肝癌组织内类阿片肽LVV-血衍吗啡素-6的作用

Role of opioid peptide Leu-Val-Val-hemorphin-6 in hepatocellular carcinoma

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.

AIM： To assay the content differences of hemorphins between hepatocellular carcinoma （HCC） and normal liver tissues, and to explore the role of hemorphins in HCC. METHODS： The tissue samples were obtained from 12 patients with HCC, and peptides were isolated by mild acid elution from the surfaces of hepatocarcinoma and normal hepatic cells. The mixtures of peptides were analyzed by high performance liquid chromatography （HPLC） and mass spectrometer （MS）. A differential comparison was made on the fractions of peptides between HCC and normal liver cells to screen some tumor-specific peptides. Finally, human hepatocarcinoma cell line HLE was treated with different concentrations of the obtained peptides （0, 10^-7, 10^-8, 10^-9, 10^-10, 10^-11, 10^-12 mol/L）, and the growth and apoptosis of HLE cells were detected by MTT assay and flow cytornetry （FCM）, respectively.RESULTS： An opioid peptide Leu-Val-Val （LVV）-hemorphin-6 with M1160.76 was identified on the surfaces of HCC cells in 7 patients. LVV-hemorphin-6 inhibited the growth of HLE cells at different concentrations, especially at 10.7 and 10^-8mol/L （24 h： 10^-7 vs 10^-8： P = 0.044, 10^-8 vs 10^-9： P = 0.047; 48 h： 10^-7 vs 10^-8： P = 0.031, 10^-8 vs 10^-9： P = 0.040）. Meanwhile, LVV-hemorphin-6 induced apoptosis of HLE cells and the apoptosis rates were 0.38% ± 0.09%, 20.23%±1.25%, 12.64% ± 2.15%, 1.65% ±0.34%, when 0, 10^-7, 10^-8, 10^-9 mol/L LVV-hemorphin-6 were used, respectively. However, naloxone could inhibit the above effect and the apoptosis rates were 0.41%± 0.11%, 1.23% ± 0.45%, 0.98% ± 0.55%, 1.34% ± 0.43%, when the corresponding concentrations were used. The apoptosis rates were signifi- cantly different between the two groups （P 〈 0.05） as 10.7 and 10~ mol/L LVV-hemorphin-6 were used. CONCLUSION： LVV-hemorphin-6 may be a product in the pathophysiological process of hepatocellular carcinoma, and it has suppressive effects on HCC ceils by activating opiate receptors.