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目的:分析卵巢癌细胞株(SKOV3)与卵巢癌淋巴结定向高转移细胞株(SKOV3-PM4)分泌蛋白的差异,寻找与卵巢癌淋巴定向转移密切相关的潜在诊断分子标志。方法:分别收集SKOV3与SKOV3-PM4培养上清,利用双向凝胶电泳(2-DE)及基质辅助激光解吸电离飞行时间质谱(MALDI_T0F-MS/MS)技术筛选及鉴定差异表达的蛋白点,对其进行生物学分析后采用蛋白质印迹法验证挑选目标蛋白在细胞分泌蛋白中的表达情况。结果:二维凝胶电泳显示,SK-OV3细胞分泌蛋白质点数约为569±156,SKOV3-PM4细胞分泌蛋白质点数约为598±175。选取差异较大的7个差异蛋白点进行MS+MS/MS质谱鉴定,分别为乙二醛酶1(GLO1)、翻译后调节肿瘤蛋白(TPTC)、二磷酸核苷激酶B(ND-PK-B)、膜联蛋白(ANXA1)、热休克蛋白27(HSP)、过氧化物氧化还原(PRDX1)和磷脂酰乙醇胺结合蛋白Ⅰ(PEBP-Ⅰ)。挑选NDPK-B、ANXA1和HSP蛋白经蛋白质印迹法验证发现,SKOV3-PM4细胞中ANXA1和HSP27表达高于SKOV3细胞,而NDPK-B表达低于SKOV3细胞。与质谱分析的结果基本一致。结论:在卵巢癌细胞与卵巢癌淋巴结定向高转移细胞之间分泌蛋白存在很大的差异,其中已被鉴定的ANXA1和HSP27与卵巢癌淋巴结转移密切相关,有望成为诊断或预测卵巢癌淋巴结转移患者血清检测的标志。
OBJECTIVE: To analyze the difference of secreted proteins between ovarian cancer cell line SKOV3 and ovarian cancer cell line SKOV3-PM4 and to find potential diagnostic molecular markers closely related to lymphatic metastasis of ovarian cancer. Methods: The supernatants of SKOV3 and SKOV3-PM4 were collected and the differentially expressed protein spots were screened by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF-MS / MS) After biological analysis, the protein expression of the target protein in the cell secretory protein was verified by Western blotting. Results: Two-dimensional gel electrophoresis showed that the number of protein spots secreted by SK-OV3 cells was about 569 ± 156 and the number of protein spots secreted by SKOV3-PM4 cells was about 598 ± 175. Seven differential protein spots were selected for MS / MS / MS mass spectrometry identification, which were glyoxalase 1 (GLO1), posttranslational regulation of tumor protein (TPTC), nucleoside diphosphate kinase B (ND- B), annexin (ANXA1), heat shock protein 27 (HSP), peroxide redox (PRDX1) and phosphatidylethanolamine binding protein I (PEBP-I). The NDPK-B, ANXA1 and HSP proteins were selected by Western blot and found that the expression of ANXA1 and HSP27 in SKOV3-PM4 cells was higher than that in SKOV3 cells, while the expression of NDPK-B was lower than that in SKOV3 cells. The results of mass spectrometry are basically the same. CONCLUSIONS: There are great differences in the secretion of proteins between ovarian cancer cells and highly metastatic lymph nodes in ovarian cancer. The identified ANXA1 and HSP27 are closely related to the lymph node metastasis of ovarian cancer and are expected to become the diagnostic or prognostic indicators for patients with lymph node metastasis of ovarian cancer Serum testing mark.