本文關鍵詞： 斑馬魚 connexin43 morpholino 心臟 血管 血液 發(fā)育 原位雜交　出處：《復旦大學》2008年碩士論文　論文類型：學位論文

【摘要】： Cx43是第一個在心肌和其他組織發(fā)現并且是表達最廣泛的一個連接蛋白。已經發(fā)現cx43的基因突變與許多人類的發(fā)育缺陷有關,包括耳聾、外周神經病和先天性的心臟病以及血液系統(tǒng)疾病。研究cx43基因在個體發(fā)育中的作用有助于理解各種相關先天性疾病特別是心血管疾病的發(fā)生的機制,為治療提供新的思路。在已有的研究中已經證實,cx43的基因敲除小鼠在胚胎期表現出明顯的心臟發(fā)育缺陷,主要是右心室和流出道的畸形,但是其確切的機制還有待于進一步的闡明。 由于cx43的基因敲除導致哺乳動物的胚胎致死或出生后不久死亡,難以深入研究其對心血管系統(tǒng)的作用。而斑馬魚是近年來出現的一種良好的模式生物,其體型較小可以通過滲透作用提供氧氣和營養(yǎng),所以即使心血管有嚴重的畸形也可以存活較長的時間。此外斑馬魚胚胎發(fā)育期,通體透明便于觀測,且心臟發(fā)育在進化上與哺乳動物具有高度的保守性,因此斑馬魚被認為是研究心血管早期發(fā)育良好的模式生物。所以利用斑馬魚研究cx43在心血管系統(tǒng)發(fā)育中的作用具有獨特的優(yōu)勢。 本文第一部分:利用生物信息學的方法證明cx43在脊椎動物的進化過程中,結構和蛋白的功能結構域上非常保守。并且用RNA全胚胎原位雜交的方法和RT-PCR技術對cx43基因在斑馬魚早期胚胎發(fā)育過程中表達譜進行了研究。 本文第二部分:我們設計了兩個嗎林修飾的反義寡核苷酸干擾cx43翻譯起始位點,建立了cx43基因表達下調的斑馬魚模型。為了驗證嗎啉修飾的反義寡核苷酸的有效性,我們將其與編碼cx43-EGFP融合蛋白的cx43-EGFP DNA共注射,結果顯示cx43-MO抑制了cx43-EGFP融合蛋白的表達,從而也驗證了cx43-mo能夠有效抑制內源性的cx43的表達;同時也驗證了cx-43-mo的特異性。 本文第三部分:cx43基因表達下調后用原位雜交和原位免疫熒光檢測心臟標志基因的表達以及心臟的表型,同時利用顯微熒光造影和原位雜交檢測血管的發(fā)育情況。用心室心房的標志基因vmhc和amhc反義RNA探針進行的原位雜交結果顯示vmhc表達下調,而amhc表達上調;原位免疫熒光顯示與原位雜交一致的結果:心房擴張心室縮小,并且心臟環(huán)化不全。用血管標志基因flk-1的RNA探針原位雜交和顯微熒光造影表明cx43基因表達下調的斑馬魚胚胎血管無明顯缺陷。此外cx43基因表達下調的斑馬魚胚胎心臟功能也有明顯改變,包括心臟搏動無力,有血液回流現象。但是下調cx43基因的表達對胚胎血管系統(tǒng)發(fā)育無明顯干擾作用。 本文第四部分:本研究證明:cx43在內細胞團(ICM)有明顯表達。Cx43基因表達下調導致紅細胞生成嚴重減少,和血細胞堆積。原位雜交顯示cx43基因表達下調對造血祖細胞沒有明顯影響但是會導致造血干細胞數量顯著減少。Cx43基因表達下調導致ICM和第四腦室的明顯水腫。因此可以認為cx43是斑馬魚胚胎早期正常原始造血功能所必須的。
[Abstract]:Cx43 is the first in the myocardium and other tissues and is an expression of connexin most widely. It has been found that Cx43 gene mutation related with many human developmental defects including deafness, peripheral neuropathy and congenital heart disease and blood system diseases. Research on Cx43 gene in the ontogeny of the role of help to understand the various congenital diseases, especially cardiovascular disease mechanism, to provide new ideas for treatment. In the previous studies have confirmed that Cx43 gene knockout mice in the embryonic heart development showed obvious defects, mainly the right ventricle and outflow tract malformations, but the exact mechanism is still to be further clarified.
Because the Cx43 knockout lead to mammalian embryos to death or died shortly after birth, it is difficult to study its role in the cardiovascular system. The zebrafish is a good model organism in recent years, the small size can be through osmosis with oxygen and nutrients, so even if there are serious cardiovascular malformations can also survive for a long time. In addition, zebrafish embryogenesis, transparent and convenient observation, heart development is highly conserved in mammals and in evolution, therefore zebrafish is considered an early model of biological development good cardiovascular research. So using zebrafish Cx43 in the development of cardiovascular system function has a unique advantage.
The first part of this paper: by using bioinformatics methods to prove Cx43 in the evolution of vertebrates is conserved domain structure and protein. And the expression of Cx43 gene in early embryonic development in zebrafish was studied by RT-PCR method and technology of RNA whole embryo in situ hybridization.
The second part: we design two: Lin modified antisense oligonucleotides interfering Cx43 translation initiation site, established the Cx43 gene expression in zebrafish model. In order to cut the effectiveness of morpholino antisense oligonucleotides verification, we will with the encoding cx43-EGFP fusion protein cx43-EGFP DNA white co injection, the results showed that cx43-MO inhibited expression of cx43-EGFP fusion protein, which also verified the expression of cx43-mo can effectively inhibit the endogenous Cx43; also verified the specificity of cx-43-mo.
The third part: downregulation of Cx43 gene expression by in situ hybridization and in situ immunofluorescence detection of cardiac marker gene expression and cardiac phenotype, and the development of micro fluorescence angiography and in situ hybridization detection of blood vessels. The results of in situ hybridization with marker genes vmhc and amhc atrial ventricular antisense RNA probe showed down-regulation of vmhc expression the increased expression of amhc; fluorescence in situ display is consistent with the results of in situ hybridization: atrial ventricular dilation and reduced cardiac insufficiency with vascular ring. The marker gene Flk-1 RNA probe in situ hybridization and microscopic fluorescence imaging showed that Cx43 gene expression in zebrafish embryonic vascular down without obvious defects. The down regulated expression of Cx43 gene in zebrafish embryos cardiac function also has obvious changes, including heart weakness, blood backflow phenomenon. But the expression level of Cx43 gene on embryonic blood vessel There was no obvious interference in the development of the system.
The fourth part: the study shows that the Cx43 inner cell mass (ICM) was expressed.Cx43 gene expression leads to the generation of red blood cells was severely reduced, and blood cell accumulation. In situ hybridization showed that Cx43 gene expression had no obvious effect on hematopoietic progenitor cells but leads to a significant reduction in the number of hematopoietic stem cells in down-regulation of.Cx43 gene caused obvious edema ICM and the expression of the fourth ventricle. So it can be concluded that Cx43 is required for early embryonic zebrafish normal primitive hematopoietic function.

【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R346

【共引文獻】

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本文編號：1479256