脂質界面基因轉殖法之效力分析與應用

基因轉殖為研究基因之功能的方法之一，能否高效率轉殖基因且不對細胞造成毒害性，是目前基因轉殖所面臨之挑戰，利用本實驗室研發之界面基因轉殖法 ( surfection )，可有效解決細胞毒性問題，而且，界面基因轉殖能有效遞送三種基因，實驗之操作程序較傳統基因轉殖技術簡單且快速。本實驗利用正價脂質進行界面基因轉殖之分析研究，實驗操作方法係利用正價脂質塗附之界面，與正價離子與 pEGFP 綠色螢光質體基因之溶液進行混合，並利用 COS-7 細胞進行基因轉殖效率之分析，經一系列不同之正價離子實驗分析發現，濃度 100 mM 之氯化鈉、 10 mM 之氯化鎂與濃度 0.3 mM 之硫酸鋅溶液，利用 2.5×105 數量之 COS-7 細胞，於脂質界面基因轉殖時，具有較高之基因遞送效率，且不產生細胞毒性。此外，本實驗亦設立基因轉殖材料之最佳使用時間，例如：脂質塗附之界面為 2 星期、質體基因與氯化鎂混合溶液為 1 星期、混合溶液界面為 1星期，更進一步，本實驗利用牛血清白蛋白 ( BSA ) ，成功保存乾燥後界面之基因轉殖效力，完成脂質界面基因轉殖快速化之目標。而在脂質界面基因轉殖之應用上，也有諸多突破，本實驗利用脂質界面基因轉殖法，已成功對老鼠骨髓幹細胞、神經細胞與肝臟星狀細胞，進行高效率之基因轉殖。綜合以上，本實驗成功建立脂質界面基因轉殖之標準操作程序，並依此操作程序對較難轉殖之細胞（例如：幹細胞、神經細胞與肝臟星狀細胞），進行高效率之基因遞送；另一方面，實驗批次材料之最佳使用時間、界面基因轉殖效力之保存方法，本實驗也都建立了系統性的管理，未來可望以脂質界面基因轉殖法為平檯，對細胞進行多功能性基因之遞送，以利訊息傳遞領堿之拓展研究。

關鍵字

脂質；界面基因轉殖；轉助基因轉殖；基因轉殖效率；穩定細胞系；氯化鈉；氯化鎂；硫酸鋅；肝臟星狀細胞；神經細胞；幹細胞

並列摘要

Transfection is one way to study functions of genes. Current transfection methods are in the face of overcoming challenges to have high transfection efficiency and low cell cytotoxicity. Using surfection can solve the problem of cell cytotoxicty. Besides, surfection protocol is faster and easier than traditional transfection which can deliver three genes effectively in the meanwhile. We took cationic lipids, DOTAP, for surfection analysis. Lipid surfection protocol is seeding COS-7 cell number 2.5×105/ well onto lipid coating surface soaking with cation and plasmid DNA mixture solution. After serial analysis by lipid surfection with different cation solutions, we found that cation solutions in specific concentration, which is 100 mM NaCl, 10 mM MgCl2 and 0.3 mM ZnSO4, will give better transfection efficiency and low cell cytotoxicity. Besides, we setup preservation deadlines of surfection materials, such as lipid coating surface, mixture solutions, and lipid coating surface with mixture solutions. Their suggested deadlines are 2 week, 1 week and 1 week, respectively. Moreover, we developed instant lipid surfection protocol. Adding 1% BSA solution onto surfection surface will preserve transfection efficacy. There are lots of breakthroughs in lipid surfection application, too. For example, high-efficiency transfection of genes to rat bone marrow stem cells ( rBMSC ) neuron cells, and hepatic stellate cells ( HSC ) is achieved by lipid surfection. In conclusion, we setup the standard operation protocols of lipid surfection ( SOP ). Next, we delivered genes to rBMSC, neuron cells and HSC in a high-efficiency manner by lipid surfection SOP. Furthermore, we established systemic management for deadlines of batch materials and preservation protocols of surfection efficacy. In the future, we may use lipid surfection as a platform to transfect multi-functional genes to cells for signal transduction advanced study.

並列關鍵字

lipid ； surfection ； substrate-mediated transfection ； transfection efficiency ； stable cell line ； sodium chloride ； magnesium chloride ； zinc sulfate ； hepatic stellate cells (HSC) ； neuron cells ； stem cells

參考文獻

1. Downward, J. (2004) Use of RNA interference libraries to investigate oncogenic signalling in mammalian cells. Oncogene, 23, 8376-8383.

2. Gao, X., Kim, K.S. and Liu, D. (2007) Nonviral gene delivery: what we know and what is next. Aaps J, 9, E92-104.

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5. Shea, L.D., Smiley, E., Bonadio, J. and Mooney, D.J. (1999) DNA delivery from polymer matrices for tissue engineering. Nature biotechnology, 17, 551-554.

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脂質界面基因轉殖法之效力分析與應用

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