醫(yī)學免費論文:NFκB及其相關(guān)炎性因子致大鼠移植肝冷保存損傷的機制
【摘要】 目的 通過大鼠移植肝在不同冷保存時間再灌注后核因子κB(nuclear transcription factorκB, NFκB)及其相關(guān)炎性因子的激活情況,判斷冷保存時間、NFκB激活以及移植肝功能損傷三者之間的聯(lián)系。方法 用Wistar大鼠建立原位肝移植模型,假手術(shù)組(A組)作為對照組,觀察大鼠移植肝在4℃ UW液中保存45min(B組)、120min(C組)、180min(D組)以及再灌注3h后肝組織、血清中NFκB、腫瘤壞死因子α(TNFα)、白細胞介素1β(IL1β)和肝損傷指標丙氨酸轉(zhuǎn)氨酶(ALT)、天冬氨酸轉(zhuǎn)氨酶(AST)和肝細胞凋亡的變化情況。結(jié)果 隨著移植肝冷保存時間的延長NFκB逐漸激活(P<0.05),并使肝組織內(nèi)TNFα、IL1β水平上調(diào)(P<0.05);再灌注后,NFκB近一步激活(P<0.05),肝組織中TNFα、IL1β進一步上調(diào)(P<0.05),ALT、AST和肝細胞凋亡指數(shù)明顯升高(P<0.05),肝功能損害加重。結(jié)論 NFκB在供肝的冷保存階段隨時間的延長呈誘導性激活,再灌注后呈爆發(fā)式激活,并通過上調(diào)其靶基因TNFα、IL1β的轉(zhuǎn)錄產(chǎn)生肝臟損傷,這可能是肝臟冷保存再灌注損傷發(fā)生的重要機制。
【關(guān)鍵詞】 肝移植;器官保存;冷保存再灌注損傷;核因子κB
The effect of NFκB and correlated inflammatory factors
in rat donor liver after cold preservation
JIANG An, LIU Feng, LIU Chang, SONG Yulong, MENG Kewei, L Yi
1. Department of Hepatobiliary Surgery, the First Affiliated Hospital, Medical School of Xian Jiaotong University, Xian 710061; 2. Department of General Surgery,
the Second Affiliated Hospital, Medical School of Xian Jiaotong University, Xian 710004;3. Department of Hepatobiliary Surgery, Qianfoshan Hospital, Jinan 250014;
4. Department of Anesthesiology, Shaanxi Provincial Peoples Hospital, Xian 710068;5. Department of Hepatobiliary Surgery, Yuhuangding Hospital, Yantai 264000, China
ABSTRACT: Objective To establish a model of rat orthotopic liver transplantation and investigate the relationship among cold preservation time, activation of nuclear transcription factorκB (NFκB) and donor preservation injury after liver transplantation. Methods Orthotopic liver transplantation was performed in Wistar rats which were randomly divided into the following groups according to the different duration of liver cold storage in UW solution: group A (sham operation, n=10), group B (45min cold storage group, n=10), group C (120min cold storage group, n=10), and group D (180min cold storage group, n=10). The expression of NFκB in liver before and after transplantation was measured by electrophoretic mobility shift assays; protein expression of tumor necrosis factoralpha (TNFα) and interleukin1 beta (IL1β) in the liver was measured by immunohistochemistry; the serum TNFα and IL1β, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hepatic cell apoptosis were examined. Results With extended cold storage duration, the activity of NFκB in the donor liver increased (P<0.05, group D vs. groups A, B and C). TNFα and IL1β levels also increased (P<0.05, group D vs. groups A, B and C). Donor liver reperfusion injury was gradually aggravated with the prolonging of graft cold preservation. Both the serum TNFα and IL1β levels increased highly (P<0.05 group A vs. groups B, C and D),and the expression of NFκB in the liver significantly increased (P<0.05, group A vs. groups D, B and C) with gradual prolonging of graft cold preservation time. The serum ALT and AST level and apoptosis index level elevated greatly (P<0.05, group A vs. groups D, B and C). Conclusion NFκB of donor liver was activated inductively in cold preservation phase and activated explosively in reperfusion phase. The longer cold preservation time was, the higher NFκB level in donor liver became. NFκB led to the expression of TNFα and IL1β in donor liver. Inflammatory factors are one of the most important mechanisms for the donor liver injury after liver transplantation醫(yī).學.全.在.線quanxiangyun.cn.
KEY WORDS: liver transplantation; organ preservation; cold preservationwarm reperfusion injury; nuclear transcription factorκB
移植肝冷保存再灌注損傷(cold preservationwarm reperfusion injury, P/RI)是移植肝功能不良(poor graft function, PGF)和原發(fā)性無功能(primary nonfunction, PNF)的直接原因[1]。PGF及其嚴重形式PNF的發(fā)生率分別占移植總數(shù)的5%~10%[2]和1.4%~8.5%[3]。PNF占術(shù)后1周內(nèi)再次肝移植病因的81%和再次肝移植總病因的21.7%[1],危害嚴重。冷保存時間超過20h,PNF發(fā)生率是保存4h以內(nèi)的4倍[4],所以冷保存時間是PNF的獨立預測因子[5]。而且,P/RI還可導致多器官功能不全和膽管非吻合口狹窄等并發(fā)癥[6],嚴重影響患者預后。因此,如何最大限度地減輕供肝冷保存損傷、提高移植物成活率是當今肝移植領(lǐng)域面臨的重要課題。本實驗通過研究移植肝P/RI過程中核轉(zhuǎn)錄因子κB(nuclear transcription factorκB, NFκB)及其相關(guān)炎性因子的表達變化,探討NFκB在移植肝冷保存中的作用機制。
1 材料與方法
1.1 動物模型的制作
清潔級健康成年封閉群雄性Wistar大鼠70只(第四軍醫(yī)大學實驗動物中心提供),體重(272±31)g。術(shù)前禁食12h,不禁飲,清潔手術(shù)。氯胺酮100mg/kg體重腹腔注射麻醉。采用改良兩袖套法建立大鼠全血供原位肝移植模型[7]。
1.2 實驗動物分組
依據(jù)供肝冷保存時間的不同,將Wistar大鼠隨機分為4組,每組10只:A組,假手術(shù)組;B組,冷保存45min再灌注3h組;C組,冷保存120min再灌注3h組;D組,冷保存180min再灌注3h組。供肝以4℃ UW液保存,冷保存結(jié)束前,留取乳頭葉腹側(cè)葉片。移植肝植入3h后,留取乳頭葉背側(cè)葉片,并取血留作標本。
1.3 觀察指標及檢測方法
1.3.1 肝組織NFκB水平的測定