【作者】 尤颢；

【导师】 廖崇先； 陈道中；

【作者基本信息】 福建医科大学 ， 外科学， 2003， 博士

【摘要】 目的：利用大鼠异位心脏移植模型，探讨转染入心肌细胞中的NF-decoy诱饵双链核苷酸在心脏移植中的作用，并对其可能的作用机制作一探讨。 方法：在建立Wistar大鼠→SD大鼠颈部异位心脏移植模型基础上，用不同浓度的Lipofectmine2000与NF-decoy诱饵ds ODN混合物处理供心，比较其在心肌细胞中存活时间的长短和移植后供心在Langendorff-Neeley离体心脏顺灌左心做功模型在功能表现，测定心脏组织损伤生化学指标；半定量RT-PCR法测定ICAM-1、VCAM、iNOS mRNA的表达。用10OD诱饵ds ODN脂质体混合物处理供心，观察移植术后供心存活时间，心室组织病理切片排斥反应分级的差别和抗ICAM-1免疫组织化学染色情况。 结果：Lipofectamine2000可将ds ODN转染入心肌细胞内，存在时间与ds ODN的浓度有关。乱序ds ODN不能对供心提供缺血再灌注的保护作用，而用5 OD及10 OD的ds ODN处理的供心表现出较佳的心脏功能，减轻心肌组织中损伤的生化学指标以及心肌mRNA中ICAM-1、VCAM、iNOS的表达。10 OD ds ODN能延长供心的存活时间与亚治疗剂量(2.5mg／kg)免疫抑制剂CsA所获得的效果类似，而与亚治疗剂量CsA合用可进一步延长供心的存活时间，减轻病理切片中排斥反应的分级和免疫组化染色ICAM—1的表达。 结论：1)转染入心肌细胞的NF-kB诱饵双链核苷酸可有效减轻移植术后的供心的缺血再灌注损伤，其机理与抑制ICAM-1、VCAM、iNOS的表达有关。2)NF-kB ds ODN还能减轻移植术后的急性排斥反应，延长供心的存活时间，减少了心肌细胞中ICAM-1的表达可能是其机理之一。更多还原

【Abstract】 Objectives: By using a rat heterotopical heart transplantation model, we investigate the effect of the lipofectin-mediated delivery double-strand oligonuide(ds ODN) decoy to NF-kB binding site in the heart transplantation.Methods: First, we developed a new heterotopic heart transplantation model by using cuff technique. Then, the donor hearts were treated with 1 OD decoy, 1OD decoy Iipofectamine2000 compound or 5 OD decoy Iipofectamine2000 compound. Transfection efficiency was determined with FITC-labeled ODN and fluorescent microscopy. Reperfusion injury was assessed by Langendorff-Neeley reperfusion system and the content of MDA, MPO in heart tissure and NO in right atrial. Reverse transcription-PCR was used to analyze intercellular adhesion molecule 1, vascular celluar adhesion molecule and inducible NOS mRNA expression. Acute rejection was determined by daily palpation. Histopathological and immunohistochemical study for measurment ICAM-1 were examined after 5 days of transplantation.Results: The heterotopic heart transplantation using cuff technique got the similar results as classical heterotopic heart transplantation. Without Lipofectamine2000, the ds ODN can not transfected into the donor heart cell. The transfected 5 OD ds ODN can lasted 14 days after heart transplantation. In the reperfusion experiment, the 5 OD ds ODN treated group had better donor heart function, less tissure MDA, myeloperoxidase(MPO) content and blood nitric oxide (NO) concentration (P<0.01 vs the control group). And the 10 OD ds ODN treated group had more efficient heart preservation effect when compared to the 5 OD treated group( P<0.01 ). The NF-kB decoy(5 OD and 10 OD), not the scramble ds ODN inhibited the donor heart ICAM-1, VCAM, iNOS mRNA expression in vivo after transplantation as assessed by semi-quantitative RT-PCR analysis(P< 0.01 vs controls). The 10 OD ds ODN treatment also prolonged donor heart survival over the controls (10.88 ± 1.55 vs. 6.75 ± 089 080.01), and had the similar results as the low-dose cyclosporine A (2.5 mg/kg) treatment.When decoy employment combined with the the low-dose cyclosporine A treatment can further prolonged the allografts survival. The histopathology showed less rejection scores after 5 days of transplantation while treated with the NF-kB decoy. The immunohistochemical study also showed decoy ds ODN can alleviated the expression of ICAM-1 in allograft rejection tissure .Conclusion: Our results suggested that Lipofectamine2000 could effectively deliver the ds ODN into the donor heart during preservation period. The NF-kB decoy ds ODN treatment can reduce the reperfusion injury and block the donor heart ICAM-1, VCAM, iNOS expression after transplantation. And the NF-kB decoy can also attenuate the acute rejection of the donor heart, the possible mechanism may contribute to the suppression of ICAM-1 expression. This highly targeted therapy may be a clinically viable strategy for the heart transplantation.更多还原