基础研究 Open Access
Copyright ©The Author(s) 2005. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2005-09-28; 13(18): 2197-2200
在线出版日期: 2005-09-28. doi: 10.11569/wcjd.v13.i18.2197
肝移植前受体亚低温对急性肝衰大鼠移植肝脏的保护作用
王志东, 韩德恩, 崔云甫, 姜明山, 张新宇, 曾兆林
王志东, 韩德恩, 崔云甫, 姜明山, 张新宇, 曾兆林, 哈尔滨医科大学附属第二医院肝胆外科 黑龙江省哈尔滨市 150086
王志东, 男, 1975-04-28生, 黑龙江省兰西县人, 汉族, 2005年哈尔滨医科大学博士, 主治医师, 主要从事急性肝衰竭肝移植的临床与实验研究工作.
基金项目: 黑龙江省科技攻关资助项目, No. GB02C142-02.
通讯作者: 王志东, 150086, 黑龙江省哈尔滨市, 哈尔滨医科大学附属第二医院肝胆外科. wzd98y2@sohu.com
电话: 0451-86605117
收稿日期: 2005-07-28
修回日期: 2005-07-31
接受日期: 2005-08-03
在线出版日期: 2005-09-28

目的: 观察肝移植术前应用亚低温对急性肝衰竭大鼠移植肝脏的影响.

方法: 采用肝大部分切除加部分缺血建立大鼠急性肝衰竭模型. 术后12 h分别对亚低温处理组(35.0 ℃)和常温组(37.5 ℃)衰竭大鼠施行原位肝移植, 比较移植前后TNF-α浓度变化、移植后供肝凋亡和再灌注损伤情况.

结果: 亚低温组移植前后血清TNF-α浓度明显低于对照组(19.3±5.9 vs 43.4±9.0 µg/L, t = 5.008, P = 0.007, 97.7±18.3 vs 137.1±23.3 µg/L, t = 9.471, P = 0.001), 移植后供肝过氧化损伤(MDA)(407.1±49.4 vs 598.2±61.8 nmol/L, t = 34.46, P = 0.0009)和凋亡指标(18.3±3.9% vs 23.6±4.3%, t = 29.63, P = 0.0007)均明显优于常温组.

结论: 肝移植前应用亚低温能够降低急性肝衰竭大鼠血清TNF-α浓度并减轻移植后供体肝脏的再灌注损伤程度.

关键词: 急性肝衰竭; 亚低温; 肝移植; 大鼠
引文著录: 王志东, 韩德恩, 崔云甫, 姜明山, 张新宇, 曾兆林. 肝移植前受体亚低温对急性肝衰大鼠移植肝脏的保护作用. 世界华人消化杂志 2005; 13(18): 2197-2200
Moderate hypothermia therapy for acute liver failure in rats before liver transplantation
Zhi-Dong Wang, De-En Han, Yun-Fu Cui, Ming-Shan Jiang, Xin-Yu Zhang, Zhao-Lin Zeng
Zhi-Dong Wang, De-En Han, Yun-Fu Cui, Ming-Shan Jiang, Xin-Yu Zhang, Zhao-Lin Zeng, Department of Hepatobilliary Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin 150086, Heilongjiang Province, China
Correspondence to: Department of Hepatobilliary Surgery, the Second Affiliated Hospital of Harbin Medical University, Harbin 150086, Heilongjiang Province, China. wzd98y2@sohu.com
Received: July 28, 2005
Revised: July 31, 2005
Accepted: August 3, 2005
Published online: September 28, 2005

AIM: To observe the effect of moderate hypothermia on the donated livers in rats with acute liver failure (ALF) before liver transplantation.

METHODS: ALF rats model were established by hepatectomy and hepatic devascularization. In situ liver transplantations were carried out for hypothermia treated group (35.0ºC) and the control group (37.5ºC) 12 h after the transplantation. Blood and tissue samples were collected just before and 6 or 24 h after transplantation. The concentrations of TNF-α were compared before and after the transplantation. The content of malondialdehyde (MDA) and the apoptotic rate of hepatic cells after the transplantation were observed. The apoptotic rate and MDA contents in donated liver were also assayed. The morphological changes of liver tissues were observed under microscope.

