摘要目的 探讨粒细胞集落刺激因子(G-CSF)抑制大鼠急性肝衰竭(ALF)肝细胞凋亡的作用及机制.方法 D-氨基半乳糖(D-GalN)制备SD大鼠急性肝衰竭模型.治疗组皮下注射重组人粒细胞集落刺激因子(rhG-CSF)50 μg/kg共3 d,安慰剂组皮下注射生理盐水共3 d.建模后观察动物生存率,分别于6 h、12 h、1 d、3 d取肝脏,流式细胞仪检测肝细胞凋亡率,免疫组化法检测肝脏Bcl-2、半胱氨酸蛋白水解酶(caspase)-3表达,图像分析系统半定量分析.结果 治疗组生存率高于安慰剂组(53.3% vs 33.3%,P=0.027).两组肝细胞凋亡率、肝组织Bcl-2、caspase-3表达量均随时间而升高.1 d时治疗组肝细胞凋亡率(29%±7%)低于安慰剂组(44%±12%),差异有统计学意义(P=0.026).治疗组12 h、1 d时肝组织Bcl-2灰度值均低于安慰剂组,差异有统计学意义(分别为152±37 vs 161±7,P=0.012;150±12 vs 159±9,P=0.018),表示Bcl-2表达量高于安慰剂组;治疗组1 d、3 d时肝组织caspase-3灰度值均高于安慰剂组,差异有统计学意义(分别为189.6±4.6 vs 169.6±15.7,P=0.000;184.7±4.8 vs 160.0±5.0,P=0.000),治疗组caspase-3表达量低于安慰剂组.结论 肝细胞凋亡在ALF发病过程中发挥了重要作用.G-CSF通过促进肝细胞Bcl-2表达和减少caspase-3表达,延缓并减少肝细胞凋亡的发生,提高ALF大鼠生存率.

abstractsObjective To explore the effects of granulocyte colony-stimulating factor(G-CSF)on hepatocyte apoptosis in acute liver failure(ALF)and possible mechanism thereof. Methods One hundred and sixty SD rats underwent intraperitoneAI injection of D-gaiactosamine(D-GalN)1. 4 g/kg so as to establish AFL models and then were randomly divided into 2 equal groups: G-CSF therapy group, injected consecutive days, and placebo control group, injected hypodermically with normal saline for 3 consecutive days. Liver samples were collected from 6 rats of each group 6 h, 12 h, 1 day, and 3 days after D-GalN injection respectively. Another six normal rats were used as normal control group. Hepatoeyte apoptosis rate was measured by flow cytometry. Immunohistochemistry was used to detect the expression of Bcl-2 and caspase-3, a proapoptosis protein, in the liver sections. Results The survival rate of the G-CSF therapy group was 53. 3%, significantly higher than that of the placebo control group(33. 3%, P=0. 027). The hepatocyte apoptosis rate and expression rates of Bcl-2 and caspase-3 in the liver sections after D-GalN injection increased along with time. The hepatoeyte apoptosis rate peaked 1 day after the D-GalN injection in both groups. The maximum hepatocyte apoptosis rate of the G-CSF group was 29%±7%, significantly lower than that of the placebo control group(44%±12%, P=0. 026). The gray scale of Bcl-2 in liver sections at hour 12 of the G-CSF group was 152±37, significandy lower than that of the placebo control group(161±7, P=0. 012). and the gray scale of Bcl-2 on day 1 of the G-CSF group was 150±12, significantly lower than that of the placebo control group(159±9, P=0. 018). The gray seales ofcaspase-3 on days 1 and 3 of the G-CSF group were 189. 6±4. 6 and 184. 7±4. 8 respectively, both significantly higher than those of the placebo control group(169. 6±15. 7 and 160. 0±5. 0, both P=0. 000). Conclusion Apoptosis is a key mechanism contributing to ALF. G-CSF prevents ALF induced by D-GaIN, thus raising the survival rate. GGSF shows an inhibitory efficacy on hepatocytes apoptosis by probably up-regulating Bcl-2 and reducing caspase-3 expression.