| Kaposi’s sarcoma (KS) is an angioproliferative neoplasm of high lethality which is common in AIDS patients. The Kaposi’s sarcoma-associated herpesvirus (KSHV) is the direct etiology for KS. G-protein coupled receptor (vGPCR) encoded by the virus is a kind of oncogenic protein which is bound up with the appearance of KSHV-associated malignancies. Many results have proved that expression of vGPCR in cells can activate different signaling pathways and influence many trancription factors, thereby increase the production of proinflammatory cytokines and angiogenesis cytokines. The expression of vGPCR in nude mice can led to tumorigenesis and in vGPCR transgenic mice it can also develop the human-like KS disease.Zebrafish is an important model organism which was widely used in the research of mechanism for embryonic development and the establishment of various disease models. Zebrafish, as a model organism for the study of human virus, was rarely reported. Currently, no report on studying KHSV which adopt zebrafish has been found. In the research we for the first time study the oncogene vGPCR in KHSV and identify its influence on embryonic development of zebrafish at early stage.In this research we adopt microinjection to import vGPCR genes into the oosperm of zebrafish. In order to analyse the influences of vGPCR on embryonic development of zebrafish at early stage, we have used the methods of morphological observation, detection of the expression level of mRNA, quantitative analysis of angiogenesis and vascular staining. Contents and results of the study in this paper are shown as below:1. Complete the construction of vGPCR recombinant plasmid named CDH-CMV-HA-vGPCR-EF1-copGFP based on the FRT/TO-cDNA5vector and CDH-CMV-MCS-EF1-copGFP vector.2. Transfected the vGPCR into the HEK293T cell by calcium phosphate transfection and detected the protein expression level by Western blotting. The results showed that the expression vector is capable of expressing protein of interest in the HEK293T cells.3. After the microinjection, the survival rates and GFP expression rates of experimental group were50.80%and53.58%respectively, while in the control group were55.02%and48.16%. The morphological observation results showed that the development process of the two groups are basically the same, which indicated that vGPCR would not have obvious impact on the early embryonic morphogenesis in zebrafish. We used RT-PCR assay to detect the embryos which injected vGPCR and expressed GFP successfully, the results showed that there exists low level of vGPCR mRNA expression in the zebrafish embryos.4. By quantitative detection of alkaline phosphatase, we analysed the group in which we inject vGPCR and the control group respectively. The results of EAP assay showed that there was little difference of the alkaline phosphatase content between the experimental group and the control group, which indicated that vGPCR have little significant effect on the angiogenesis level of zebrafish embryos.5. We used the method of alkaline phosphatase staining on embryonic blood vessel after72h injection of vGPCR into it, we observed the new blood vessels of zebrafish embryos were affected by the injection of vGPCR in varying degrees. The injection mainly lead to distortion of subintestinal veins and disordered arrangement of blood vessels, which showed that vGPCR can cause developmental malformation of vessels at early stage in zebrafish.In summury, this study demonstrated that vGPCR genes can be successfully expressed in zebrafish embryos. The introduction of exogenous vGPCR genes does not cause significant impact on its morphological development, but can cause developmental malformations in early embryonic angiogenesis in zebrafish. Accordingly, we suppose that zebrafish can be the most suitable biological model in the research on vGPCR and KSHV and it paves a way for the new study about pathogenic mechanisms of vGPCR and ohter KSHV genes. |
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