RESULTS: In the hypothermia treated group, the concentrations of TNF-α before and after the transplantation were significantly lower than those in the control group (19.3±5.9 vs 43.4±9.0 µg/L, t= 5.008, P = 0.007; 6 h, 97.7±18.3 vs 137.1±23.3 µg/L, t = 9.471, P = 0.001); the MDA contents and apoptotic rate after transplantation were also significantly lower than those in the control group (407.1±49.4 vs 598.2±61.8 nmol/L, t = 34.46, P = 0.001; 18.3±3.9% vs 23.6±4.3%, t = 29.63, P = 0.001).

CONCLUSION: Moderate hypothermia can decrease TNF-α level and relieve the damages of the donated liver in rats with ALF.

Key Words: Acute liver failure; Moderate hypothermia; Liver transplantation; Rats
0 引言

原位肝移植是挽救重症急性肝衰竭的最有效措施[1-4]. 近年来有人将亚低温应用于急性肝衰竭治疗, 取得良好效果[5-12]. 目前, 亚低温用于急性肝衰竭治疗尚处于研究阶段, 其对随后肝移植的影响更少见报道. 我们利用改进的大鼠急性肝衰竭模型观察亚低温对衰竭大鼠肝移植后移植肝脏的影响.

1 材料和方法
1.1 材料

供受体均为雄性Wistar大鼠, 由哈尔滨医科大学动物实验中心提供, 受体质量300±20 g, 供体质量270±20 g. 将大鼠随机分成3组: A组为亚低温衰竭移植组, B组为常温衰竭移植组, C组为假手术移植对照组, 每组16只. 急性肝衰竭模型采用肝脏大部切除加部分缺血模型. 乙醚麻醉后正中切口开腹, 游离并结扎通往右肝叶和两个前叶的管道系统, 按照Higgins and Anderson方法[13]切除两个前叶. 原位肝移植采用改良的Kamada二袖套法[14], 未重建肝动脉. 灌洗液和保存液使用乳酸林格液(大冢制药有限公司). 供肝冷保存时间控制在40 min, 门静脉阻断时间控制在18-20 min, 受体手术时间40±5 min. 根据前述方法将A, B组大鼠制成急性肝衰竭模型, 模型完成后, 通过置入直肠内的温度传感器(灵敏度为0.1 ℃,上海仪表有限公司XMTD-2001E)和自制温度控制装置(图1)自动控制大鼠体温在设定范围内. 控温期间禁食, 为防止出现脱水和低血糖, 每4 h生理盐水和50 g/L葡萄糖SC各5 mL[15]. A组体温控制在35 ℃±0.1 ℃, B组体温控制在37.5±0.1 ℃, C组按照A, B组手术步骤, 但只游离不结扎和切除肝叶, 体温控制在37.5±0.1 ℃.

图1
图1 温度控制装置示意图.
1.2 方法

三组大鼠第1次手术后12 h分别接受原位肝移植手术, 术前股静脉采血0.5 mL. 移植后6 h每组随机选择5只大鼠, 股静脉采血0.5 mL.ELISA法检测血清TNF-α浓度(上海森雄科技实业有限公司试剂盒 BS6002). 移植后6 h每组随机选择5只大鼠, 开腹取肝脏左外叶组织, 制备成100 g/L组织匀浆, 按照南京建成生物技术有限公司试剂盒说明书用比色法测定MDA浓度. 移植后24 h处死所有大鼠, 取肝脏组织做病理检查和凋亡检测. 肝脏组织切片常规染色. 脱氧核苷酸末端转移酶介导缺口末端标记法(TUNEL)检测凋亡. 肝脏组织常规石蜡包埋, 切取厚5 µm切片, 按照Boehringer Mannheim公司TUNEL试剂盒说明书操作, 最后用苏木素复染. 细胞核被染成棕黄色者并有凋亡特征者为阳性细胞. 400倍光镜下随机选取4个视野, 计算平均阳性细胞百分率, 即凋亡指数(AI).

统计学处理 实验数据用mean±SD来表示, 两两均数间比较用t检验, P<0.05有显著性差异.

