不同大鼠心脏死亡器官捐献模型肝脏损伤比较研究

A comparative study on the damage against liver in different donation after cardiac death rat models

目的探讨不同大鼠心脏死亡器官捐献模型供肝损伤机制。方法将15只SD大鼠按随机数字表法分为氯化钾注射组、膈肌剪断组、心尖夹闭组,每组5只。氯化钾注射组通过背部浅表层静脉注射10%氯化钾溶液诱导心脏停跳;膈肌剪断组将膈肌剪断导致大鼠低血压和缺氧,诱导心脏停跳;心尖夹闭组开腹后用血管夹夹住左、右心室顶端,诱导心脏停跳。记录各组大鼠心脏停跳时间。3组大鼠心脏停跳后立即下腔静脉采血,检测血清ALT、AST、葡萄糖、乳酸脱氢酶(LDH)含量。取各组大鼠肝脏组织行HE染色,光镜下观察肝脏组织病理学变化。检测各组大鼠肝脏组织中过氧化氢、一氧化氮含量。实时荧光定量PCR法检测大鼠肝脏组织B细胞淋巴瘤/白血病-2相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)、半胱氨酸天冬氨酸特异性蛋白酶-3(Capase-3)转录情况。蛋白质印迹法检测大鼠肝脏组织磷酸化Akt(p-Akt)、Bax和Bcl-2蛋白表达情况。各组大鼠建模后心脏停跳时间、血清肝功能指标、肝脏组织一氧化氮和过氧化氢含量、凋亡相关分子(Bax、Bcl-2、Capase-3)转录水平及相关蛋白表达量比较均采用单因素方差分析。P<0.05为差异有统计学意义。结果膈肌剪断组和心尖夹闭组建模后心脏停跳时间分别为(720±137)、(1 020±142)s,均长于氯化钾注射组[(111±10)s,P均<0.05];氯化钾注射组和膈肌剪断组血清ALT分别为(29±14)、(49±11)U/L,均低于心尖夹闭组[(71±13)U/L,P均<0.05];心尖夹闭组血清AST、葡萄糖和LDH分别为(293±61)、(26.7±6.6)和(2 534±907)U/L,均高于氯化钾注射组[(131±67)、(15.8±8.5)、(1 528±1 170)U/L,P均<0.05]。HE染色结果显示心尖夹闭组和膈肌剪断组较氯化钾注射组有严重的充血和红细胞渗入,而心尖夹闭组与膈肌剪断组差异不明显。氯化钾注射组和膈肌剪断组肝脏组织过氧化氢含量分别为(13.8±3.7)、(13.7±1.8)mmol/L,均低于心尖夹闭组[(19.1±4.1)mmol/L,P均<0.05];心尖夹闭组一氧化氮含量为(13.3±3.2)mmol/L,高于膈肌剪断组[(6.9±0.4)mmol/L,P<0.05]。氯化钾注射组Bax转录量低于膈肌剪断组,心尖夹闭组Bax表达量高于氯化钾注射组,氯化钾注射组P-Akt 473和P-Akt 308表达量显著高于心尖夹闭组(P均<0.05)。结论氯化钾注射组对肝脏损伤影响最小,可能与细胞凋亡减少、P-akt相关信号通路激活有关。

Objective To investigate the damage against liver in different donation after cardiac death rat models and explore the underlying mechanisms. Methods Fifteen rats were equally divided into three groups： potassium chloride injection（PCI）,diaphragmatic cutting（DC）,apical clipping（AC） group. The PCL group was induced by intravenous injection of 10% potassium chloride solution to cardiac arrest through superficial dorsal vein of the penis. The DC group was made by clipping diaphragm to induce hypotension and hypoxia,and finally cardiac arrest. The AC group was made by clamping top of left and right ventricle of the heart with vascular clamp until cardiac arrest.The time of cardiac arrest was recorded. Serum was collected through postcava after cardiac death to evaluate liver function. Liver samples were collected and HE staining was performed. The level of nitric oxide（NO） and hydrogen peroxide（H2 O2） were determined. The mRNA level of Bax,Bcl-2 and Caspase-3 were detected by RT-PCR and the protein level of p-Akt,Bax and Bcl-2 were detected by western blot. One-way ANOVA was used for comparing the time of cardiac arrest、liver function indexes、NO and H2 O2 content in liver tissue、transcription levels of apoptosis-related molecules（Bax,Bcl-2,Capase-3） and related protein expressions between different groups. P〈0. 05 was considered statistically significant. Results The time of cardiac arrest of DC and AC group were（720 ± 137）,（1 020 ± 142） s respectively,longer than that in PCI group [（111 ± 10） s,P all 0. 05]. The serum levels of ALT in the PCI and DC group were（29 ± 14） 、（49 ± 11） U/L,respectively,lower than that in AC group [（71 ± 13） U/L,P all 0. 05]. The serum levels of AST、glucose and lactate dehydrogenase in the AC group were（293 ± 61）,（26. 7 ± 6. 6）,（2 534 ± 907） U/L,respectively,higher than those in PCI group [（131 ± 67）,（15. 8 ± 8. 5）,（1 528 ± 1 170） U/L,P all 0. 05].The result of HE staining showed that the AC and DC groups suffered more serious congestion and red blood cell infiltration than that in PCI group,while there were no significant differences between the AC and DC group. The contents of H2 O2 of PCI and DC group were（13. 8 ± 3. 7）,（13. 7 ± 1. 8） mmol/L,lower than that in AC group [（19. 1 ± 4. 1） mmol/L,P all 0. 05]. The NO content in the AC group was（13. 3 ± 3. 2） mmol/L,higher than that in DC group [（6. 9 ± 0. 4） mmol/L,P all 0. 05]. The mRNA level of Bax was found higher in DC group than that in PCL group. The expression of Bax in AC group was higher than that in the PCL group. There were marked increase of P-Akt 473 and P-Akt 308 expression in PCL group than AC group（P all 0. 05）. Conclusion The PCL group was considered the less damage to liver and its mechanism was probably associated with the cell apoptosis reduction and P-Akt pathway activation.