砷剂对实验性鼠肝癌血管生成的影响

Anti-hepatoma effect and its mechanism of arsenic trioxide on experimental liver cancer

目的:研究三氧化二砷(As2O3)对大鼠体内实验性肝癌的治疗作用及其作用机制. 方法:选择健康成年封闭群(?) SD大鼠(2月龄),以含0.5g/L 2-乙酰氨基芴(2-FAA)进行喂饲制备大鼠肝癌模型.治疗组以两种浓度的As2O3溶液与复方苦参分别注射于大鼠腹腔,1次/d,2 wk后改为2次/wk,总给药时间为4 wk; 同时设生理盐水对照组(NS).治疗4 wk末处死大鼠,获取肝脏组织和血清;称肝湿重;记录肝表面肿瘤结节分布隋况; 肝肿瘤组织HE染色后在光镜下观察肝脏组织形态学变化; 流式细胞仪检测肿瘤细胞动力学变化;并分别应用VEGF多克隆抗体和FⅧRAg多克隆抗体染色的免疫组化方法检测致癌组织的血管内皮生长因子(VEGF)和微血管密度(MVD) 表达情况;并测定血清丙氨酸氨基转移酶(ALT)、谷丙转氨取AST)及总胆红素(TBi)和结合胆红素(DBi)水平. 结果:As2O3治疗组,肝肿瘤结节明显少于生理盐水对照组,尤以中剂量As2O3治疗组表现最为显著(P<0.01).免疫组化显示As2O3治疗组VEGF表达强度及相应的MVD明显低于生理盐水对照组(P<0.001).复方苦参和生理盐水对照组上述各项指标相比差异无显著性(P>0.05).各治疗组的血清AST活性明显低于生理盐水对照组(P<0.05),但各组之间无显著性差异(P>0.05);血清ALT、TBi及DBi水平各组之间以及与生理盐水对照组差异均无显著性(P>0.05). 结论:As2O3对大鼠肝细胞肝癌生长具有明显抑制作用,并目.对肝功能又无明显毒副作用.作用机制可能与抑制VEGF 合成间接抗肿瘤血管形成有关.复方苦参无明显治疗作用.

AIM: To study the synergistic anti-hepatoma efficacy of arsenic trioxide (As2O3) and matrine in the treatment of experimental rat hepatocellular carcinoma (HCC) induced by 2-acetamidofluorene (2-FAA) and to elucidate the possible mechanisms. METHODS: SD rats (2 months old) were fed with 2-FAA for 8 weeks to induce HCC, then treated with As2O3 and matrine. On day 29, we weighed the liver and counted liver tumors. The histological changes of the liver were observed under microscope, and the cellular dynamic parameters were studied by flow cytometry. Immunohis-tochemistry (two-step method) was used to observe the expression of vascular endothelial growth factor (VEGF) and to show micro-vessel density (MVD) on consecutive sections. The pathological parameters were analyzed at the same time. The levels of serum AST, ALT, TBi and DBi were also investigated. RESULTS: The number of liver tumors decreased significantly in groups treated with As2O3, especially in middle-dose (1 mg/kg) group (P <0.01). Treatment with As2O3 caused HCC cell death via apoptosis. Necrosis was seldomly found and apoptosis was common when the dose was appropriate (1 mg/kg). Proliferation index (PI) decreased sharply in middle-dose (1 mg/kg) group (P<0.01), but not in another (0.2 mg/kg) group (P>0.05). However, S phase fraction (SPF) decreasd dramatically in the two groups, it reached the top only when the dose (1 mg/kg) was appropriciate (P<0.01), and it was obviously accompanied with accumulation of cells in G0/G1 (G0/G1 restriction). The inhibitory effects of As2O3 on HCC cells were not signifi- cantly enhanced by matrine. Comparied with single treatment with As2O3 1 mg/kg or 0.2 mg/kg, G0/G1 phase cells in the group of combination treatment with As2O3 1 mg/kg or 0.2 mg/kg and matrine 4.2 g/kg did not increased, and apoptostic cells with fractional DNA content did not increased either. The expression of VEGF and MVD was significantly lower in middle-dose (1 mg/kg) group (P<0.01) than that in normal saline group. In comparison with normal saline group, administration of AS2O3 or/and matrine lowered the levels of AST in serum (P<0.05), but had no effect on the amount of serum AST, TBi and DBi (P>0.05). CONCLUSION: Arsenic trioxide inhibits growth of experimental hepatocellular carcinoma in rats induced by 2-acetamidofluorene, but As2O3 has no obvious effect on the normal hepatic cells. The mechanisms may involved in decreasing cell mitosis, accumulating cells in G0/G1 phrase, inducing apoptosis of turner cells, and having inhibitory effects on the angiogeninase through inhibiting vascular endothelial growth factor. Matrine with As2O3 has not significantly synergistic anti-hepatoma effects.