放射性肝损伤的血清比较蛋白质组学分析

Serum comparative proteomic analysis of radiation-induced hepatic injury in cirrhotic rats

目的:通过分离、鉴定及验证SD肝硬化大鼠肝脏放疗前后血清差异蛋白质的表达,探讨可能用于检测及监控放射性肝损伤的早期生物学指标。方法:8只健康成年SD大鼠皮下注射四氯化碳(CCl4),连续6周,建立肝硬化模型后随机分组,每组4只,即对照组和实验组。对照组大鼠(CCl4组)未给予放疗,实验组大鼠(CCl4+放疗组)行半肝照射,单次剂量15 Gy。6 h后分别提取2组大鼠的血清总蛋白,进行比较蛋白质组学分析。采用双向凝胶电泳分离蛋白,经图像分析识别2组大鼠放疗前后差异表达的蛋白质。应用基质辅助激光解析电离飞行时间质谱(MALD-TOF-MS)鉴定差异蛋白质。应用Western blotting对血清中乙酰肝素酶和肝细胞生长因子受体的表达水平进行验证。结果:获得了分辨率和重复性均较好的凝胶蛋白图谱。共筛选出33个在实验组明显差异表达的蛋白点,其中12个蛋白质鉴定成功。在这12个蛋白质中,实验组中5个表达上调,7个表达下调。Westernblotting进一步证实了实验组乙酰肝素酶表达上调,肝细胞生长因子受体表达下调。结论:成功鉴定了12个与放射性肝损伤相关的蛋白。乙酰肝素酶和肝细胞生长因子受体可能作为预测及监控放射性肝损伤的早期指标。临床上要将二者作为可能的生物学标志物,其敏感性和特异性还有待进一步研究。

AIM： To investigate the differential expression of serum proteins in cirrhotic SD rats for exploring the pathogenesis and identifying the potential biomarkers of radiation-induced hepatic injury.METHODS： Liver cirrhosis was induced in 8 healthy SD rats with subcutaneous injection of carbon tetrachloride（CCl4） for 6 weeks and then the animals were randomly divided into 2 groups（4 rats in each group）： control group（CCl4 alone） and experimental group（CCl4 plus radiation）.The latter received hemi-liver radiation with single dose of 15 Gy while the former did not receive radiation.Total serum proteins of the 2 groups were extracted 6 h after radiation.Two-dimensional gel electrophoresis（2-DE） were performed to look for differentially expressed proteins.These proteins were then analyzed and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF-MS）.Western bloltting was used to validate the expression of heparanase and hepatocyte growth factor receptor in all independent series of serum samples.RESULTS： Two-dimensional gel images were acquired with good resolution and repetition.Thirty-three differentially expressed proteins were selected and 12 proteins were successfully identified by MS,in which 5 were up-regulated while 7 were down-regulated.The increased level of heparanase and decreased level of hepatocyte growth factor receptor were further confirmed by Western blotting.CONCLUSION： Twelve proteins associated with radiation-induced hepatic injury are successfully characterized by serum comparative proteomics.Heparanase and hepatocyte growth factor receptor might be useful for detecting and monitoring radiation-induced hepatic injury.