糖基转移酶β4GalT1在膀胱癌中的初步功能研究
发布时间：2019-02-15 08:38
【摘要】：β1,4-半乳糖基转移酶1(β1,4-galactosyltransferase 1,β4GalT1)属于β-1,4-半乳糖基转移酶家族,是合成糖蛋白上N-乙酰乳糖胺(Galβ1-4Glc NAc)的关键酶。目前已报道β4GalT1在乳腺癌中高表达并增强细胞的迁移能力,但该酶在膀胱癌发生发展中的作用却鲜有研究。本文以人源正常膀胱上皮细胞HCV29和浸润性膀胱癌细胞YTS1为研究对象。首先用Cy3标记的鸡冠刺桐凝集素对细胞染色检测两个细胞内N-乙酰乳糖胺表达。实验发现N-乙酰乳糖胺在YTS1细胞中表达明显高于HCV29。通过蛋白免疫印迹检测比较两株细胞内半乳糖基转移酶的转录和蛋白水平的表达,实验结果发现β4GalT1在YTS1细胞中表达明显高于HCV29,与上述结果相符另外。,膀胱癌组织切片的结果也同样证实肿瘤细胞内β4GalT1的表达高于正常细胞。利用转化生长因子(transforming growth factor beta,TGF-β)诱导HCV29发生上皮间质转化(epithelial-mesenchymal transition,EMT)处理细胞后,细胞形态由上皮变成间质细胞的纤维状,在EMT过程中N-乙酰乳糖胺结构与β4GalT1的表达显著升高。β4GalT1是一种II型跨膜蛋白,它以两种形式——长型和短型广泛存在于细胞中。本课题将来源于YTS1的长型β4GalT1和短型β4GalT1基因分别克隆于真核表达载体p LVX上,在HCV29细胞中转染获得过表达长型和短型β4GalT1的稳定转染细胞株HCV29/B4-L和HCV29/B4-S。通过细胞免疫荧光染色,发现短型β4GalT1主要定位在高尔基体上,而长型β4GalT1除定位在高尔基体上还有少部分布在细胞膜表面,该结果提示两者的生物学功能可能存在差异。利用流式细胞术检测细胞周期、增殖以及凋亡能力,发现过表达长型的β4GalT1促进细胞增殖,而过表达短型β4GalT1抑制细胞增殖;在细胞周期方面,过表达短型β4GalT1的细胞更多的阻滞在S期,这可能是短型β4GalT1抑制细胞增殖的原因;细胞凋亡方面,过表达长型β4GalT1和过表达短型β4GalT1对细胞本身的凋亡情况无明显影响,但用诱导细胞凋亡的药物喜树碱处理细胞后,HCV29/B4-L和HCV29/B4-S细胞凋亡率不同,长型β4GalT1促进细胞凋亡,而短型β4GalT1抑制细胞凋亡。此外,过表达长型β4GalT1和短型β4GalT1均能增强细胞的迁移能力。β4GalT1的两种结构对细胞表型的差异影响表明了其对细胞内信号通路分子机制调控的复杂性,有待于更深入的研究。
[Abstract]:尾 _ 1C _ 4-galactosyltransferase _ 1 (尾 _ 4GalT1) belongs to the 尾 -1C _ 4-galactosyltransferase family and is the key enzyme for the synthesis of Gal 尾 1-4Glc NAc in glycoproteins. Up to now, it has been reported that 尾 4GalT1 is highly expressed in breast cancer and enhances cell migration, but the role of 尾 4GalT1 in the development of bladder cancer has not been studied. Human normal bladder epithelial cells (HCV29) and invasive bladder cancer cells (YTS1) were studied. The expression of N- acetyllactamine in two cells was detected by Cy3 labeled agglutinin staining. It was found that the expression of N-acetyllactamine in YTS1 cells was significantly higher than that in HCV29. cells. The transcriptional and protein levels of galactosyltransferase were compared by Western blot analysis. The results showed that the expression of 尾 4GalT1 in YTS1 cells was significantly higher than that in HCV29, cells. The expression of 尾-4GalT1 in bladder cancer cells was also higher than that in normal cells. Transforming growth factor (transforming growth factor beta,TGF- 尾 (TGF- 尾) was used to induce HCV29 to produce epithelial interstitial transformation (epithelial-mesenchymal transition,EMT). After the cells were treated with TGF- 尾, the morphology of the cells changed from epithelium to fibrous form of mesenchymal cells. During EMT, the structure of N-acetyllactamine and the expression of 尾 4GalT1 increased significantly. 尾 4GalT1 is a transmembrane protein of II type, which is widely distributed in cells in two forms, long and short. In this study, the long 尾 4GalT1 and short 尾 4GalT1 genes derived from YTS1 were cloned into the eukaryotic expression vector p LVX, and the stable transfection lines HCV29/B4-L and HCV29/B4-S. were obtained by transfection of long and short 尾 4GalT1 in HCV29 cells. By immunofluorescence staining, it was found that the short 尾 4GalT1 was mainly located on Golgi body, while the long type 尾 4GalT1 was located on the surface of cell membrane except Golgi body. This result suggested that the biological function of the two groups might be different. Flow cytometry was used to detect cell cycle, proliferation and apoptosis. It was found that overexpression of long type 尾 4GalT1 promoted cell proliferation, while over-expression of short 尾 4GalT1 inhibited cell proliferation. In cell cycle, cells overexpression of short 尾 4GalT1 were more blocked in S phase, which may be the reason of short 尾 4GalT1 inhibiting cell proliferation. In terms of apoptosis, overexpression of long 尾 4GalT1 and overexpression of short 尾 4GalT1 had no significant effect on the apoptosis of the cells themselves, but the apoptotic rates of HCV29/B4-L and HCV29/B4-S cells were different after treated with camptothecin, a drug that induced cell apoptosis. Long 尾 4GalT1 promotes apoptosis, while short 尾 4GalT1 inhibits apoptosis. In addition, overexpression of long 尾 4GalT1 and short type 尾 4GalT1 can enhance the migration ability of cells. The difference of phenotypic effect of 尾 4GalT1 suggests the complexity of the regulation of the molecular mechanism of intracellular signaling pathway, which needs to be further studied.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14
