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目的筛查具有早期诊断价值的人胰腺癌相关膜抗原。方法采用膜生物学技术抽提多株胰腺癌细胞的膜蛋白;双向聚丙烯酰胺凝胶电泳(2-DE)分离膜蛋白,将其转移至 PVDF 膜上;利用纯化的胰腺癌患者血清 IgG 与分离的膜蛋白进行免疫印迹杂交,比对膜蛋白2-DE 图谱,筛查胰腺癌特异的免疫杂交阳性斑点;MALDI-TOF 质谱鉴定杂交阳性斑点;采用 RT-PCR 技术检测膜抗原基因在多株胰腺癌细胞中的表达;利用免疫组化染色技术检测膜抗原在人胰腺癌组织中的表达情况。结果成功提取了胰腺癌细胞株的膜蛋白;经比对膜蛋白2-DE 图谱,筛查出免疫杂交阳性斑点;经MALDI-TOF 质谱鉴定,免疫杂交阳性点分别为 VDAC-2、VDAC-1、CHCHD3、SLP-2、TOM40;RT-PCR 结果显示上述膜抗原基因在多株胰腺癌细胞中均有表达;免疫组织化学染色证实,SLP-2在胰腺癌组织中呈现显著高表达。结论 SLP-2、VDAC-1、VDAC-2、CHCHD3、TOM40是新发现的与人胰腺癌相关的具有免疫原性的膜抗原,利用这些膜抗原检测胰腺癌高危人群外周血中的特异性自身抗体将有助于实现胰腺癌的早期诊断。
Objective To screen human pancreatic cancer associated membrane antigen with early diagnostic value. Methods The membrane proteins of pancreatic cancer cells were extracted by membrane biotechnology. The membrane proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) and transferred to PVDF membrane. Using the purified IgG from patients with pancreatic cancer Western blot hybridization was performed on the membrane proteins, and the specific immunoprecipitation spots specific to pancreatic cancer were screened by 2-DE map of membrane proteins. Hybridization positive spots were identified by MALDI-TOF mass spectrometry. RT- The expression of membrane antigen in human pancreatic cancer tissues was detected by immunohistochemical staining. Results The membrane proteins of pancreatic cancer cell lines were successfully extracted. Immunofluorescence positive spots were screened by 2-DE map of membrane proteins. The positive spots of immunospecific hybridization were identified as VDAC-2 and VDAC-1 by MALDI-TOF mass spectrometry, respectively , CHCHD3, SLP-2 and TOM40. The results of RT-PCR showed that the above membrane antigen genes were expressed in many pancreatic cancer cells. Immunohistochemical staining confirmed that SLP-2 was highly expressed in pancreatic cancer tissues. CONCLUSIONS: SLP-2, VDAC-1, VDAC-2, CHCHD3 and TOM40 are newly discovered membrane antigen with immunogenicity in human pancreatic cancer. These membrane antigens are used to detect the specificity of peripheral blood in high-risk pancreatic cancer patients Antibodies will help in the early diagnosis of pancreatic cancer.