胰腺癌相关免疫原性膜抗原的筛查和鉴定

目的 应用蛋白质组学技术筛查、鉴定胰腺癌相关免疫原性膜抗原.方法 培养、提取并纯化胰腺癌细胞株SW1990的膜蛋白,将膜蛋白通过双向凝胶电泳(2-DE)进行分离,平行的3块2-DE凝胶分别行考马斯亮蓝染色和免疫印迹杂交.收集并纯化临床上66例胰腺癌和24例慢性胰腺炎患者血清中的IgG,分别与膜蛋白2-DE平行凝胶进行免疫印迹杂交.应用基质辅助激光解吸离子化飞行时间质谱分析杂交阳性蛋白位点,并经肽指纹库鉴定.应用RT-PCR、实时荧光定量PCR(real-time PCR)、Western blot方法对筛查出的膜抗原进行验证,并比较不同胰腺癌细胞株、正常胰腺组织中目的 膜抗原的基因及蛋白表达水平的差异.结果 胰腺癌细胞株SW1990膜蛋白与胰腺癌患者血清IgG免疫印迹杂交共出现9个阳性点,与同慢性胰腺炎IgG杂交所出现的2个阳性点无重复,经质谱分析鉴定出电压依赖性离子通道(VDAC)2可能是有潜力的候选膜抗原.经RT-PCR和real-time PCR验证,VDAC2基因在胰腺癌细胞株SW1990、AsPc、P3中均有表达.Western blot实验结果表明,VDAC2在胰腺癌细胞株中的蛋白表达明显高于正常胰腺组织.结论 胰腺癌细胞膜蛋白VDAC2可能是具有免疫原性的胰腺癌相关膜抗原,其基因在胰腺癌细胞株SW1990、AsPc、P3中均有表达,并且在胰腺癌细胞株中的蛋白表达水平明显高于正常胰腺组织.

Research of immunogenic membrane antigens of pancreatic cancer

Objective To screen and obtain the validate immunogenic membrane antigens in pancreatic cancer. Methods Pancreatic cancer cell line SW1990 membrane protein was extracted and separated by two-dimensional gel electrophoresis (2-DE). One of the three parallel 2-DE gels underwent Coomassie blue staining while the other two underwent immunoblot. Serum IgG was purified from clinically collected sera of 66 pancreatic cancer patients and 24 chronic pancreatitis patients and used as the primary antibody of the immunoblot. Positive dots of immunoblot were identified by MALDI-TOF mass spectrometry and PMF matching. The candidate membrane antigens were further validated respectively in cell lines and tissues by RT-PCR, renl-time PCR, Western blot, and their different expression level of gene and protein between pancreatic caner cell line and normal pancreatic tissue were contrastly studied. Results The immunoblot of SW1990 membrane protein with serum IgG from cancer patients showed nine positive dots which were not the same as those from immunoblot with serum IgG from chronic pancreatitis patients. One talent dot was identified with MALDI and PMF as VDAC2. RT-PCR and real-time PCR showed that the gene of VDAC2 was expressed in the pancreatic cancer ceLl line. Western blot showed that the expression of protein level of VDAC2 in the pancreatic cancer cell line was obviously higher than in normal pancreatic tissue. Conclusions VDAC2 might be the candidate immunogenic membrane antigens of pancreatic cancer, and its gene is all expressed in the pancreatic cancer cell line SW1990,AsPc and P3. The protein level of VDAC2 is significantly overexpressed in pancreatic cancer cell line than in normal pancreatic tissue.