【摘要】：β1,4-半乳糖基轉(zhuǎn)移酶1(β1,4-galactosyltransferase 1,β4GalT1)屬于β-1,4-半乳糖基轉(zhuǎn)移酶家族,是合成糖蛋白上N-乙酰乳糖胺(Galβ1-4Glc NAc)的關(guān)鍵酶。目前已報(bào)道β4GalT1在乳腺癌中高表達(dá)并增強(qiáng)細(xì)胞的遷移能力,但該酶在膀胱癌發(fā)生發(fā)展中的作用卻鮮有研究。本文以人源正常膀胱上皮細(xì)胞HCV29和浸潤(rùn)性膀胱癌細(xì)胞YTS1為研究對(duì)象。首先用Cy3標(biāo)記的雞冠刺桐凝集素對(duì)細(xì)胞染色檢測(cè)兩個(gè)細(xì)胞內(nèi)N-乙酰乳糖胺表達(dá)。實(shí)驗(yàn)發(fā)現(xiàn)N-乙酰乳糖胺在YTS1細(xì)胞中表達(dá)明顯高于HCV29。通過蛋白免疫印跡檢測(cè)比較兩株細(xì)胞內(nèi)半乳糖基轉(zhuǎn)移酶的轉(zhuǎn)錄和蛋白水平的表達(dá),實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)β4GalT1在YTS1細(xì)胞中表達(dá)明顯高于HCV29,與上述結(jié)果相符另外。,膀胱癌組織切片的結(jié)果也同樣證實(shí)腫瘤細(xì)胞內(nèi)β4GalT1的表達(dá)高于正常細(xì)胞。利用轉(zhuǎn)化生長(zhǎng)因子(transforming growth factor beta,TGF-β)誘導(dǎo)HCV29發(fā)生上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)處理細(xì)胞后,細(xì)胞形態(tài)由上皮變成間質(zhì)細(xì)胞的纖維狀,在EMT過程中N-乙酰乳糖胺結(jié)構(gòu)與β4GalT1的表達(dá)顯著升高。β4GalT1是一種II型跨膜蛋白,它以兩種形式——長(zhǎng)型和短型廣泛存在于細(xì)胞中。本課題將來(lái)源于YTS1的長(zhǎng)型β4GalT1和短型β4GalT1基因分別克隆于真核表達(dá)載體p LVX上,在HCV29細(xì)胞中轉(zhuǎn)染獲得過表達(dá)長(zhǎng)型和短型β4GalT1的穩(wěn)定轉(zhuǎn)染細(xì)胞株HCV29/B4-L和HCV29/B4-S。通過細(xì)胞免疫熒光染色,發(fā)現(xiàn)短型β4GalT1主要定位在高爾基體上,而長(zhǎng)型β4GalT1除定位在高爾基體上還有少部分布在細(xì)胞膜表面,該結(jié)果提示兩者的生物學(xué)功能可能存在差異。利用流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期、增殖以及凋亡能力,發(fā)現(xiàn)過表達(dá)長(zhǎng)型的β4GalT1促進(jìn)細(xì)胞增殖,而過表達(dá)短型β4GalT1抑制細(xì)胞增殖;在細(xì)胞周期方面,過表達(dá)短型β4GalT1的細(xì)胞更多的阻滯在S期,這可能是短型β4GalT1抑制細(xì)胞增殖的原因;細(xì)胞凋亡方面,過表達(dá)長(zhǎng)型β4GalT1和過表達(dá)短型β4GalT1對(duì)細(xì)胞本身的凋亡情況無(wú)明顯影響,但用誘導(dǎo)細(xì)胞凋亡的藥物喜樹堿處理細(xì)胞后,HCV29/B4-L和HCV29/B4-S細(xì)胞凋亡率不同,長(zhǎng)型β4GalT1促進(jìn)細(xì)胞凋亡,而短型β4GalT1抑制細(xì)胞凋亡。此外,過表達(dá)長(zhǎng)型β4GalT1和短型β4GalT1均能增強(qiáng)細(xì)胞的遷移能力。β4GalT1的兩種結(jié)構(gòu)對(duì)細(xì)胞表型的差異影響表明了其對(duì)細(xì)胞內(nèi)信號(hào)通路分子機(jī)制調(diào)控的復(fù)雜性,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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.14

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