人肝癌细胞系的糖蛋白质组学研究

Glycoproteomics Investigation of Human Hepatocellular Carcinoma Cell Lines

糖基化是最重要的蛋白质翻译后形式之一,糖基化蛋白的糖链部分影响着蛋白质的折叠和稳定性以及其生物学功能.许多恶性肿瘤组织与正常组织相比已显示出蛋白质糖基化的差异.采用蛋白质组学分析方法结合先进的糖蛋白荧光染色技术,研究了正常人肝细胞系(ChangLiver)和人肝癌细胞系(Hep3B)糖蛋白糖基化的差异.首先用细胞裂解法提取细胞总蛋白质,进行双向电泳(2-DE),然后用pro-QEmerald488糖蛋白荧光染料进行糖蛋白染色,得到两种细胞系糖基化蛋白表达谱,经2-DE分析软件Dymension分析2-DE图像,比较糖蛋白的糖基化程度,并对糖基化蛋白进行质谱鉴定.结果显示正常人肝细胞表达(74±2)个(n=3),而人肝癌细胞系表达(78±3)个糖蛋白(n=3).两者匹配的糖蛋白质点31个,Hep3B表达而ChangLiver不表达的糖蛋白质点47个,ChangLiver表达而Hep3B不表达的糖蛋白质点43个.两种细胞系糖基化程度存在明显差异,与正常人肝细胞相比,肝癌细胞发生糖基化改变的糖蛋白有25个,其中糖基化水平上调的有10个,下调的有15个,质谱鉴定出12个发生糖基化改变的糖蛋白.这些结果显示蛋白质糖基化改变可能在肝癌的发生和发展中起一定作用.

Glycosylation is one of the most important forms of post-translational modification of proteins. Glycan influences folding and stability of proteins and their biological functions. A berrant glycosylation has been associated with many malignant tumor. Proteomic analysis methods combined with Pro-Q Emerald glycoprotein staining technology was applied to investigate the glycosylation difference of glycoprotein between normal human liver cell lines and hepatocellular carcinoma cell lines. Total proteins were extracted by using cell-splitting methods, then subjected to two-dimension electophoresis（2-DE）. After that, 2-DE gel were stained with Pro-Q Emerald glycoprotein stain. Glycoprotein patterns of both cell lines were obtained. Glycosylation status of glycoproteins were quantificationally compared by using Dymention software and glycoproteins with altered glycosylation were identified with mass spectrometry. Results showed that （74±2） glycoproteins （n =3） were detected in normal human liver cell lines whereas （78±3） glycoproteins （n=3） were detected in human hepatocellular carcinoma cell lines. There were 31 matched glycoproteins between both cell lines. Glycoprotein patterns had distinct difference between normal human liver cells and human hepatocellular carcinoma cells. 25 glycoproteins presented glycoslytion changes in human hepatocellular carcinoma cells comparing with normal human liver cells. Among such glycoproteins, glycosylation level of 10 glycoproteins were up-regulated whereas glycosylation level of 15 glycoproteins were down-regulated and 12 of these glycoproteins had been identified by mass spectrometry, These results imply that glycosylation changes may play key roles in occurrence and development of liver cancer.