靶向斑马鱼VEGF的shRNA表达载体的构建及其对斑马鱼胚胎血管发育的影响

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	靶向斑马鱼VEGF的shRNA表达载体的构建及其对斑马鱼胚胎血管发育的影响
其他题名	Construction of shRNA expression vector against zebrafish VEGF and the effect on vascular development in zebrafish embryos
	杨少丽
学位类型	博士
导师	林秀坤
	2007-06-15
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
学位专业	海洋生物学
关键词	斑马鱼 Vegf基因血管形成 Shrna Nrp1基因
摘要	血管内皮生长因子(vascular endothelial growth factor, VEGF)是一种多功能的细胞因子，其主要作用是促进血管内皮细胞增殖和增加血管通透性，是肿瘤及正常组织血管生成的中心调控因素，以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。RNAi是近年来新发展的一项反向遗传学技术，是一种研究基因功能的有力工具。斑马鱼作为一种重要的模式生物，被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。然而在斑马鱼中运用RNAi技术进行基因功能研究是一个相对较新的领域，研究资料较少，并且目前进行的斑马鱼RNAi实验中，siRNA大都是通过化学方法或体外转录合成的。体外合成的siRNA在进入体内后会被降解而无法达到持久阻抑基因表达的目的。因此本研究旨在探讨VEGF特异性siRNA表达载体对斑马鱼VEGF基因的沉默作用，通过分析表型及相关细胞因子的变化，阐明VEGF对斑马鱼胚胎血管生成的影响及作用机制。研究通过计算机辅助设计软件，针对斑马鱼VEGF mRNA不同位点设计合成了4段含siRNA特异序列的DNA单链，经退火，克隆入pSilencer 4.1-CMV neo载体CMV启动子下游，构建了重组质粒pS1-VEGF、pS2-VEGF、pS3-VEGF及pS4-VEGF。通过显微注射的方法将载体导入1-2细胞期斑马鱼体内，于胚胎发育的48 h采用RT-PCR的方法检测VEGF基因的表达量，研究不同干扰序列对VEGF基因表达的干涉作用。结果显示，针对不同位点的表达载体对VEGF基因表达的抑制效率有显著差异。它们对VEGF mRNA的抑制率分别为80.5%，42.8%，12.5%，40.7%。通过筛选我们得到了一条具有高效抑制作用的载体pS1-VEGF，该载体的相应序列靶向斑马鱼两个主要异构体VEGF165和VEGF121的共有外显子序列。形态学检测结果显示，注射了pS1-VEGF的胚胎出现了心包膜水肿、血流速度减慢、循环红细胞堆积等症状。定量碱性磷酸酶染色显示，注射pS1-VEGF能够抑制斑马鱼胚胎新生血管的形成，当注射剂量为0.4 ng时，血管生成的抑制率为31.8%。NBT/BCIP血管染色显示，注射该载体后72 h，50%的斑马鱼肠下静脉、节间血管以及其它血管的发育受到不同程度的抑制。随着注射剂量的加大，血管发育受抑制的情况也随之加重，当注射剂量为1 ng时，只有心脏、头部及卵黄有血液循环。对干扰效果的特异性进行了研究，结果表明pS1-VEGF对斑马鱼内源基因胸苷酸合成酶（thymidylate synthase, TS）基因的表达没有明显的抑制作用。针对TS基因的shRNA表达载体及与斑马鱼没有同源性的对照载体对VEGF基因表达也没有明显的抑制作用。浓度梯度实验表明在0-1.2 ng的范围内干扰效果具有剂量依赖性。以胚胎整体原位杂交的方法检测质粒对VEGF基因受体NRP1基因表达的影响，发现VEGF特异性shRNA表达载体能够引起NRP1基因表达的降低，说明斑马鱼中VEGF所介导的血管生成作用至少在部分上是依赖于NRP通路所调节的。本研究工作为进一步研究斑马鱼基因功能、VEGF调控网络提供了一个快速、有效的手段，为阐明斑马鱼的血管生成机制提供了新的资料，为采用RNAi技术，以VEGF为靶点，以斑马鱼为模型对肿瘤进行基因治疗研究奠定了基础。
其他摘要	VEGF (vascular endothelial growth factor) is a highly specific endothelial cell mitogen and it is critical for vascularization of tissues, including tumors. The central role of VEGF in angiogenesis makes it an attractive target for anti-tumor therapy. Recently, zebrafish has been generally considered as an excellent model in case of drug screening, disease model establishment, and vertebrate embryonic development study. However, almost all studies to date in zebrafish used chemically synthesized siRNA or in vitro transcribed siRNA, and the application of shRNA in zebrafish remains highly limited. To study the potential of shRNA for gene knockdown in zebrafish, we used a vector-based siRNA expression system, which overcomes the limitation of transience and instability of synthetic siRNA, to specially inhibit zebrafish VEGF gene expression. The potential shRNA target sites in the zebrafish were determined using the siRNA design program. Altogether four siRNAs targeting different locations of VEGF gene were designed. The forward and reverse synthetic oligonucleotides were annealed and inserted into the downstream of CMV promotor of the pSilencer 4.1-CMV neo vector to construct shRNA expression vectors, pS1-VEGF, pS2-VEGF, pS3-VEGF, and pS4-VEGF。 The shRNA vectors were microinjected into zebrafish embryos at 1-2cell stages and total RNA was isolated from embryos at 48 hpf (hours post fertilization). RT-PCR analysis indicated that the vectors could down regulate the expression of VEGF to various degrees. VEGF mRNA levels were decreased by 80.5%, 42.8%, 12.5% and 40.7%, respectively. pS1-VEGF, which transcribing an shRNA against the common extron of VEGF165 and VEGF121 gene, was proved to be the most efficient one. Erythrocyte accumulation and pericardial edema, with reduced numbers of circulating red blood cells were observed following the injection of pS1-VEGF. Quantitative EAP assay showed that the shRNA could cause up to 31.8% reduction of zebrafish angiogenesis at the dose of 0.4 ng. Alkaline phosphatase staining using NBT and BCIP indicated that pSI-V1 could cause specific vascular defects in zebrafish in a dose-dependent manner. Absences of SIV and intersegmental vasculature were observed in 50% embryos following the injection of pS1-VEGF. A phenotypic class of embryos only with heart, yolk and head blood vessels was observed at the dose of 1 ng. Further study found that the expression of TS (thymidylate synthase) gene was not significantly affected after the injection of pS1-VEGF. Similarly, the expression of VEGF gene was not down regulated by control vector and siRNA expression vector against TS gene, as determined by RT-PCR. Dose-dependent study was then performed, and the result showed that pS1-VEGF could cause specific gene silencing in a dose-dependent manner at the concentration of 0-1.2 ng. In situ hybridization analysis indicated that silencing VEGF gene expression by shRNA resulted in down regulation of NRP1, a potent VEGF receptor. This provided evidence that the VEGF-mediated agiogenesis in zebrafish is dependent in part on NRP1 expression. The siRNA expressing vector in zebrafish have the potential to overcome the limitations of relying on transient regulation of gene targeting, and opening up the possibility of using zebrafish for functional gene analysis and anti-tumor therapy studies.
页数	114
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/771
专题	海洋环流与波动重点实验室
作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	杨少丽. 靶向斑马鱼VEGF的shRNA表达载体的构建及其对斑马鱼胚胎血管发育的影响[D]. 海洋研究所. 中国科学院海洋研究所,2007.