大鼠供肝缺血预处理对肝移植后急性排斥反应的影响研究
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叶启发 E-mail:wangyg122008@163.com

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The relationship between ischemic preconditioning and acute rejection after allogenic orthotopic liver transplantation in rats 
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    摘要:

    目的:探讨肝移植时缺血预处理(IP)对不同程度缺血再灌注损伤(IRI)后急性排斥反应(AR)的影响及机制。
    方法:将大鼠分为6个实验组,每组供、受者各12只。同系移植组(2组)供肝热缺血时间分别为0~2 min,同种移植组(4组)供肝热缺血时间分别为0~2,15,0~2, 15 min,各组冷缺血时间均为80 min;另设假手术组仅在开腹并游离肝脏后关腹。同种移植3,4组先采用入肝血流阻断法进行供肝热缺血预处理5 min。肝移植采用改良Kamada二袖套法。术后1,3,5,7 d每组分别处死3只受者,取移植肝组织,行病理学检查及观察IRI程度,用Banff系统分级标准进行AR评分,用免疫组化法和PCR法检测移植肝组织Fas,穿孔素及颗粒酶 B的蛋白和mRNA表达,用TUNEL法检测移植肝细胞的凋亡率。
    结果:同种移植1~4组移植肝病理改变及IRI程度依次加重。术后1,3,5,7 d,假手术组、同系移植组未发现AR,同种移植1~4组AR明显;同种移植3组AR评分较同种移植1组,同种移植4组较同种移植2组随术后时间延长而降低(P<0.05)。术后各时点,假手术组、同系移植组移植肝中穿孔素和颗粒酶B mRNA均无表达,Fas mRNA仅有低表达;同种移植1~3组较假手术组、同系移植组、同种移植4组的表达明显升高(P<0.05);术后相同时点,同种移植3组表达量较同种移植1组,同种移植4组表达量较同种移植2组下降(P<0.05)。
    结论:IP能相应减轻肝移植相同冷、热缺血时间所致IRI程度及AR的程度,其机制是抑制细胞凋亡调控基因Fas及穿孔素/颗粒酶 B mRNA和蛋白的表达。

    Abstract:

    Objective:To investigate the relationship between ischemic preconditioning (IP) and acute rejection (AR) after allogenic orthotopic liver transplantation (OLT) in rats.
    Methods:By using random digits table,the rats were divided into Sham group (group A), similar gene OLT group (group B), OLT with ischemia reperfusion (IR) group (group C, D), and ischemic OLT with IR and with or without IP group(group E, F).  The rats were killed at 1,3,5 and 7 day after the models and sham operation were constructed respectively. Plasma samples were assessed for the severity of IR injury (IRI). The liver tissue was paraffin-embedded to examine the liver histological changes (HE), and rejection was classificed by rejection activity index of Banff. Apoptosis was measured by TUNEL, and the protein and mRNA expression of Fas, perforin and granzyme B were detected by using immunohistochemistry and real-time PCR.
    Results:The severity of acute rejection in hepatic tissue of groups C-F was paralleled with the degree of IRI.No rejection occurred in group A and B. The extent of disease and degree IRI and the classification of rejection activity index were lower in group E than in group  C, and group F than group D (P<0.05). The mRNA and protein expression levels of Fas, perforin and granzyme B in group C were higher than group  E (P<0.05),  and group  D were higher than group F (P<0.05) at 1,3,5 and 7 days after graft reperfusion.
    Conclusions: IP relieved the severity of IRI and the classification of acute rejection of the same cold and warm ischemia time, which is correlated with modulation of apoptosis and modulation of mRNA and protein expression of Fas, perforin and granzyme B. 

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王永刚|叶启发|时军|戴小明.大鼠供肝缺血预处理对肝移植后急性排斥反应的影响研究[J].中国普通外科杂志,2010,19(7):739-742.
DOI:10.7659/j. issn.1005-6947.2010.07.006

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  • 收稿日期:2010-03-09
  • 最后修改日期:2010-05-28
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  • 在线发布日期: 2010-07-15