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目的:分析肝癌组织与正常肝组织中血衍吗啡素类肽含量的差异并探究其意义.方法:肝癌患者12例,利用弱酸洗涤法从肝癌细胞和肝细胞表面提取肽段,然后用高压液相(HPLC)和质谱分析这些肽段并进行差异比较,筛选肿瘤特异性肽段.用这些肽段(0,10-7,10-8,10-9,10-10,10-11,10-12mol/L)处理人肝癌细胞株HLE,采用MTT法、流式细胞仪法观察其对肝癌细胞的生长、细胞凋亡的影响.结果:从7例患者的肝癌细胞表面成功检测出一条天然的类阿片肽——Leu-Val-Val(LVV)-血衍吗啡素-6(Mr1160.76).不同浓度LVV-血衍吗啡素-6均对HLE细胞有抑制作用,并且当浓度为10-7和10-8mol/L时有显著意义(24h:10-7vs10-8:P=0.044,10-8vs10-9:P=0.047;48h:10-7vs10-8:P=0.031,10-8vs10-9:P=0.040).同时,LVV-血衍吗啡素-6可诱导HLE细胞发生凋亡(浓度为0,10-7,10-8,10-9mol/L时的凋亡率分别为0.38%±0.09%,20.23%±1.25%,12.64%±2.15%,1.65%±0.34%),而阿片受体阻断剂naloxone能够逆转这种抑制效应(以上对应浓度时凋亡率分别为0.41%±0.11%,1.23%±0.45%,0.98%±0.55%,1.34%±0.43%).两组在LVV-血衍吗啡素-6浓度为10-7和10-8mol/L时有显著差异(P<0.05).结论:LVV-血衍吗啡素-6为肝癌细胞的病理产物,可激活阿片受体对肝癌细胞产生抑制作用.
OBJECTIVE: To analyze the differences of the blood levels of morphininoid peptides in hepatocellular carcinoma tissues and normal liver tissues.Methods: Twelve patients with hepatocellular carcinoma (HCC) were treated with weak acid washing method to extract peptides from hepatocellular carcinoma cells and hepatocytes, These peptides were analyzed by phase-contrast (HPLC) and mass spectrometry and compared for differences to screen for tumor-specific peptides. Using these peptides (0, 10-7, 10-8, 10-9, 10-10, 10-11, -12mol / L) were used to treat human hepatocellular carcinoma cell line HLE.MTT assay and flow cytometry were used to observe the effect of HLE on the growth and apoptosis of hepatoma cells.Results: A natural Leu-Val-Val (LVV) -symorphin-6 (Mr1160.76) .Various levels of LVV-heme oxygenated morphin-6 all inhibited HLE cells, and when the concentration was 10 -7 and 10-8mol / L, respectively (24h: 10-7 vs 10-8: P = 0.044,10-8 vs 10-9: P = 0.047; 48h: 10-7 vs 10-8: P = 0.031,10-8 vs10 -9: P = 0.040) .At the same time, the apoptosis of HLE cells was induced by LVV-blood-derived morphin-6 (the apoptosis rates were 0,10-7,10-8 and 10-9mol / L respectively 0.38% ± 0.09%, 20.23% ± 1.25%, 12.64% ± 2.15%, 1.65% ± 0.34%), while opioid receptor blockade naloxone could reverse the inhibitory effect (the apoptotic rates were 0.41% ± 0.11%, 1.23% ± 0.45%, 0.98% ± 0.55%, 1.34% ± 0.43%, respectively) (P <0.05) .Conclusion: LVV-HSA is a pathological product of hepatocarcinoma cells and can activate opioid receptor on the production of hepatoma cells Inhibition.