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肝特異的転写因子の過剰発現による肝機能誘導型遺 伝子組換えヘパトーマ細胞の開発
ジェーン, トネロ
https://doi.org/10.15017/1866257
出版情報：Kyushu University, 2017, 博士（システム生命科学）, 課程博士 バージョン：
権利関係：
（様式3）
氏 名 ：Jane Tonello
論 文 名 ：Development of genetically engineered hepatoma cell lines with inducible liver functions by overexpression of liver-enriched transcription factors
（肝特異的転写因子の過剰発現による肝機能誘導型遺伝子組換えヘパトーマ細胞の開発）
区 分 ：
甲論 文 内 容 の 要 旨
The liver is one of the largest internal organs by mass and vital in its functions for the human body. Some of these functions include metabolic and digestive processes, detoxification of the blood, hormone and protein production, and storage of nutrients. Therefore, when a patient suffers from liver damage or injury, it can result in acute liver failure. In modern medicine, the only known treatment for acute liver failure is a liver transplant. However, it is difficult for patients to be able to find and receive a donor organ in a timely manner. In such cases, a bioartificial liver system (BAL) can be used as a supportive device, while the patient waits for a donor liver to be located. BALs need to be able to perform the same functions as a natural liver, in order to alleviate patients from the ill side-effects of liver failure.
The determining factor in the effectiveness of a BAL is often the cellular component of the device. While primary hepatocytes are considered the ‘gold standard’ in research and therapeutic uses, they have several draw-backs. Primary hepatocytes have excellent liver function, but lose their ability to proliferate outside of their natural environment in the liver.
Also, just as with donor livers, primary hepatocytes are not an infinitely available cell source.
Therefore, alternative cells sources are being continuously researched for an effective cell component.
Hepatoma cells, or cells derived from liver carcinoma, are one of such alternative cells sources. Due to their ability to proliferate infinitely and easy maintenance under standard cell culture conditions, hepatoma cells would be an ideal component for BALs. However, hepatoma cells are also known to have very low expression of liver functions. Therefore, it is imperative to find new methods to increase liver function in hepatoma cells.
In a previous study, by transducing cells with inducible overexpression of liver enriched transcription factors: HNF1α, HNF1β, HNF3β, HNF4α, HNF6, which belong to the HNF family (Hepatocyte nuclear factor), and C/EBPα, C/EBPβ, C/EBPγ that belong to the C/EBP (CCAAT/Enhancer binding protein) family, mouse hepatoma cells with high levels of liver function were created. Out of the cells derived from the Hepa1-6 mouse hepatoma cells line, Hepa/8F clone #5 (Hepa/8F5) was considered to be the most efficient.
Chapter 1 introduces more about the background of the liver and current techniques used for the development of bioartificial livers. This section also briefly covers the use and development
of alternative cells sources, which is the major goal of our study.
Chapter 2 of this paper covered the studies related to the previously established murine hepatoma cell line Hepa/8F5. This includes the quantitative analyses of the cells, as well as alternate culturing techniques to improve liver function.
Chapter 3 involves the three-dimensional culture of Hepa/8F5 cells on macroporous gelatin beads. Three-dimensional culture methods improved liver function in the Hepa/8F5 cell line.
Chapter 4 describes the use of epigenetic reagents as a liver function enhancer in Hepa/8F5 cells. It shows that combination of certain small molecule drugs as well as vitamins can further improve liver functions on hepatoma cells.
Chapter 5 covers the use of the same genetic enhancement techniques used in Hepa/8F5 but on human hepatoma cell lines Huh7 and HepG2. The results suggest that due to the use of our introduction of inducible overexpression of liver-enriched transcription factors to the cells, liver function could be enhanced for both the human cell lines.
Chapter 6 is a summary of all included information on this thesis, and possible future applications for the development of genetically modified hepatoma cell lines.
