藉由體外組織培養系統探討ZFP668對胰臟內分泌細胞分化的影響

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

方議賢(Fang Yi-Hsien)

指導教授 ： 江明格

國立中正大學/理學院/生命科學系生物醫學研究所/碩士(2016年)

摘要

胰臟(Pancreas)為人體中第二大的腺體器官，其功能包括消化系統、內分泌系統(endocrine system)及外分泌系統(exocrine system)的調控。目前已知在胰臟發育過程中，需要多種轉錄因子調控胰臟各個部分的成熟，包含PDX1、Ptf1a (P48)、Ngn3；其中Ngn3主要負責調控內分泌細胞的發育及成熟，Ngn3首先表現在胰臟內分泌前驅細胞，接著誘導細胞分化成各種類型的內分泌細胞，包括α、β、ε、PP及δ細胞。
先前實驗室用酵母菌雙雜交系統(Yeast two-hybrid assay)篩選出與Ngn3有交互作用的蛋白質-鋅手指蛋白668 (ZFP668)，它是具有16個C2H2鋅手指結構的轉錄因子，目前在胰臟發育過程中扮演的角色及功能未知。先前學長已證實Ngn3與ZFP668有自主性的交互作用後，我想進一步探討ZFP668是否會影響胰臟內分泌細胞發育或其生理功能。
我的實驗共使用三種方法進行驗證，第一個方法我使用帶有shZFP668的慢病毒(Lentivirus)將胰島素瘤細胞株-NIT1中的ZFP668基因靜默(Knockdown)，並使用RT-PCR及Q-PCR觀察Ngn3下游基因是否受到影響。第二個實驗我使用小鼠胚胎胰臟體外培養模型進行驗證，實驗取胚胎期第12.5天(E12.5)小鼠胚胎胰臟做為材料，再利用慢病毒將ZFP668基因靜默後，於體外培養七天，收集樣本後利用細胞抹片(cytospin)與免疫螢光染色(immunofluorescence staining)觀察ZFP668是否影響內分泌細胞的分化。第三個實驗我使用小鼠胚胎幹細胞，並將細胞誘導分化成胰臟細胞，在誘導過程中，利用慢病毒將ZFP668基因靜默，觀察分化出的內分泌細胞數量與正常組別相比有無明顯差異。
從NIT1靜默ZFP668並分析Ngn3下游基因的結果來看，可證實ZFP668確實會與Ngn3共同調控部分下游基因，但並不是全部；在小鼠胚胎胰臟體外培養的實驗中，我也證明帶有shZFP668並表現綠螢光的慢病毒可有效感染小鼠胚胎胰臟，並可利用細胞抹片與免疫染色的方法追蹤被感染細胞的命運；在誘導胚胎幹細胞分化成胰臟細胞的實驗中，我也在幹細胞分化中期觀察到部分內分泌細胞基因的表現，而在分化後期也能看到Insulin2、Glucagon等內分泌細胞產物的表現，證實目前實驗方法確實可誘導細胞朝胰臟細胞分化。

關鍵字

鋅手指蛋白668 (ZFP668) ； 胚胎幹細胞 ； 體外胰臟培養

並列摘要

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.

並列關鍵字

Zinc finger protein 668 ； Embryonic Stem Cell ； ex vivo pancreas culture

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