况均较同种对照组明显减少,外膜炎症细胞浸润程度、管腔再狭窄率均较同种对照组降低,差异有统计学意义。结论紫杉醇能有效抑制同种大鼠移植动脉内膜增生,防止移植物动脉硬化,其作用机制可能与抑制血管平滑肌细胞增殖和减轻对移植物的免疫排斥反应有关。     

大鼠移植动脉硬化加快模型的建立

目的　建立一种简捷 ,有代表性且稳定的移植物动脉硬化模型。方法　将SD大鼠的腹主动脉分别冷缺血 1、2 4、48h行SD→SD及SD→Wistar的原位腹主动脉移植 ,观察术后植入段血管病理改变、TGF β1表达及手术前后过氧化脂质的变化。结果　SD→SD及SD→Wistar缺血1h组分别于术后 10周及 6周见内膜明显增厚 ,而缺血 2 4h组只需 2周 ;各组移植后 2h过氧化脂质均明显高于术前 ,术后 4、2 4h与术前比差异无显著性 (P >0 .0 5 ) ;强化缺血组TGF β1不论是SD→SD还是SD→Wistar均于术后 1周即出现高表达。结论　以SD/Wistar作为供 /受体行腹主动脉移植 ,强化冷缺血损伤 ,可加快移植物动脉硬化 ,可望成为新型慢排模型。     

受体细胞参与移植动脉内膜增生的研究

目的研究大鼠移植动脉新生内膜细胞Sty基因的表达，探讨受体细胞在移植动脉内膜增生中的作用。方法建立大鼠腹主动脉移植模型，分为4组：雌雄同系移植组、雌性异系移植组、雄性异系移植组、雌雄异系移植组。移植术后10周时，取移植动脉标本，进行病理组织学观察，测量其内膜厚度和中膜厚度，分析移植动脉内膜增生情况；采用显微切割技术收集新生内膜细胞，用PCR方法检测Sry基因的表达。分析新生内膜细胞与受体细胞的关系。结果异系移植组移植动脉内膜显著增生，动脉壁内膜厚度及内膜／中膜厚度比均显著高于同系移植组（P〈0．01）；而同性或异性异系移植组之间差异无统计学意义（P〉0．05）。PCR分析显示，雄性异系移植组和雌雄异系移植组在242bp处均有1条特异性扩增条带，而雌性异系移植组则无相应的核酸扩增条带。结论受体细胞作为移植物血管新生内膜细胞的来源，参与移植动脉内膜增生及移植物动脉硬化。     

基质衍生因子在大鼠腹主动脉移植术后介导的修复重构机制

目的 探讨基质衍生因子-1（SDF-1）在大鼠腹主动脉移植术后介导的修复重构机制。方法 雄性Wistar大鼠为供者，雄性SD大鼠为受者，建立腹主动脉原位移植模型。免疫组织化学技术检测受者移植动脉内皮细胞上SDF-1的表达以及血管内膜的厚度；逆转录-聚合酶链法（RT-PCR）检测移植动脉血管中SDF-1受体CXCR4的表达。结果 受者移植的腹主动脉内皮细胞上持续表达SDF-1，且其表达水平与术后血管内膜增生厚度密切相关。结论 SDF-1能动员干细胞至移植的腹主动脉，并分化成平滑肌样细胞。SDF-1可能成为预警血管硬化、慢性移植物功能丧失发生和发展的指标。     

反义寡核苷酸防治移植物动脉硬化的作用

目的　探讨防治移植物动脉硬化的途径。方法　通过大鼠胸腹主动脉移植简化模型 ,用各种增殖细胞核抗原 (PCNA)寡核苷酸在脂质体介导下转染大鼠胸主动脉后 ,移植到大鼠腹主动脉 ,于术后 15、3 0和 60d取移植动脉作病理学检查及PCNA逆转录PCR检测。结果　病理学结果示 ,在 3个时相点 ,反义寡核苷酸组移植动脉再狭窄率 (8.3 %、12 .4%、19.5 % )均显著低于对照组 (P <0 .0 5 )。逆转录PCR结果显示 ,在 3个时相点 ,反义寡核苷酸组PCNAmRNA的表达(5 9.2 4、80 .16、18.5 8)均明显低于对照组 (P >0 .0 5 )。结论　PCNA反义寡核苷酸可明显抑制移植动脉PCNA蛋白的表达 ,抑制动脉内膜增生 ,防治移植物动脉硬化。     