本文编号：2423157
[Abstract]:尾 _ 1C _ 4-galactosyltransferase _ 1 (尾 _ 4GalT1) belongs to the 尾 -1C _ 4-galactosyltransferase family and is the key enzyme for the synthesis of Gal 尾 1-4Glc NAc in glycoproteins. Up to now, it has been reported that 尾 4GalT1 is highly expressed in breast cancer and enhances cell migration, but the role of 尾 4GalT1 in the development of bladder cancer has not been studied. Human normal bladder epithelial cells (HCV29) and invasive bladder cancer cells (YTS1) were studied. The expression of N- acetyllactamine in two cells was detected by Cy3 labeled agglutinin staining. It was found that the expression of N-acetyllactamine in YTS1 cells was significantly higher than that in HCV29. cells. The transcriptional and protein levels of galactosyltransferase were compared by Western blot analysis. The results showed that the expression of 尾 4GalT1 in YTS1 cells was significantly higher than that in HCV29, cells. The expression of 尾-4GalT1 in bladder cancer cells was also higher than that in normal cells. Transforming growth factor (transforming growth factor beta,TGF- 尾 (TGF- 尾) was used to induce HCV29 to produce epithelial interstitial transformation (epithelial-mesenchymal transition,EMT). After the cells were treated with TGF- 尾, the morphology of the cells changed from epithelium to fibrous form of mesenchymal cells. During EMT, the structure of N-acetyllactamine and the expression of 尾 4GalT1 increased significantly. 尾 4GalT1 is a transmembrane protein of II type, which is widely distributed in cells in two forms, long and short. In this study, the long 尾 4GalT1 and short 尾 4GalT1 genes derived from YTS1 were cloned into the eukaryotic expression vector p LVX, and the stable transfection lines HCV29/B4-L and HCV29/B4-S. were obtained by transfection of long and short 尾 4GalT1 in HCV29 cells. By immunofluorescence staining, it was found that the short 尾 4GalT1 was mainly located on Golgi body, while the long type 尾 4GalT1 was located on the surface of cell membrane except Golgi body. This result suggested that the biological function of the two groups might be different. Flow cytometry was used to detect cell cycle, proliferation and apoptosis. It was found that overexpression of long type 尾 4GalT1 promoted cell proliferation, while over-expression of short 尾 4GalT1 inhibited cell proliferation. In cell cycle, cells overexpression of short 尾 4GalT1 were more blocked in S phase, which may be the reason of short 尾 4GalT1 inhibiting cell proliferation. In terms of apoptosis, overexpression of long 尾 4GalT1 and overexpression of short 尾 4GalT1 had no significant effect on the apoptosis of the cells themselves, but the apoptotic rates of HCV29/B4-L and HCV29/B4-S cells were different after treated with camptothecin, a drug that induced cell apoptosis. Long 尾 4GalT1 promotes apoptosis, while short 尾 4GalT1 inhibits apoptosis. In addition, overexpression of long 尾 4GalT1 and short type 尾 4GalT1 can enhance the migration ability of cells. The difference of phenotypic effect of 尾 4GalT1 suggests the complexity of the regulation of the molecular mechanism of intracellular signaling pathway, which needs to be further studied.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14
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