【作者】 秦棪楠；

【导师】 李铮；

【作者基本信息】 西北大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 目的：许多疾病的发生伴随着相关糖蛋白糖基化的改变,糖蛋白糖链结构的多样性对其生理功能起着重要的作用。目前已证明多种癌症均有细胞内糖谱变化。肝纤维化作为一种普遍多发的疾病,如不能及时诊断和治疗可发展为肝硬化。肝星状细胞(Hepatic Stellate Cells, HSCs)是肝脏合成细胞外基质(Extracellular Matrixc, ECM)的主要细胞。HSCs的活化与增殖是肝纤维化形成的中心环节。目前针对HSCs的研究多在基因层面,糖谱的研究很少涉及。因此本实验利用凝集素芯片对HSCs糖蛋白糖链进行规模化分析研究,比较“静态”HSCs和经转化生长因子β1 (transforming growth factorβ1, TGF-β1)诱导活化的HSCs的糖蛋白糖链表达差异,寻找肝纤维化相关糖蛋白糖链,分析糖基化蛋白与肝纤维化的关系,研究肝纤维化过程中蛋白糖基化修饰的改变,推测肝纤维化相关糖蛋白糖链的合成通路。方法：以LX-2细胞系为肝星状细胞系模型,采取终浓度为2ng／mL的TGF-β1诱导刺激LX-2细胞24h。半定量RT-PCR检测LX-2细胞系是否发生肝纤维化变化。提取对照组LX-2细胞和实验组LX-2细胞总蛋白,Cy3荧光标记,与凝集素芯片孵育,观察表达差异糖蛋白,分析糖蛋白糖链修饰变化,利用已公开相关基因芯片研究结果推测肝纤维化过程中糖链合成通路及其与糖基化修饰变化的关系。结论：通过半定量RT-PCR发现,TGF-β1和MMP-2基因在经转化生长因子β1(TGF-β1)诱导的LX-2细胞中高表达。证明TGF-β1诱导LX-2细胞系发生肝纤维化的细胞培养方法可行。应用凝集素芯片分析经TGF-β1诱导LX-2细胞系和“静态”LX-2细胞系糖蛋白糖链表达差异。得出以下初步结果：(1)肝纤维化的肝星状细胞中T抗原／Tn抗原糖链结构增加,T抗原或Tn抗原唾液酸化明显。(2)发生肝纤维化的肝星状细胞糖蛋白N-糖链分支结构增加显著,特别是Bisecting GlcNAc结构。(3)核心岩藻糖结构在纤维化肝星状细胞中主要以Fucosea-1,6GlcNAc(core fucose)结构存在,而且末端岩藻糖结构升高。(4)MAL-Ⅱ特异性识别的Sia2-3Galβ1-4Glc(NAc)结构主要存在于正常肝星状细胞,而SNA特异性识别的Sia2-6Galβ1-4Glc(NAc)主要存在于纤维化肝星状细胞。(5) GlcNAc、β1-4GlcNAc、Galβ-1,4GlcNAc结构在纤维化肝星状细胞中表达量升高,GalNAcβ-1,4GlcNAc结构则相对稳定。(6)ConA、LCA在纤维化肝星状细胞中信号增强,说明纤维化肝星状细胞中α-甘露糖结构增多。(7)根据WGA、PWM、STL、LEL、DSA显示结果分析,在纤维化肝星状细胞中GlcNAc及(GlcNAc)n (n=2,3)结构减少,(GlcNAc)n(≧3)结构增加。(8)根据PTL-Ⅱ、PTL-Ⅰ、BPL、VVA、SBA、SJA荧光信号强度分析,末端GalNAc和Gal结构增加,同时aGalNAc结构减少,Gal结构增多。本研究的技术路线图如下：更多还原

【Abstract】 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.更多还原