人arresten重组蛋白对移植物动脉硬化的抑制作用

目的观察人arresten重组蛋白对移植物动脉硬化的抑制作用。方法用pRSET原核表达系统表达并纯化人arresten重组蛋白。建立腹主动脉移植大鼠模型，将受体鼠分为同系动脉移植组、异系移植对照组和异系移植实验组。自术后第3天起，皮下给予arresten重组蛋白（每日5mg／kg体重）处理。8周后取移植动脉组织标本，进行病理组织学观察与免疫组织化学染色，分析移植动脉新生内膜增生及新生内膜细胞α-平滑肌肌动蛋白（α-SMA）和PCNA的表达。结果异系移植组移植动脉新生内膜中α-SMA表达阳性平滑肌细胞大量增生，致动脉内膜增厚，管腔狭窄。异系移植实验组移植动脉内膜增生受到明显抑制，新生内膜面积（0．14±0．03）mm^2。及新生内膜／中膜面积比（0．807±0．073）均显著低于异系移植对照组[（0．33±0．07）mm^2，（1．794±0．089），P〈0．01]；并且异系移植实验组移植动脉新生内膜细胞PCNA标记指数（31．72±5．26）％显著低于异系移植对照组（69．53±4．38）％（P〈0．01）。结论人arresten重组蛋白能有效抑制移植物动脉硬化的发生发展，在抗移植物慢性排斥反应方面显示出良好的应用前景。     

犬涤纶人造血管移植后不同时期的组织形态学研究

我们通过１０条犬的涤纶血管移植实验研究，连续观察分析了中口径国产涤纶人造血管移植于犬腹主动脉，术后１２周以内不同时期移植血管的组织形态学改变。结果表明２ｃｍ长国产中口径涤纶人造血管移植于腹主动脉后其新生内膜覆盖管腔内壁大约需要４周。新生内膜主要来自邻接动脉内膜的平滑肌增生爬行；内皮细胞覆盖人造血管内膜的时间较自体静脉晚得多，术后７周涤纶血管内膜尚无完整稳定的内皮细胞层；经高压蒸汽灭菌法消毒过的中口径国产涤纶人造血管不宜再次使用。     

Development of a combined cardiac and aortic transplant model to investigate the development of transplant arteriosclerosis in the mouse.

BACKGROUND: The degree of transplant arteriosclerosis in murine cardiac allografts is difficult to assess. Aortic allografts represent an alternative model for evaluating the impact of novel transplant strategies on transplant arteriosclerosis in which the vascular changes can be quantified easily. However, it remains controversial as to whether vascular lesions seen in this model are equivalent to those that develop in solid-organ transplants. The aim of this study was to develop a model of combined cardiac and aortic transplantation to allow more precise quantification of transplant arteriosclerosis and to establish a correlation between the lesions that develop in the 2 types of graft. METHODS: CBA (H2(k)) recipients received a C57BL/10 (H2(b)) cervical cardiac allograft on Day 0 and a C57BL/10 (H2(b)) abdominal aortic allograft on Day 1. Recipients were treated with anti-CD154 mAb (MR1) on Days 0, 2, and 4. We performed histology and morphometric measurements for both grafts 30 days after transplantation. RESULTS: We observed significant intimal proliferation in both the cervical cardiac and abdominal aortic allografts from recipients treated with anti-CD154 mAb (heart, 64% +/- 9%; aorta, 67% +/- 8%; n = 5). Abdominal aortic grafts transplanted alone into anti-CD154-treated recipients developed a degree of transplant arteriosclerosis equivalent to that seen in the aortic grafts of the combined group (aorta alone, 68% +/- 9%, vs aorta + heart, 67% +/- 8%; n = 5). CONCLUSIONS: This combined cardiac and aortic transplant model permitted quantitative assessment of transplant arteriosclerosis while monitoring graft survival by cardiac palpation. Furthermore, development of transplant arteriosclerosis was equivalent in abdominal aortic allografts either in the presence or absence of an additional solid- organ transplant.     

