| In 2014, the outbreak of Ebola haemorrhagic fever in West Africa had attracted wide attention in the whole world. As the contact between China and West Africa is frequent, the possibility that Ebola virus would enter China was high. The pivotal issue for prevention and control is to improve the early and accurate detection ability for Ebola virus.With the purpose of ensuring the detection capability for Ebola virus in China, especially for the important harbor cities, the related external quality assessment (EQA) was organized by National center for Clinical Laboratory in December 2014. Consideraing the high fatality rate of Ebola virus, which belongs to biosafety level 4 controled virus, the conventional quality control samples, such as inactivated virus or positive patients’serum, are inapplicable in this EQA. The recombinational MS2 virus-like particles (MS2 VLPs) encapsulated with targeted RNA could take place of the ordinary quality control samples, which have the features of RNase and DNase resistance, good stability and safety. In this study, we prepared MS2 VLPs with the whole sequences of Nucleoprotein gene, parts of Glycoprotein and Polymerase of the 2014 epidemic strains of Ebola virus, which were send to all qualified participants.The results of this EQA were set back to the National Center for Clinical Laboratory. The 20 results were returned by 19 participating agencies. Accurate detection was reported by 14 of 19 participators (73.68%). Three of 19 agencies have returned only one false negative result (15.79%), one of 19 with two false negative results (5.26%), while another with more than three false negative results (5.26%). The results for negative samples were all correct, while the slight positive sample were unsatisfied. Focused on the false negative problems existed in this EQA, we put forward the following suggestions:commercial kits, standard primers form Chinese CDC and two-target detection approachs were recommended. Additionally, standardization of operations and punctual adjustment of instruments are also necessary. Generally, the Ebola detection ability was improved by this EQA which effectively prevented the outbreak of Ebola virus in China.In the second part, in order to overcome the difficulties of low crosslinking efficiency, high cost, long time consuming and tedious procedures for chemical crosslingking in previously MS2 VLPs-based miRNA delivery system, TAT peptide was designed to display on the surface of MS2 VLP instead of chemical crosslinking by the platform of phage surface display.Firstly, we identify whether MS2 VLP displaying TAT possessed the capabilities of penetrating cytomembrane and miR-122 delivery. Next, the inhibited function of 2MS-TAT-miR122 VLPs for HCC was further tested. In order to better identify the anti-tumor effects, MS-miR122 VLPs conjugated with TAT were brought into this study for comparison. The technologies of CCK8, transwell, flow cytometry, Western blot, CT scan, HE staining and immunohistochemistry were all used in this experiment, in order to investigate the inhibition effects of MS2 VLPs in vivo and vitro for HCC.The results indicated that the prepared MS2 VLPs with pre-miR122 and displaying TAT on surface could penetrate the cell membrance and delivery miR122 effectively. In cellular level, both VLPs displaying TAT and crosslingking TAT could inhibit cell growth for Hep3B, HepG2 and Huh7. It was more obvious inhibited effects for MS2 VLPs displaying TAT. Then Hep3B cell was selected for further animal experiments. We obsevered the tumor size after tumor-burdened mice injected VLPs by tail-vein. The results indicated that above VLPs with pre-miR122 could inhibit the tumor growth, while VLPs displaying TAT was more effective. Generally, the recombinational MS2 VLPs encapsulating pre-miR122 and displaying TAT on surface could penetrate cell membranes, and have the advantages of easier preparation, shorter period and more effective for HCC compared with VLPs crosslinking TAT, which has shown an anticipated prospect for application in the future. |
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