2 结果
2.1

移植肝脏24 h HE染色显示3组供肝组织结构均发生明显病理改变: 对照组肝细胞水肿明显, 汇管区少量炎性细胞浸润, 部分肝细胞发生嗜酸性变(图2A); 常温组可见大片肝细胞坏死, 汇管区炎性细胞浸润明显(图2B-C); 低温组汇管区炎性细胞浸润少于常温组, 可见点灶状坏死(图2D).

图2
图2 移植肝组织学 HE×400. A: 假手术移植对照组; B: 常温移植组; C: 常温移植组; D: 亚低温组.
2.2 血清TNF-α水平

低温组移植0 h血清TNF-α水平低于常温组(P<0.05), 移植后6 h血清TNF-α水平仍明显低于常温组(P<0.05, 表1).

表1 肝移植前亚低温处理对移植大鼠血清TNF-α、肝脏MDA和肝脏凋亡指数(AI)的影响 (mean±SD, n = 5).
0 h TNF-α(µg/L)6 h TNF-α(µg/L)6 h MDA(nmol/L)24 h AI(%)
低温组19.3±5.9a97.7±18.3a407.1±49.4a18.3±3.9a
常温组43.4±9.0137.1±23.3598.2±61.823.6±4.3
对照组5.1±0.7a89.3±15.7a376.7±42.6a8.8±2.7a
aP<0.05, vs 常温组.
2.3 肝组织MDA检测

低温组肝移植后6 h肝组织MDA水平明显低于常温组(P<0.05, 表1).

2.4 肝细胞凋亡情况

移植后24 h对照组肝内散在分布凋亡细胞(图3A); 常温组肝内凋亡细胞多见, 广泛均匀分布(图3B), 二者差异显著(P<0.05); 低温组肝内凋亡细胞明显少于常温组(P<0.05)(图3C). 三组凋亡率见表1.

图3
图3 移植肝细胞凋亡( TUNEL ×400). A: 亚低温移植组; B: 常温衰竭移植对照; C: 健康移植对照.
3 讨论

受体循环当中可能影响供体肝脏的因素很多, 如多种促炎症因子和炎性介质、胆红素等毒性代谢产物、内毒素的直接损害等等[16-18]. 考虑到TNF-α在再灌注损伤中的重要作用和亚低温对TNF-α的影响[19-23],本部分实验重点观察了受体术前、术后循环中TNF-α水平的变化和供体肝脏损伤的情况. 本结果表明, 移植后6 h移植肝脏的过氧化损伤程度存在差别: 常温组损伤程度最重, 对照组损伤最轻, 亚低温组损伤介于两者之间, 与常温组有显著差异. 这表明受体移植前内环境差别对移植肝脏的再灌注损伤有影响, 而移植前亚低温处理能够减轻受体因素对移植肝脏的损害. 本实验移植0 h和移植6 h血清TNF-α水平检测结果显示亚低温处理组TNF-α水平明显低于常温移植对照组. 同时, 移植后6 h供肝MDA水平和24 h供肝凋亡指数都明显低于常温移植对照组. 这说明受体移植前循环中高水平TNF-α可能是影响移植肝脏损伤的受体因素之一[24-26]. 术前亚低温处理可能是通过降低循环中TNF-α水平减轻随后移植的供体肝脏的再灌注损伤[27]. 由于灌注液和保存液不够理想, 移植后24 h肝脏HE染色显示所有三组大鼠肝脏组织损伤都很严重, 但是常温组供肝肝细胞坏死和汇管区炎性细胞浸润明显重于其他两组, 显示肝实质损伤较重. 本实验结果表明肝移植前对急性肝衰竭受体大鼠进行亚低温处理能够减轻随后移植供体肝脏的再灌注损伤, 提示亚低温在急性肝衰竭围手术期的应用不仅有利于脑水肿的防治, 对减轻移植肝脏损伤也可能有效[28,29]. 本实验中还有很多问题需要进一步研究: 亚低温对肝脏再生能力的影响如何; 术前受体高炎症因子血症加重供体肝脏缺血再灌注损伤的机制如何; 肝脏再灌注损伤相关基因的表达情况以及亚低温对抑制炎症反应因子, 如IL-10的影响如何[30].

电编:张勇 编辑:潘伯荣 审读:张海宁

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