KSHV编码vGPCR对斑马鱼胚胎早期发育的影响

发布时间：2018-05-10 21:26

  本文选题：vGPCR + 斑马鱼　； 参考：《湖南师范大学》2014年硕士论文

【摘要】：卡波氏肉瘤(KS)是艾滋病患者常见的高致死性血管增生型肿瘤。卡波氏肉瘤相关疱疹病毒(KSHV)是KS的直接病因,该病毒基因组编码的G-蛋白偶联受体(vGPCR)是已知的几个KSHV致瘤蛋白之一,此蛋白与KSHV相关恶性肿瘤的发生密切相关。vGPCR在细胞中的表达能够激活多条信号通路和影响诸多转录因子从而增加促炎症细胞因子和血管生成细胞因子的生成。vGPCR在裸鼠中表达能够引起裸鼠产生肿瘤,转vGPCR基因小鼠也能形成类似人类KS的病变。 斑马鱼是一种重要的模式生物,在胚胎发育机制的研究及各种人类疾病研究中得到了广泛的应用。斑马鱼作为人类病毒研究的模式生物罕见报道,目前尚未有采用斑马鱼来研究KHSV的研究报道。本研究首次采用斑马鱼来对KHSV中致癌基因vGPCR进行研究,鉴定vGPCR对斑马鱼早期胚胎发育的影响。 本研究采用显微注射法将vGPCR基因导入斑马鱼受精卵；利用形态学观察、mRNA水平表达检测、血管生成定量分析和血管染色法分析vGPCR对斑马鱼早期胚胎发育的影响。本论文研究内容和研究结果如下： 1.以FRT/TO-cDNA5载体和CDH-CMV-MCS-EF1-copGFP载体为基础构建了表达vGPCR的重组质粒CDH-CMV-HA-vGPCR-EF1-copGFP; 2.通过磷酸钙转染法将CDH-CMV-HA-vGPCR-EF1-copGFP转入HEK293T细胞,并进行免疫印迹杂交检测,结果显示该重组质粒能够在细胞内成功表达目的蛋白； 3.采用显微注射技术将CDH-CMV-HA-vGPCR-EF1-copGFP导入斑马鱼受精卵,统计结果表明注射vGPCR的实验组胚胎存活率为50.80%,GFP表达率为53.58%；注射空载体的对照组胚胎存活率为55.02%,GFP表达率为48.16%。形态学检测结果显示,注射vGPCR的胚胎与对照组的发育进程基本一致,vGPCR对斑马鱼胚胎早期形态发育未产生明显影响；对注射了vGPCR并表达GFP的胚胎进行RT-PCR检测,结果显示vGPCR在斑马鱼胚胎中存在较低水平的mRNA表达； 4.利用碱性磷酸酶定量检测法对vGPCR注射组斑马鱼胚胎和对照组胚胎分别进行分析,结果表明两组胚胎的碱性磷酸酶含量相差不大,表明vGPCR对斑马鱼早期胚胎血管生成总量无明显影响； 5.对注射vGPCR后72小时的胚胎血管进行碱性磷酸酶染色,观察到斑马鱼胚胎新生血管发育受到了不同程度的影响,主要为肠下静脉扭曲与血管排布紊乱,说明vGPCR能够造成斑马鱼胚胎早期血管发育畸形。 综上所述,本研究证明了vGPCR基因能够在斑马鱼胚胎中表达,外源vGPCR的导入对斑马鱼早期胚胎的形态学发育并未造成明显影响,但会导致斑马鱼早期胚胎新生血管发育畸形。本研究表明斑马鱼能够成为研究vGPCR及KSHV的生物模型,为今后研究vGPCR以及其他KSHV基因的致病机制开拓了新的领域。
[Abstract]:Kaposi's sarcoma (KS) is a common highly lethal angiogenic tumor in AIDS patients. Kaposarcoma associated herpesvirus (KSHV) is the direct cause of KS, and its genome-encoded G-protein-coupled receptor (VGPCR) is one of several known KSHV oncoproteins. This protein is closely related to the occurrence of KSHV associated malignant tumors. VGPCR expression in cells can activate multiple signal pathways and affect many transcription factors, thereby increasing the production of inflammatory cytokines and angiogenic cytokines. Expression in nude mice can cause tumors in nude mice. VGPCR transgenic mice can also develop lesions similar to human KS. Zebrafish is an important model organism, which has been widely used in the study of embryonic development mechanism and various human diseases. Zebrafish, as a model of human virus research, has not been reported yet. Zebrafish have not been used to study KHSV. In this study, zebrafish were used to study the oncogene vGPCR in KHSV for the first time, and to identify the effect of vGPCR on the development of zebrafish early embryos. In this study, the vGPCR gene was introduced into zebrafish fertilized eggs by microinjection, and the effect of vGPCR on the early embryo development of zebrafish was analyzed by morphological observation, quantitative analysis of angiogenesis and vascular staining. The contents and results of this thesis are as follows: 1. Based on FRT/TO-cDNA5 vector and CDH-CMV-MCS-EF1-copGFP vector, the recombinant plasmid CDH-CMV-HA-vGPCR-EF1-copGFPwas constructed. 2. CDH-CMV-HA-vGPCR-EF1-copGFP was transfected into HEK293T cells by calcium superphosphate transfection and detected by Western blotting. The results showed that the recombinant plasmid could successfully express the target protein in the cells. 3. CDH-CMV-HA-vGPCR-EF1-copGFP was introduced into zebrafish fertilized eggs by microinjection. The results showed that the embryo survival rate of the experimental group injected with vGPCR was 53.58 and that of the control group injected with empty vector was 55.02% and 48.16% respectively. Morphological analysis showed that the developmental process of the embryos injected with vGPCR was basically consistent with that of the control group, and that the early morphogenesis of zebrafish embryos was not affected by VGPCR, and that the embryos injected with vGPCR and expressed GFP were detected by RT-PCR. The results showed that there was a low level of mRNA expression in zebrafish embryos. 4. The content of alkaline phosphatase in zebrafish embryos of vGPCR injection group and control group was analyzed by alkaline phosphatase quantitative detection method. The results showed that the content of alkaline phosphatase in the two groups was not different. The results showed that vGPCR had no significant effect on the total angiogenesis of zebrafish early embryo. 5. The embryonic vessels of zebrafish were stained with alkaline phosphatase (ALP) at 72 hours after injection of vGPCR. It was observed that the embryonic neovascularization of zebrafish was affected by different degrees, mainly the distortion of inferior intestinal vein and the disorder of vascular arrangement. These results suggest that vGPCR can cause early vascular malformation in zebrafish embryos. In conclusion, this study demonstrated that vGPCR gene can be expressed in zebrafish embryos. The introduction of exogenous vGPCR has no significant effect on the morphological development of zebrafish early embryos, but it can lead to angiogenesis malformation of zebrafish early embryos. This study shows that zebrafish can be used as a biological model for studying vGPCR and KSHV, which opens up a new field for studying the pathogenesis of vGPCR and other KSHV genes in the future.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R732.2;R512.91

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