碩士 / 國立臺灣海洋大學 / 生物科技研究所 / 102 / Ulcerative colitis (UC) and Crohn’s disease (CD) are two types of human inflammatory bowel disease (IBD). Patients with UC present thinner intestinal mucus layer which inspires us to investigate relationship between intestinal goblet cell development and human IBD using zebrafish. We used chemicals including 2,4,6-trinitrobenzene sulfonic acid (TNBS) and Dextran sulfate sodium (DSS) that used in mouse to induce IBD to treat zebrafish embryos. Embryos were treated with DSS from 72 to 120 hpf. We stained with Alcian blue and detected significant decreased intestinal goblet cell number in 120 hpf DSS treated-embryos. Whole-mount in situ hybridization revealed increased number of DSS-treated embryos expressing ectopic myeloperoxidase (mpx) at 96 hpf; this indicates inflammatory response is induced by DSS treatment. However we see no alteration of expression of PPARγ, cdx1b, foxa1 or foxa2, marker genes between DSS-treated and control embryos. We detected significant increased alcian blue-stained intestinal goblet cell number in 8 dpf embryos treated with TNBS. We observed increased number of TNBS-treated embryos showing ectopic mpx at 96 hpf, indicating that inflammatory response is induced by TNBS treatment. However we see no alteration of expression of PPARγ or cdx1b between TNBS-treated and control embryos, while we detected slightly decreased expression of foxa1 or foxa2 in 96 hpf TNBS treated-embryos. We also generated Tg(agr2endo2778 : NTR-EMD) transgenic fish line and expect to detect death of intestinal goblet cell upon treatment with meteronidazole (Mtz) after it has been converted into cytotoxin by Nitroreductase (NTR)-EMD fusion protein. However, western blot results showed major production of 26 kDa EMD protein in Tg(agr2endo2778: NTR-EMD) embryos. Therefore, no apoptosis of intestinal goblet cells can be detected upon Mtz treatment. We also generated 3 transgenic fish lines including Tg(Tol2-exo200agr2-352endo-TetA- mcherry), Tg(TRE-BI : klf4a-AcGFP) and Tg(TRE-BI : cdx1b-AcGFP). We expect to detect increased intestinal goblet cell number in embryos from intercross of Tg(Tol2-exo200agr2- 352endo-TetA-mcherry) with either Tg(TRE-BI : klf4a-AcGFP) or Tg(TRE-BI : cdx1b-AcGFP) after Doxycycline (Dox)/Dexamethasone (Dex) treatment; this is due to induced expression of klf4a or cdx1b in the intestine by Dox/Dex can increase goblet cell number in the intestine. However, the screened Tg(Tol2-exo200agr2-352endo- TetA-mcherry) embryos did not express transactivator in the intestine but in the otic vesicles and resulted EGFP expression in the otic vesicles; this may be due to the effect of integration site of vector on the chromosome.
keywords : inflammatory bowel disease, mucus layer, goblet cells, chemical induction
| Identifer | oai:union.ndltd.org:TW/102NTOU5613028 |
| Date | January 2014 |
| Creators | Yen, Tzu-Chun, 顏自君 |
| Contributors | Hwang, Sheng-Ping, 黃聲蘋 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 40 |
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