本文選題：斑馬魚 + β細(xì)胞��； 參考：《貴陽(yáng)中醫(yī)學(xué)院》2008年碩士論文

【摘要】： 目的:本課題旨在利用轉(zhuǎn)基因技術(shù),構(gòu)建活體的胰島β細(xì)胞綠色熒光可示蹤的斑馬魚系,從而為在此基礎(chǔ)上進(jìn)一步破壞胰島β細(xì)胞,建立活體的胰島β細(xì)胞可示蹤的,可供高通量藥物篩選的糖尿病胰島β細(xì)胞破壞模型作出前期準(zhǔn)備。 方法:通過(guò)轉(zhuǎn)基因技術(shù),PCR技術(shù)、分子克隆等方法,建立GFP標(biāo)記胰島B細(xì)胞的遺傳學(xué)轉(zhuǎn)基因斑馬魚品系;活體呈像動(dòng)態(tài)示蹤胰島B細(xì)胞的起源和成熟;全胚胎原位雜交和基因組PCR證實(shí)GFP陽(yáng)性細(xì)胞與內(nèi)源性胰島B細(xì)胞共定位。 結(jié)果:1.獲得INS部分啟動(dòng)子的克隆和重組質(zhì)粒INS:GFP,顯微鏡注射建立生殖系胰島素轉(zhuǎn)基因斑馬魚。 2.通過(guò)生殖系轉(zhuǎn)基因斑馬魚的篩選和熒光觀察,24hpf GFP標(biāo)記的胰腺β細(xì)胞群被探測(cè)到位于第二、三體節(jié)下方中線兩側(cè)的單一的橢圓形實(shí)體。隨著斑馬魚胚胎的發(fā)育,胰腺逐漸增大并向右遷移,72hpf可以觀察到胰腺β-細(xì)胞位于腹中線的右側(cè)。卵黃囊消失后,120hpf明顯看到胰腺右偏,同時(shí)下移到魚鰾下方的十二指腸背側(cè)。 3.全基因組PCR證實(shí)GFP在基因組中的穩(wěn)定整合,全胚胎原位雜交說(shuō)明了活體轉(zhuǎn)基因斑馬魚的GFP細(xì)胞為胰島β-細(xì)胞。 結(jié)論:1.利用轉(zhuǎn)基因技術(shù),構(gòu)建了胰島β細(xì)胞可示蹤斑馬魚系。 2.通過(guò)PCR和全胚胎原位雜交的方法證實(shí)所建立的胰島β細(xì)胞可示蹤斑馬魚系是完全成功的。 3.本研究的成功為進(jìn)一步建立可供高通量藥物篩選的胰島β細(xì)胞破壞斑馬魚疾病模型奠定了研究基礎(chǔ)。
[Abstract]:Objective: to construct a living zebrafish line with green fluorescent tracer of islet 尾 cells by using transgenic technology, so as to further destroy the islet 尾 cells and establish in vivo islet 尾 cells traceable. Diabetic islet 尾-cell destruction model for high-throughput drug screening was prepared. Methods: genetic transgenic zebrafish strains labeled with GFP were established by means of transgene technique and molecular cloning, and the origin and maturation of islet B cells were traced in vivo. Whole embryo in situ hybridization and genomic PCR confirmed that GFP positive cells were colocated with endogenous islet B cells. The result is 1: 1. The partial promoter of INS was cloned and the recombinant plasmid ins: GFP was obtained, and the reproductive line insulin transgenic zebrafish was established by microscope injection. 2. By screening and fluorescence observation of transgenic zebrafish, a single elliptical entity located at the middle line of the second and third ganglia was detected by 24hpf GFP labeled pancreatic 尾 cell group. With the development of zebrafish embryos, the pancreas gradually enlarged and migrated to the right of 72hpf. It was observed that the pancreatic 尾-cells were located on the right side of the middle line of abdomen. At 120h after the disappearance of yolk sac, the pancreas could be seen to the right and move down to the dorsal side of the duodenum below the swim bladder. 3. The whole genome PCR confirmed the stable integration of GFP in the genome. The in situ hybridization of whole embryo indicated that the GFP cells of transgenic zebrafish were islet 尾-cells. Conclusion 1. Islet 尾-cells traceable zebrafish lines were constructed by transgenic technique. 2. The establishment of islet 尾 cells was proved to be completely successful by PCR and whole embryo in situ hybridization. 3. The success of this study laid a foundation for the further establishment of islet 尾 -cell destruction model for zebrafish disease.
【學(xué)位授予單位】：貴陽(yáng)中醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R587.1;R-332

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