摘要
目的对前期蛋白质组学筛查、鉴定的人胰腺癌相关免疫原性候选膜抗原进行有效验证。方法前期实验中，将胰腺癌患者血清提取的IgG与人胰腺癌细胞株SW1990的膜蛋白进行免疫印迹杂交，共获得了9个阳性蛋白位点，现应用基质辅助激光解吸离子化飞行时间质谱分析与肽质指纹库对5号和6号位点进行鉴定。应用RT．PCR、real-timePCR验证二者在不同的人胰腺癌细胞株中的基因表达水平。分别提取人正常胰腺组织和胰腺癌组织的mRNA，应用real—timePCR检测prohibitin2在人正常胰腺组织中和胰腺癌组织中的基因表达差异。结果5号和6号阳性蛋白位点经质谱分析鉴定分别是prohibitin2和prohibitin。RT—PCR和real—timePCR证明，候选膜抗原prohibitin2和prohibitin在人胰腺癌细胞株SWl990、AsPc、P3的细胞中均有明确的基因表达，尤其在P3细胞中mRNA表达水平最高（t=7．442，P〈0．01）。并且prohibitin2在人胰腺癌组织中的基因表达水平明显高于正常胰腺组织（t=0．893，P〈0．01）。结论人胰腺癌细胞膜蛋白prohibitin2和prohibitin在人胰腺癌细胞株SWl990、AsPc、P3中均有明确的基因表达，并且prohibitin2在人胰腺癌组织中的基因表达水平明显高于人正常胰腺组织，二者可能是具有免疫原性的胰腺癌相关膜抗原。
Objective To validate those obtained immunogenic membrane antigens candidate of human pancreatic cancer in the performed research. Methods In the pre-studies, serum IgG purified from clinically collected sera of pancreatic cancer patients underwent immunoblot with human pancreatic caner cell line SW1990 membrane protein, totally obtained 9 positive protein spots. Number 5 and 6 positive dots of immunoblot were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting matching. The candidate membrane antigens were further validated in cell lines by RT-PCR and real-time PCR. RNA of human normal pancreatic tissue and pancreatic cancer tissue was extracted respectively, different gene expression level of prohibitin 2 was studied by real-time PCR. Results Number 5 and 6 positive dots were identified as prohibitin 2 and prohibitin. RT-PCR and real-time PCR all showed that gene of prohibitin 2 and prohibitin were expressed in the human pancreatic cancer cell line SW1990, AsPc and P3 respectively, especially in P3 cell with highest expression（ t = 7.442,P 〈 0.01 ）. In addition, gene expression level of prohibitin 2 was significant higher in human pancreatic cancer than that of normal pancreatic tissue （ t = 0. 893, P 〈 0. 01 ）. Conclusions Prohibitin 2 and prohibitin are both differently expressed in the pancreatic cancer cell line SW1990, AsPc and P3. Prohibitin 2 is obvious highly expressed in human pancreatic cancer tissue. Prohibitin 2 and prohibitin might be the candidate immunogenic membrane antigens of human pancreatic cancer.
出处
《中华外科杂志》
CAS
CSCD
北大核心
2012年第3期260-263,共4页
Chinese Journal of Surgery
