Investigating the influence of ZFP668 on pancreatic endocrine cell differentiation using ex vivo tissue culture system

• Main Authors: Fang Yi-Hsien, 方議賢
• Other Authors: Chiang Ming-Ko
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/55928346031382170753

碩士 === 國立中正大學 === 生物醫學研究所 === 104 === The pancreas is a glandular organ in the digestive system and endocrine system of vertebrates. It controls blood glocuse stability. Transcription factors such as PDX1、Ptf1a(P48) and Ngn3 function coordinately to regulate pancreas development during embryonic development.. Neurogenin3 (Ngn3) is a member of the basic helix-loop-helix family. It controls the development and formation of the endocrine cells ‚such as α cells、β cells、ε cells、δ cells、PP cells. Previous study has shown that Zinc finger protein 668 (ZFP668)，which contains 16 C2H2 type zinc finger motif can interact with Ngn3. ZFP668 has been identified to interact with Ngn3 by GST-pull down assay and co-immunoprecipitation (Co-IP) experiment. Our previous study has also shown that ZFP668 could regulate the expression of Ngn3’s downstream target genes such as NeuroD1 and Insm1. The aim of my project, is to clarify whether ZFP668 could modulate endocrine cells differentiation. I used β cell line- NIT1 and ex vivo embryonic pancreas bud culture as my experimental model. First‚ I suppress the ZFP668 expression by lentivirus infection in both model. After ZFP668 is knocked down, I analysed the Ngn3 downstream genes expression in NIT1 cells. On the other hand‚ I also dissected the embryonic pancreas bud from mouse embryos, infected with lentivirus and then cultured the cells for seven days. Finally, I used immunostain to detect the lentivirus infected cells which co-expressed endocrine cell markers. Then, I investigated whether knockdown ZFP668 can affect endocrine cells differentiation. In order to further confirm whether ZFP668 can affect endocrine cell differentiation, I established the ES cells differentiation model. I induced ES cells to differentiate into insulin-producing cells and analysed the expression of pancreatic endocrine markers during differentiation. The current results show that endoderm markers can be detected, and some endocrine cell markers can also be deceted during ES cell differentiation process. These results confirmed that ES cell can be induced to differentiate into pancreas like cell. However, different conditions for the induction, including culture medium and different combination of growth factors for treating ES cell, need to be explored to improve the efficiency of pancreatic cell differentiation at final stage.