【摘要】： 目的：許多疾病的發(fā)生伴隨著相關糖蛋白糖基化的改變,糖蛋白糖鏈結(jié)構(gòu)的多樣性對其生理功能起著重要的作用。目前已證明多種癌癥均有細胞內(nèi)糖譜變化。肝纖維化作為一種普遍多發(fā)的疾病,如不能及時診斷和治療可發(fā)展為肝硬化。肝星狀細胞(Hepatic Stellate Cells, HSCs)是肝臟合成細胞外基質(zhì)(Extracellular Matrixc, ECM)的主要細胞。HSCs的活化與增殖是肝纖維化形成的中心環(huán)節(jié)。目前針對HSCs的研究多在基因?qū)用?糖譜的研究很少涉及。因此本實驗利用凝集素芯片對HSCs糖蛋白糖鏈進行規(guī)�；治鲅芯�,比較“靜態(tài)”HSCs和經(jīng)轉(zhuǎn)化生長因子β1 (transforming growth factorβ1, TGF-β1)誘導活化的HSCs的糖蛋白糖鏈表達差異,尋找肝纖維化相關糖蛋白糖鏈,分析糖基化蛋白與肝纖維化的關系,研究肝纖維化過程中蛋白糖基化修飾的改變,推測肝纖維化相關糖蛋白糖鏈的合成通路。 方法：以LX-2細胞系為肝星狀細胞系模型,采取終濃度為2ng／mL的TGF-β1誘導刺激LX-2細胞24h。半定量RT-PCR檢測LX-2細胞系是否發(fā)生肝纖維化變化。提取對照組LX-2細胞和實驗組LX-2細胞總蛋白,Cy3熒光標記,與凝集素芯片孵育,觀察表達差異糖蛋白,分析糖蛋白糖鏈修飾變化,利用已公開相關基因芯片研究結(jié)果推測肝纖維化過程中糖鏈合成通路及其與糖基化修飾變化的關系。 結(jié)論：通過半定量RT-PCR發(fā)現(xiàn),TGF-β1和MMP-2基因在經(jīng)轉(zhuǎn)化生長因子β1(TGF-β1)誘導的LX-2細胞中高表達。證明TGF-β1誘導LX-2細胞系發(fā)生肝纖維化的細胞培養(yǎng)方法可行。應用凝集素芯片分析經(jīng)TGF-β1誘導LX-2細胞系和“靜態(tài)”LX-2細胞系糖蛋白糖鏈表達差異。得出以下初步結(jié)果：(1)肝纖維化的肝星狀細胞中T抗原／Tn抗原糖鏈結(jié)構(gòu)增加,T抗原或Tn抗原唾液酸化明顯。(2)發(fā)生肝纖維化的肝星狀細胞糖蛋白N-糖鏈分支結(jié)構(gòu)增加顯著,特別是Bisecting GlcNAc結(jié)構(gòu)。(3)核心巖藻糖結(jié)構(gòu)在纖維化肝星狀細胞中主要以Fucosea-1,6GlcNAc(core fucose)結(jié)構(gòu)存在,而且末端巖藻糖結(jié)構(gòu)升高。(4)MAL-Ⅱ特異性識別的Sia2-3Galβ1-4Glc(NAc)結(jié)構(gòu)主要存在于正常肝星狀細胞,而SNA特異性識別的Sia2-6Galβ1-4Glc(NAc)主要存在于纖維化肝星狀細胞。(5) GlcNAc、β1-4GlcNAc、Galβ-1,4GlcNAc結(jié)構(gòu)在纖維化肝星狀細胞中表達量升高,GalNAcβ-1,4GlcNAc結(jié)構(gòu)則相對穩(wěn)定。(6)ConA、LCA在纖維化肝星狀細胞中信號增強,說明纖維化肝星狀細胞中α-甘露糖結(jié)構(gòu)增多。(7)根據(jù)WGA、PWM、STL、LEL、DSA顯示結(jié)果分析,在纖維化肝星狀細胞中GlcNAc及(GlcNAc)n (n=2,3)結(jié)構(gòu)減少,(GlcNAc)n("g3)結(jié)構(gòu)增加。(8)根據(jù)PTL-Ⅱ、PTL-Ⅰ、BPL、VVA、SBA、SJA熒光信號強度分析,末端GalNAc和Gal結(jié)構(gòu)增加,同時aGalNAc結(jié)構(gòu)減少,Gal結(jié)構(gòu)增多。 本研究的技術(shù)路線圖如下：
[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，

本文編號：2225860