含TLR4 siRNA的心脏保存液对小鼠供心冷保存心脏移植的保护作用的研究

Despite major achievements in heart transplantation, we still face some urgent problems such as: prolonging the preservation period of donor heart,.improving myocardial function and metabolism, and reducing myocardial ischemia/ reperfusion injury. To compare the role of Toll-like receptor-4(TLR4)in heart transplantation using cold preservation solution,two types of mice were used in this study:free wild type mice(C3H/HeN)and TLR-4 gene mutation type(C3H/HeJ)on the two periods of static cold storage and reperfusion after transplantation.The changes of myocardial apoptosis, inflammatory responses and oxidative stress and so on are observed. We establish the model of isolated heart Langendorff perfusion and the model of heterotopic heart transplantation. We develope the preservation solution consisting of siRNA that targets TLR4,and perfuse donor hearts with the siRNA solution and silence TLR4 gene in the preservation period. The effectiveness of siRNA uptake by heart tissues is determined. We can discuss the protection on the donor heart cold ischemic and reperfusion injury from cardiac function and myocardial injuries. This study will demonstrate a new hypothesize and method for donor heart protection, and can potentially have significance in clinical application.

延长供心冷保存时间，提高保存质量，减轻心肌缺血再灌注损伤是目前心脏移植领域迫切需要解决的临床问题。本课题观察TLR4基因突变小鼠在供心冷保存和移植后再灌注两个阶段，心肌细胞凋亡、炎症反应和氧化应激等方面的变化，探讨TLR4在冷保存心脏移植中的作用。建立小鼠离体心脏Langendorff 灌注模型和异位心脏移植模型，通过含有TLR4 siRNA的冷保存液灌注并保存供心，转染心肌后抑制TLR4基因表达，明确 TLR4 siRNA 冠脉灌注法对供体心脏基因转染的效率，从心脏功能和心肌损伤角度探讨其对供心冷缺血性和再灌注损伤的保护作用。为今后临床心脏移植心肌保护的发展提供一种新的方法和理论依据。

目的：缺血再灌注损伤会导致移植物功能障碍和移植后免疫反应。心脏热IR损伤研究较多，但心脏移植中供心冷IR损伤描述较少，我们相信TLR4信号通路参与其中。.方法：以TLR4基因突变C3H / HeJ小鼠与野生型C3H / HeN小鼠为实验对象，建立异位心脏移植模型，从细胞凋亡、炎症、氧化应激等方面探讨TLR4在心脏移植中对冷IR心肌的影响。另一实验中，在细胞水平采用H9C2心肌细胞系，建立心肌细胞缺氧复氧模型。运用基因工程技术，合成TLR4 siRNA，转染H9C2细胞，观察对H9C2细胞TLR4 mRNA及蛋白水平的抑制效果。在细胞水平验证TLR4 siRNA对TLR4 信号通路抑制效果，为后期在体实验打基础。.结果：冷缺血及再灌注后，TLR4突变鼠与野生型小鼠心肌的TLR4mRNA与蛋白表达均较正常值升高。但野生鼠升高尤为明显，与突变鼠差异显著。（P＜0.05）。TLR4下游信号TNF-α及NF-κB的蛋白表达在野生型小鼠与TLR4蛋白的变化趋势一样，与TLR4突变鼠比较有显著差异。TLR-4 突变鼠与野生型小鼠比较，能提高心肌中SOD 活性，降低心肌中MDA、MPO 水平。.结论：证实TLR4信号通路促成心脏移植冷I/R后的炎症应答，在心脏移植冷IRI中起着重要作用。