摘要
目的采用血清蛋白质组分析技术(SERPA)筛选、鉴定肝癌自发抗体。方法双向电泳分离肝癌细胞系HCCLM3的总蛋白后将其转膜,肝癌、肝炎和正常组血清各8份与膜免疫印迹,图像分析确定不同血清免疫印迹图谱间的差异点及其与双向电泳图谱的对应关系,最后用基质辅助激光解吸飞行时间质谱进行鉴定。结果建立了高重复性HCCLM3的双向电泳图谱及其肝癌、肝炎及正常组血清的免疫印迹图谱,图谱均点数分别为603、70.75±24.25、68.50±23.44和41.38±15.05,肝癌、肝炎组图谱点数明显多于正常组,但肝癌与肝炎组差异无统计学意义。质谱鉴定确定了核蛋白、细胞骨架、代谢酶及热休克蛋白等五类肝癌自发抗体。结论SERPA是一种高通量筛选、鉴定肿瘤自发抗体的新技术,大量肝癌自发抗体的发现为肝癌进一步的免疫诊断及治疗奠定了基础。
Objective To screen hepatocellular carcinoma （HCC） autoantibodies as diagnostic biomarkers or therapy targets by serologic proteome analysis （SERPA）. Methods Total proteins extracted from human HCC cell line HCCLM3 were separated by two-dimensional electrophoresis （2-DE） and then transferred onto PVDF membranes, which were subsequently incubated with sera from HCC, hepatitis B virus （HBV） infected patients or healthy volunteers. All immuno-reactive protein spots on blot films were matched to those on 2-DE gel maps by image analysis and identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry （MALDI-TOF-MS/MS）. Results 2-DE gel maps of HCCLM3 and corresponding blot films of good quality and reproducibility were established. The number of spots on HCCLM3 2-DE reference gel totaled 603 and those on HCC, HBV and healthy sera blotted films were 70.75 ± 24.25, 68.5 ± 23.44 and 41.38 ± 15.05, respectively. Blot films of HCC and HBV groups had more spots than those of the healthy group （P 〈0.05） while no significance was found between films of HCC and HBV groups. By identification, those HCC autoantibodies could be classified as nuclear proteins, cytoskeleton proteins, heat shock proteins and metabolic enzymes. Conclusion Serological proteome analysis is a high throughput technique for screening tumor autoantibodies. Those newly identified HCC associated tumor antigens and corresponding autoantibodies can be used in the early diagnosis or immuno-therapy of HCC.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2005年第11期832-835,共4页
Chinese Journal of Hepatology
基金
国家973项目(001CB510202)863项目(2002BKC11A11)十五攻关项目(2004BA763802)