The cathepsin-S/protease-activated receptor-(PAR)-2 axis drives chronic allograft vasculopathy and is a molecular target for therapeutic intervention

BackgroundCathepsin S (CatS) and proteinase-activated receptor (PAR)-2 are involved in the remodelling of vascular walls and neointima formation as well as in alloantigen presentation and T-cell priming. Therefore, we hypothesized that CatS/PAR-2 inhibition/deficiency would attenuate chronic allograft vasculopathy.MethodsHeterotopic aortic murine transplantation was performed from C57BL/6J donors to C57BL/6J recipients (syngeneic control group), Balb/c to C57BL/6J without treatment (allogenic control group), Balb/c to C57BL/6J with twice daily oral CatS inhibitor (allogenic treatment group) and Balb/c to Par2−/− C57BL/6J (allogenic knockout group). The recipients were sacrificed on day 28 and the grafts were harvested for histological analysis and RT-qPCR.ResultsAfter 28 days, mice of the allogenic control group exhibited significant neointima formation and massive CD8 T-cell infiltration into the neointima while the syngeneic control group showed negligible allograft vasculopathy. The mRNA expression level of CatS in allografts was 5-fold of those in syngeneic grafts. Neointima formation and therefore intima/media-ratio were significantly decreased in the treatment and knockout group in comparison to the allogenic control group. Mice in treatment group also displayed significantly fewer CD8 T cells in the neointima compared with allogeneic controls. Additionally, treatment with the CatS inhibitor and PAR2-deficiency decreased mRNA-levels of interleukins and cytokines.ConclusionIn conclusion, our data indicate that inhibiting CatS and PAR-2 deficiency led to a marked reduction of neointima formation and associated inflammation in a murine heterotopic model for allograft vasculopathy.     

Attenuation of transplant arteriosclerosis by oral feeding of major histocompatibility complex encoding chitosan-DNA nanoparticles

One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the alloimmune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation. Therefore, we performed fully allogeneic mouse abdominal aortic transplants using C57BL/6 (H2^b) mice as donors and CBA.J (H2^k) mice as recipients. Aortic grafts were analyzed by histology and morphometry on day 30 after transplantation, levels of circulating alloantibodies were detected by FACS analysis. Pre-treatment of recipient mice with chitosan-DNA nanoparticles encoding for K^b, one of the MHC-I molecules of the donor, resulted in a significant reduction of intimal proliferation compared to untreated controls. When Ovalbumin was fed instead of K^b encoding nanoparticles (K^b-NP) or Balb/c (H2^d) grafts were used instead of C57BL/6 (H2^b) grafts as antigen controls, both groups showed no reduction of intimal thickness indicating an antigen-specific mechanism. In addition, analysis of peripheral blood of the transplanted mice showed significant suppression of alloantibody formation in the K^b-NP fed group compared to all other allogeneic transplanted groups suggesting modulation of the humoral immune response. These results demonstrate the potential of chitosan-DNA nanoparticles to induce K^b-specific tolerance and to reduce the development of transplant arteriosclerosis.     

Regulation of transplant arteriosclerosis by CD25+CD4+ T cells generated to alloantigen in vivo

BACKGROUND: CD25+CD4+ regulatory T cells have been shown to suppress alloimmunity in various experimental settings. Here, we hypothesized that alloantigen-reactive regulatory T cells would reduce the severity of transplant arteriosclerosis. METHODS: CD25+CD4+ T cells from CBA mice that were pretreated with C57BL/6 (B.6) blood (donor-specific transfusion, DST) and nondepleting anti-CD4 Ab (YTS 177) were cotransferred with na?ve CBA CD25-CD4+"effector" T cells into CBA-rag-/- mice. These animals received aorta transplants from B.6 CD31-/- donors. CBA wild-type recipients of B.6 aorta grafts were pretreated with 177/DST directly. Some animals received 6x10(5) CD25+CD4+ T cells from pretreated mice to augment regulation on day -1. Grafts were harvested on day 30. RESULTS: Luminal occlusion of the graft caused by neointima formation was 29.3+/-19.4% (n=5) after transfer of effector T cells only. Co-transfer of CD25+CD4+ regulators reduced occlusion significantly (2.4+/-3.3%, n=3; P=0.009). This effect was partially abrogated in the presence of a CTLA4 blocking Ab (11.1+/-4.7%, n=4; P=0.008). Pretreating immunocompetent CBA recipients of B.6 aortic allografts with 177/DST did not reduce transplant arteriosclerosis significantly (43.0+/-15.7%, n=5 vs. 56.6+/-16.8%, n=5; 177/DST vs. controls; P=0.22). However, when pretreated primary CBA recipients received an additional transfer of 6 x 10(5) CD25+CD4+ T cells procured from other mice pretreated with 177/DST before transplantation, luminal occlusion of the graft was markedly reduced (33.0+/-7.6%, n=5; P=0.002). CONCLUSION: Regulatory T cells generated in vivo to alloantigen can prevent CD25-CD4+ T-cell-mediated transplant arteriosclerosis. In immunocompetent recipients, these cells have potential to be used as cellular immunotherapy to control transplant arteriosclerosis.     

