- 學位論文
增加MDCK及Vero細胞表面唾液酸含量的方法研究
Glycoengineering of cell surface sialic acid contents in MDCK and Vero cells
摘要
醣修飾(glycosylation)是蛋白質轉譯後修飾的一種，可以分成N鍵結與O鍵結兩種。在哺乳類動物細胞中，佔據醣蛋白的N鍵結以及O鍵結醣修飾功能基末端的，通常是唾液酸(sialic acid)。唾液酸具有許多功能，例如可作為流行性感冒病毒(influenza virus)入侵宿主細胞的結合受器 (virus receptor)。由於使用受精雞蛋製造流感疫苗會造成一些問題，因此新一代的流感疫苗多朝向使用動物細胞來生產的方向。但是目前使用動物細胞製造流感病毒的產率 (yield) 不夠高。如何提高生產的病毒量達到可以工業化量產的條件，是亟待克服的問題。 根據流感病毒感染宿主細胞的路徑，Hatakeyama等人於2005年證明若是在MDCK細胞 (Madin -Darby canine kidney cell) 中大量表現 (overexpress) α2,6-唾液酸轉移酶(α2,6-sialyltransferase)，不但會增加MDCK細胞表面的α2,6-唾液酸含量，同時也會使其生產的病毒產率提升。 根據此結果，在本研究中將嘗試用生物工程的方法提升兩個製造流感疫苗常用的細胞株，MDCK及Vero (Africa Green Monkey kidney cell) 細胞表面的唾液酸含量來增加病毒產量。實驗方法為在MDCK以及Vero細胞的培養基中分別添加兩種唾液酸的前驅物，N-乙醯甘露糖胺 (ManNAc) 以及N-乙醯葡萄糖胺(GlcNAc)，之後以高解析液相層析儀 (HPLC) 和流式細胞儀 (flow cytometer) 測量細胞中唾液酸含量。研究結果顯示，加入N-乙醯甘露糖胺會使細胞內的唾液酸含量增加，但是加入N-乙醯葡萄糖胺卻沒有任何影響。N-乙醯葡萄糖胺在唾液酸合成路徑中位於N-乙醯甘露糖胺的上游，由這個結果可推測在MDCK以及Vero細胞的唾液酸合成路徑中，N-乙醯甘露糖胺的生成可能是速率決定步驟 (rate-limiting step)之一。這個推測與Keppler等人在1999所得到的結論相符合。另一方面，若在MDCK及Vero細胞中加入N-乙醯甘露糖胺的同時大量表現一磷胞苷酸生成酶(CMP-sialic acid synthetase)會使得這兩個細胞株中的唾液酸含量更明顯地增加。這個結果暗示一磷胞苷酸生成酶可能也是唾液酸生成的調控因素。 本研究建立了可以在MDCK以及Vero細胞中提升其唾液酸含量的工程方法，同時也提供了研究這兩種細胞株唾液酸合成路徑的重要線索。這個方法也可以使用在其他的哺乳類動物細胞中。增加細胞唾液酸含量有許多用途，例如應用在流感疫苗或是重組蛋白質(recombinant protein)的生產。而這些有潛力的應用值得投入更多的研究。
並列摘要
Glycosylation is one of the protein post-translational modifications, which plays important roles in normal cell physiology. There are two types of glycosylation: N- and O-linked glycosylation. In mammalian cells, the most common monosaccharides found in the terminus of N- and O-linked oligosaccharides of glycoproteins and glycolipids are sialic acids. Sialic acids have many important functions, including acting as receptors for influenza viruses to invade host cells. Influenza virus vaccines are manufactured in the embryonated chicken eggs, but many problems are involved in this method. Thus, many researches focus on how to produce next generation of influenza vaccines in cultured mammalian cell lines. But, the yields of influenza viruses in cell lines are usually very low compared to those in chicken eggs. In order to decrease the production cost of vaccines produced in cultured mammalian cells, virus yields need to be optimized. According to the infection pathway of influenza viruses, Hatakeyama et al. showed that α2,6-sialyltranseferase overexpressed MDCK cells, which had higher surface sialic acid contents, had higher virus yields when they infected by viruses This result suggested that increase in sialic acid levels may improve the virus yields. In this study, N-acetylmannosamine (ManNAc) and N-acetylglucosamine (GlcNAc) were fed in the culture medium for the purpose of raising sialic acid contents in MDCK and Vero cells. Sialic acid contents increased upon ManNAc supplementation in both MDCK and Vero cells. But, GlcNAc supplement did not affect sialic acid levels in those two cells. These results showed that ManNAc supplementation was a good method for raising surface sialic acid contents in MDCK and Vero cells and also implied that UDP-GlcNAc 2-epimerase regulates rate-limiting step of the pathway, namely biosynthesis of ManNAc, in MDCK and Vero cells. This speculation was supported by the study of Keppler et al. [1] Besides, supplement of ManNAc to CMP-sialic acid synthetase expressed MDCK or Vero cells led to even higher increase in sialic acid level. This result implied that CMP-sialic acid synthetase may also regulate another bottleneck of sialic acid synthesis pathway. The developed method applied in MDCK and Vero cells may have many applications such as production of recombinant proteins or influenza vaccines. Further investigations of these sialylation-increased cells are needed.