Glycoprotein Glycan Analysis Of TGF-β1 Induced Human Hepatic Stellate Cells

Purpose:The occurrences of many diseases are accompanied by changes in glycosylation of related glycoprotein markers. The multiplicity of glycoprotein sugar chain structure is playing the vital role to its physiological function. Currently it has been proven that many kinds of cancers have changes of their glycan profiles. The hepatic fibrogenesis is a kind of disease happens generally,which may develop to hepatic cirrhosis if it couldn't been diagnosed and treated promptly.Hepatic stellate cells (HSCs) are the main cells which synthesis extracellular matrix. The HSCs activation and proliferation are the center links which the hepatic fibrogenesis forms. At present many research aim at HSCs are mainly in the field of gene,the glycan profile research very little involves. Therefore this experiment analysed the HSCs glycoprotein glycan chains by the lectin microarry, comparing the "quiescent" HSCs with the"activated"HSCs induced by transforming growth factorβ1 (TGF-β1) on differences of glycoprotein expression, seeking for the hepatic fibrogenesis related glycoproteins, analysing the relations of glycosylation protein and the hepatic fibrogenesis, studying the changes of protein glycosylation modification during the hepatic fibrogenesis process, using already publiced gene chip findings speculate the hepatic fibrogenesis related glycoprotein glycan chain's synthesis pathways.Method:This experiment taked the LX-2 cells as the hepatic stellate cell line model,.and cultured LX-2 cells in the presence of 2 ng/mL TGF-β1 for 24h. Semi-quantitative RT-PCR examined the LX-2 cell line whether had the hepatic fibrogenesis change.The total proteins of the induced LX-2 cells and its control were extracted, labled with Cy3 fluorescent dye,then incubated with the lectin microarray.Conclusion:Semi-quantitative RT-PCR result showed that the expression of transforming growth factorβ1 (TGF-β1) and metalloproteinase-2(MMP-2) were increased in LX-2 cells induced by TGF-β1.That demonstrated the method of LX-2 cells induced by TGF-β1 was feasible. The lectin microarray was used to hybridizate with the induced LX-2 cells and its control.The results were as follows:(1)The T antigen/Tn antigen glycan chain structure increased in the induced LX-2 cells.(2)The branches of N-glycans significantly increased,especially biantennary N-glycans.(3) Core fucose structure was existed as form as Fucosea-1,6GlcNAc(core fucose) in the induced LX-2 cells.(4) Sia2-3Galβ1-4Glc the (NAc) specificitily recognised by MAL-Ⅱmainly existed in the control cells group; Sia2-6Galβ1-4Glc(NAc) specificitily recognised by SNA mainly existed in the induced LX-2 cells.(5) GlcNAc,β1-4GlcNAc and Galβ-1,4GlcNAc were increased in the induced LX-2 cells.(6)α-mannose was increased in the induced LX-2 cells.(7) GlcNAc and (GlcNAc) n (n=2,3)were reduced, while (GlcNAc) n(≧3) were increased in the induced LX-2 cells.(8) In the induced LX-2 cells, terminal GalNAc and gal were increased.