Rosiglitazone attenuates transplant arteriosclerosis after allogeneic aorta transplantation in rats

BACKGROUND: Transplant arteriosclerosis is a leading cause of chronic transplant dysfunction and is characterized by occlusive neointima formation in intragraft arteries. Development of transplant arteriosclerosis is refractory to conventional immunosuppressive drugs and adequate therapy is not available. In this study, we determined the efficacy of the synthetic peroxisome proliferator-activated receptor (PPAR)-gamma agonist rosiglitazone to attenuate the development of transplant arteriosclerosis in rat aortic allografts. METHODS: Lewis aortic allografts were transplanted into Brown Norway recipient rats. Recipient rats received either approximately 5 mg rosiglitazone/day (starting 1 week before transplantation until the end of the experiment) or were left untreated. Transplant arteriosclerosis was quantified using morphometric analysis. Alloreactivity was measured in vitro using mixed lymphocyte reactions. Regulatory T cell frequency and function were analyzed using flow cytometry and in vitro suppression assays, respectively. Intragraft gene expression was analyzed using real-time polymerase chain reaction. Finally, medial and neointimal vascular smooth muscle cell proliferation was analyzed in vitro. RESULTS: Rosiglitazone significantly reduced transplant arteriosclerosis development 8 weeks after transplantation (P<0.01 vs. nontreated). Rosiglitazone reduced T cell alloreactivity which was not mediated through modulation of CD4+CD25+FoxP3+ regulatory T cells. Reduced development of transplant arteriosclerosis coincided with reduced intragraft expression of stromal-derived factor-1alpha and platelet-derived growth factor receptor-beta. Finally, rosiglitazone reduced growth-factor-driven proliferation of both medial and neointimal vascular smooth muscle cells in vitro, which was not mediated through PPARgamma. CONCLUSION: PPARgamma agonists may offer a new therapeutic strategy in clinical transplantation to attenuate the development of transplant arteriosclerosis and thereby chronic transplant dysfunction.     

灵长类动物预致敏后肾移植加速排斥反应模型的建立

目的 建立灵长类动物预致敏后肾移植加速排斥反应模型.方法 取血型相容的正常猕猴配对,预先将供者腹部全层皮肤移植到受者背部,使受者预致敏.2周后再将同一供者的左侧肾脏移植到受者腹腔内,间时切除受者自体双肾,术后予以环孢素A、霉酚酸酯和泼尼松治疗(致敏用药组),不用免疫抑制剂者为对照(致敏对照组),以未致敏的肾移植作为对照组.术后观察受者血肌酐变化、移植物存活时间及病理特点.结果 对照组的4只移植肾存活时间分别为9、18、8、7 d;致敏对照组的3只移植肾存活时间分别为3、3、4 d;致敏用药组的3只移植肾存活时间分别为2、3、4 d.移植皮肤于术后10 d出现排斥反应,至术后14 d被完全排斥.对照组于肾移植1周以后才发牛排斥反应,而致敏者均在肾移植后3 d左右发生较严重的排斥反应.结论 受者被供者皮肤预致敏后再行肾移植,可以加速移植物的排斥,且不能被环孢素A、霉酚酸酯及泼尼松所组成的三联免疫抑制方案逆转.