本文選題：vGPCR + 斑馬魚�。� 參考：《湖南師范大學(xué)》2014年碩士論文

【摘要】：卡波氏肉瘤(KS)是艾滋病患者常見的高致死性血管增生型腫瘤�？úㄊ先饬鱿嚓P(guān)皰疹病毒(KSHV)是KS的直接病因,該病毒基因組編碼的G-蛋白偶聯(lián)受體(vGPCR)是已知的幾個KSHV致瘤蛋白之一,此蛋白與KSHV相關(guān)惡性腫瘤的發(fā)生密切相關(guān)。vGPCR在細(xì)胞中的表達能夠激活多條信號通路和影響諸多轉(zhuǎn)錄因子從而增加促炎癥細(xì)胞因子和血管生成細(xì)胞因子的生成。vGPCR在裸鼠中表達能夠引起裸鼠產(chǎn)生腫瘤,轉(zhuǎn)vGPCR基因小鼠也能形成類似人類KS的病變。 斑馬魚是一種重要的模式生物,在胚胎發(fā)育機制的研究及各種人類疾病研究中得到了廣泛的應(yīng)用。斑馬魚作為人類病毒研究的模式生物罕見報道,目前尚未有采用斑馬魚來研究KHSV的研究報道。本研究首次采用斑馬魚來對KHSV中致癌基因vGPCR進行研究,鑒定vGPCR對斑馬魚早期胚胎發(fā)育的影響。 本研究采用顯微注射法將vGPCR基因?qū)氚唏R魚受精卵；利用形態(tài)學(xué)觀察、mRNA水平表達檢測、血管生成定量分析和血管染色法分析vGPCR對斑馬魚早期胚胎發(fā)育的影響。本論文研究內(nèi)容和研究結(jié)果如下： 1.以FRT/TO-cDNA5載體和CDH-CMV-MCS-EF1-copGFP載體為基礎(chǔ)構(gòu)建了表達vGPCR的重組質(zhì)粒CDH-CMV-HA-vGPCR-EF1-copGFP; 2.通過磷酸鈣轉(zhuǎn)染法將CDH-CMV-HA-vGPCR-EF1-copGFP轉(zhuǎn)入HEK293T細(xì)胞,并進行免疫印跡雜交檢測,結(jié)果顯示該重組質(zhì)粒能夠在細(xì)胞內(nèi)成功表達目的蛋白； 3.采用顯微注射技術(shù)將CDH-CMV-HA-vGPCR-EF1-copGFP導(dǎo)入斑馬魚受精卵,統(tǒng)計結(jié)果表明注射vGPCR的實驗組胚胎存活率為50.80%,GFP表達率為53.58%；注射空載體的對照組胚胎存活率為55.02%,GFP表達率為48.16%。形態(tài)學(xué)檢測結(jié)果顯示,注射vGPCR的胚胎與對照組的發(fā)育進程基本一致,vGPCR對斑馬魚胚胎早期形態(tài)發(fā)育未產(chǎn)生明顯影響；對注射了vGPCR并表達GFP的胚胎進行RT-PCR檢測,結(jié)果顯示vGPCR在斑馬魚胚胎中存在較低水平的mRNA表達； 4.利用堿性磷酸酶定量檢測法對vGPCR注射組斑馬魚胚胎和對照組胚胎分別進行分析,結(jié)果表明兩組胚胎的堿性磷酸酶含量相差不大,表明vGPCR對斑馬魚早期胚胎血管生成總量無明顯影響； 5.對注射vGPCR后72小時的胚胎血管進行堿性磷酸酶染色,觀察到斑馬魚胚胎新生血管發(fā)育受到了不同程度的影響,主要為腸下靜脈扭曲與血管排布紊亂,說明vGPCR能夠造成斑馬魚胚胎早期血管發(fā)育畸形。 綜上所述,本研究證明了vGPCR基因能夠在斑馬魚胚胎中表達,外源vGPCR的導(dǎo)入對斑馬魚早期胚胎的形態(tài)學(xué)發(fā)育并未造成明顯影響,但會導(dǎo)致斑馬魚早期胚胎新生血管發(fā)育畸形。本研究表明斑馬魚能夠成為研究vGPCR及KSHV的生物模型,為今后研究vGPCR以及其他KSHV基因的致病機制開拓了新的領(lǐng)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.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R732.2;R512.91

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