TGF-β1诱导下人肝星状细胞糖蛋白糖谱的研究
发布时间：2018-09-06 09:03
【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("g3)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：
[Abstract]:Objective: the occurrence of many diseases is accompanied by the changes of glycosylation of related glycoproteins. The diversity of glycoprotein glycosylation plays an important role in its physiological function. It has been proved that many cancers have intracellular glucose profile changes. Liver fibrosis, as a common disease, can develop into cirrhosis without timely diagnosis and treatment. Hepatic stellate cell (Hepatic Stellate Cells, HSCs) is the main cell of hepatic extracellular matrix (Extracellular Matrixc, ECM). The activation and proliferation of hepatic stellate cell (Hepatic Stellate Cells, HSCs) is the central link of hepatic fibrosis. At present, most of the studies on HSCs are at the gene level, and the study of glycometry is seldom involved. In this study, lectin chip was used to analyze the glycosyl chain of HSCs glycoprotein in order to compare the difference between "static" HSCs and transforming growth factor 尾 _ 1 (TGF- 尾 _ 1) -induced expression of glycoprotein sugar chain in activated HSCs. The relationship between glycosylated protein and hepatic fibrosis was analyzed. The changes of glycosylation modification during hepatic fibrosis were studied. The synthesis pathway of glycosylated glycoprotein chain associated with liver fibrosis was inferred. Methods: LX-2 cell line was used as the model of hepatic stellate cell line and LX-2 cells were stimulated with TGF- 尾 1 at the final concentration of 2ng/mL for 24 h. The changes of liver fibrosis in LX-2 cell line were detected by semi-quantitative RT-PCR. Total protein Cy3 was labeled by fluorescent staining in LX-2 cells of control group and LX-2 cells of experimental group, then incubated with lectin chip to observe the expression of differentially expressed glycoproteins, and to analyze the changes of glycosylation of glycoproteins. Based on the published results, the relationship between glycosylation and glycosylation modification in hepatic fibrosis was inferred. Conclusion: the overexpression of TGF- 尾 1 and MMP-2 genes in LX-2 cells induced by transforming growth factor 尾 1 (TGF- 尾 1) was detected by semi-quantitative RT-PCR. The results showed that TGF- 尾 1 induced hepatic fibrosis in LX-2 cell line was feasible. Lectin chip was used to analyze the difference of glycoprotein sugar chain expression between LX-2 cell line and "static" LX-2 cell line induced by TGF- 尾 _ 1. The following preliminary results were obtained: (1) in hepatic stellate cells with hepatic fibrosis, the structure of T antigen / T n antigen increased significantly, and the salivary acidification of T antigen or Tn antigen was significantly increased. (2) the branching structure of hepatic stellate cell glycoprotein N-sugar chain increased significantly in hepatic fibrosis. In particular, Bisecting GlcNAc structure. (3) the core fucose structure mainly existed as Fucosea-1,6GlcNAc (core fucose) structure in hepatic stellate cells, and the terminal fucose structure increased. (4) the Sia2-3Gal 尾 1-4Glc (NAc) structure specifically recognized by MAL- 鈪，
本文编号：2225860
[Abstract]:Objective: the occurrence of many diseases is accompanied by the changes of glycosylation of related glycoproteins. The diversity of glycoprotein glycosylation plays an important role in its physiological function. It has been proved that many cancers have intracellular glucose profile changes. Liver fibrosis, as a common disease, can develop into cirrhosis without timely diagnosis and treatment. Hepatic stellate cell (Hepatic Stellate Cells, HSCs) is the main cell of hepatic extracellular matrix (Extracellular Matrixc, ECM). The activation and proliferation of hepatic stellate cell (Hepatic Stellate Cells, HSCs) is the central link of hepatic fibrosis. At present, most of the studies on HSCs are at the gene level, and the study of glycometry is seldom involved. In this study, lectin chip was used to analyze the glycosyl chain of HSCs glycoprotein in order to compare the difference between "static" HSCs and transforming growth factor 尾 _ 1 (TGF- 尾 _ 1) -induced expression of glycoprotein sugar chain in activated HSCs. The relationship between glycosylated protein and hepatic fibrosis was analyzed. The changes of glycosylation modification during hepatic fibrosis were studied. The synthesis pathway of glycosylated glycoprotein chain associated with liver fibrosis was inferred. Methods: LX-2 cell line was used as the model of hepatic stellate cell line and LX-2 cells were stimulated with TGF- 尾 1 at the final concentration of 2ng/mL for 24 h. The changes of liver fibrosis in LX-2 cell line were detected by semi-quantitative RT-PCR. Total protein Cy3 was labeled by fluorescent staining in LX-2 cells of control group and LX-2 cells of experimental group, then incubated with lectin chip to observe the expression of differentially expressed glycoproteins, and to analyze the changes of glycosylation of glycoproteins. Based on the published results, the relationship between glycosylation and glycosylation modification in hepatic fibrosis was inferred. Conclusion: the overexpression of TGF- 尾 1 and MMP-2 genes in LX-2 cells induced by transforming growth factor 尾 1 (TGF- 尾 1) was detected by semi-quantitative RT-PCR. The results showed that TGF- 尾 1 induced hepatic fibrosis in LX-2 cell line was feasible. Lectin chip was used to analyze the difference of glycoprotein sugar chain expression between LX-2 cell line and "static" LX-2 cell line induced by TGF- 尾 _ 1. The following preliminary results were obtained: (1) in hepatic stellate cells with hepatic fibrosis, the structure of T antigen / T n antigen increased significantly, and the salivary acidification of T antigen or Tn antigen was significantly increased. (2) the branching structure of hepatic stellate cell glycoprotein N-sugar chain increased significantly in hepatic fibrosis. In particular, Bisecting GlcNAc structure. (3) the core fucose structure mainly existed as Fucosea-1,6GlcNAc (core fucose) structure in hepatic stellate cells, and the terminal fucose structure increased. (4) the Sia2-3Gal 尾 1-4Glc (NAc) structure specifically recognized by MAL- 鈪，
本文编号：2225860
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