缺血再灌注损伤促进裸鼠肝癌生长及癌旁组织VEGF和MMP-9表达

Ischemia-reperfusion promotes hepatic cancer growth and expression of VEGF and MMP-9 in adjacent tissues of cancer in nude mice

目的:观察缺血再灌注损伤对裸鼠肝癌生长及癌旁组织转移复发相关基因(VEGF、MMP-9)表达的影响。方法:将人肝癌细胞Hep3B原位种植于裸鼠肝脏制备荷肝癌裸鼠模型,荷瘤裸鼠随机分为缺血再灌注后1 h、6 h、5 d、7 d及假手术组(n=8),缺血再灌注组荷肝癌裸鼠采用肝门阻断法制备缺血再灌注损伤模型,假手术组不阻断肝门血供.其余同缺血再灌注组。缺血再灌注后1 h、6 h检测裸鼠肝功能(ALT、AST)的变化(n=8);实时荧光定量PCR检测瘤旁组织VEGF和MMP-9 mRNA的表达,并与正常对照组作比较(n=6)。缺血再灌注后5 d、7 d取肝组织,H—E染色观察肝组织病理学变化,计算肿瘤周边变性坏死区域(n=6)。剩余荷瘤裸鼠于缺血再灌注后2周处死取肿瘤,测定缺血再灌注组及假手术组裸鼠肿瘤体积及质量。结果:术后2周缺血再灌注组肿瘤体积(mm^3)及质量(g)明显高于假手术组[(209.6±25.74)vs(330.6±32.01).(0.214±0.036)vs(0.374±0.045),P<0.01]。缺血再灌注后1、6 h裸鼠血浆ALT和AST增高,明显高于假手术组(P<0.01)。H-E染色结果表明再灌注后5 d肿瘤周边组织炎症细胞浸润、变性坏死区域较假手术组更明显(P<0.05);再灌注后7 d肿瘤周边变性坏死区域较5 d减少(P<0.05),但变性坏死区域被肿瘤细胞浸润代替。荧光定量结果表明,缺血再灌注后1 h和6 h癌旁组织VEGF和MMP-9 mRNA表达明显高于假手术组(P<0.01)。且二者表达具有一定相关性(r=0.418,P<0.01)。结论:肝门阻断所导致的缺血再灌注损伤加速了裸鼠肝肿瘤细胞的生长,促进了癌旁组织中与转移复发相关基因(VEGF、MMP-9)的表达。

Objective：To observe the influence of ischemia-reperfusion （I/R） on hepatoma growth and on the expression of genes associated with tumor metastasis and recurrence （VEGF and MMP-9） in the adjacent tissues of cancer in nude mice. Methods： BALB/c nude mouse model bearing Hep3B-tumor in the liver was established and the model mice were evenly randomly into 5 groups： sham group and ischemia/reperfusion 1 h, 6 h, 5 d, and 7 d groups （n=8）. I/R models were established by blocking porta hepatic; the sham group underwent the same treatment as the I/R model group except for blocking of porta hepatic. ALT and AST were detected in I/R 1 h and 6 h groups. Real-time-PCR was employed to detect the change of VEGF and MMP-9 in the adjacent tissues of cancer and the results were compared with that of the control group （n = 6）. Histopathological changes of liver were studied by H-E staining and necrotic areas were calculated in I/R 5 d and 7 d groups （n=6）. The remnant tumor bearing mice were sacrificed 2 weeks after I/R to measure the volume and mass of the tumors. Results： Two weeks later, the tumor volume and mass in I/R group were increased compared with those in the sham group （[209.6±25.74] mm^3 vs[330.6±32.01] mm3 ,[0. 214±0. 036] g vs [0. 374±0. 045] g, P〈0.01）. Levels of ALTandAST were significantly elevated in I/R 1 h and 6 h groups compared with those in the sham group （P〈0.01）. H-E staining showed that the infiltration of inflammatory cells around the tumors and the areas of necrosis became more prominent 5 days after I/R compared with the sham operation group （P〈0.05）. The necrosis area was reduced 7 days after I/R compared with 5 days after I/R （P〈 0. 05）; however, the necrosis area was replaced by invasion of tumor cells （P〈 0. 05）. It was found that the expression of VEGF and MMP-9 mRNA was higher in the adjacent tissues of cancer after I/R than that in the sham group （P〈 0.01）, and the expression of the 2 was correlated with each other （r=0. 418, P〈0.01）. Conclusion： I/R induced by blocking porta hepatic can accelerate tumor cell growth and promote expression of VEGF and MMP-9 in the adjacent tissues of cancer.