Connexin43在斑马鱼胚胎心血管系统发育中的作用

发布时间：2018-01-31 14:17

本文关键词： 斑马鱼 connexin43 morpholino 心脏血管血液发育原位杂交　出处：《复旦大学》2008年硕士论文　论文类型：学位论文

【摘要】： Cx43是第一个在心肌和其他组织发现并且是表达最广泛的一个连接蛋白。已经发现cx43的基因突变与许多人类的发育缺陷有关,包括耳聋、外周神经病和先天性的心脏病以及血液系统疾病。研究cx43基因在个体发育中的作用有助于理解各种相关先天性疾病特别是心血管疾病的发生的机制,为治疗提供新的思路。在已有的研究中已经证实,cx43的基因敲除小鼠在胚胎期表现出明显的心脏发育缺陷,主要是右心室和流出道的畸形,但是其确切的机制还有待于进一步的阐明。由于cx43的基因敲除导致哺乳动物的胚胎致死或出生后不久死亡,难以深入研究其对心血管系统的作用。而斑马鱼是近年来出现的一种良好的模式生物,其体型较小可以通过渗透作用提供氧气和营养,所以即使心血管有严重的畸形也可以存活较长的时间。此外斑马鱼胚胎发育期,通体透明便于观测,且心脏发育在进化上与哺乳动物具有高度的保守性,因此斑马鱼被认为是研究心血管早期发育良好的模式生物。所以利用斑马鱼研究cx43在心血管系统发育中的作用具有独特的优势。本文第一部分:利用生物信息学的方法证明cx43在脊椎动物的进化过程中,结构和蛋白的功能结构域上非常保守。并且用RNA全胚胎原位杂交的方法和RT-PCR技术对cx43基因在斑马鱼早期胚胎发育过程中表达谱进行了研究。本文第二部分:我们设计了两个吗林修饰的反义寡核苷酸干扰cx43翻译起始位点,建立了cx43基因表达下调的斑马鱼模型。为了验证吗啉修饰的反义寡核苷酸的有效性,我们将其与编码cx43-EGFP融合蛋白的cx43-EGFP DNA共注射,结果显示cx43-MO抑制了cx43-EGFP融合蛋白的表达,从而也验证了cx43-mo能够有效抑制内源性的cx43的表达;同时也验证了cx-43-mo的特异性。本文第三部分:cx43基因表达下调后用原位杂交和原位免疫荧光检测心脏标志基因的表达以及心脏的表型,同时利用显微荧光造影和原位杂交检测血管的发育情况。用心室心房的标志基因vmhc和amhc反义RNA探针进行的原位杂交结果显示vmhc表达下调,而amhc表达上调;原位免疫荧光显示与原位杂交一致的结果:心房扩张心室缩小,并且心脏环化不全。用血管标志基因flk-1的RNA探针原位杂交和显微荧光造影表明cx43基因表达下调的斑马鱼胚胎血管无明显缺陷。此外cx43基因表达下调的斑马鱼胚胎心脏功能也有明显改变,包括心脏搏动无力,有血液回流现象。但是下调cx43基因的表达对胚胎血管系统发育无明显干扰作用。本文第四部分:本研究证明:cx43在内细胞团(ICM)有明显表达。Cx43基因表达下调导致红细胞生成严重减少,和血细胞堆积。原位杂交显示cx43基因表达下调对造血祖细胞没有明显影响但是会导致造血干细胞数量显著减少。Cx43基因表达下调导致ICM和第四脑室的明显水肿。因此可以认为cx43是斑马鱼胚胎早期正常原始造血功能所必须的。
[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.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

【共引文献】