ABSTRACT
BACKGROUND: Exosomes have become more and more popular in the field of tumor research in recent years. They can carry a large number of bioactive molecules directly to the receptor cells and participate in the. information exchange between cells. As the highest content and most kinds of non-coding RNA in exosomes, miRNAs play an important role in the proliferation, invasion, drug resistance and immunity of glioma. In addition, miRNAs have potential application value in the auxiliary diagnosis, treatment orientation and prognosis evaluation of glioma. OBJECTIVE: To describe the mechanism of exosome miRNAs sorting, the regulatory role in gliomas and its clinical application, to provide literature and theoretical basis for further exploring the relationship between exosome miRNAs and gliomas, and to exert positive significance in the diagnosis and treatment of gliomas. METHODS: The first author searched CNKI, WanFang database, PubMed database and EI database. The key words were “glioma, exosomes, miRNAs, sorting mechanism, biomarkers” in Chinese and English. Totally 118 literatures were retrieved. According to the inclusion and exclusion criteria, and literature supplement, totally 52 literatures were selected for summary and induction, mainly regarding the sorting mechanism of exosome miRNAs, the regulatory role in glioma and clinical application. RESULTS AND CONCLUSION: At present, the effective treatment of gliomas cannot meet the clinical needs even though a variety of high-intensity combined treatment schemes are adopted in this study. In this case, it is an important supplement for the clinical treatment of gliomas to propose the application of exosomes-mediated miRNAs in gene therapy of gliomas. Exosomes-mediated miRNAs cannot only play a regulatory role in the occurrence and development of glioma, invasion and metastasis, radiochemotherapy resistance, and immune regulation, but also play a potential role as a biomarker in the grading diagnosis, prognosis evaluation, treatment and other aspects of glioma. This will provide a solid theoretical basis for us to further explore the relationship between exosome miRNAs and glioma, and also has a positive clinical significance in the innovative diagnosis and treatment of glioma.
ABSTRACT
AIM: To solve the two difficulties of bone resorption and inflammation in rheumatoid arthritis, clone the recombinant human osteoprotegerin (OPG) and mycobacteria heat shock protein 70 (HSP70) functional gene,and study the expression and activity of OPG-HSP70 fusion protein in E.coli. METHODS: Experiments were performed at the Laboratory of Department of Immunology, Capital Medical University from May 2006 to September 2007. Complementary DNA encoding full length OPG protein was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from human osteosarcoma cell line MG63 and cloned into pGEMT-Easy vectors. Then, using the recombinant plasmid as the template,the DNA encoding the fusion protein OPG-HSP 70 was amplified by PCR,and was inserted into prokaryotic expression vector pET-28a. Construct pET-28a-OPG-HSP 70 was used to transform into competent E.coli. BL21(DE3) which were induced by isopropyl B-D-thiogalactopyranoside (IPTG),and the fusion protein from above E.coli was collected. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western-blotting were performed to identify OPG-HSP 70 fusion protein. The experiments of osteoclast inhibition and restraining inflammation were used to detect the bioactivity of fusion protein. RESULTS: ①The complementary DNA encoding full length OPG protein was obtained. OPG-HSP 70 fusion gene obtained in this experiment was successfully inserted into pET-28a vector. OPG-HSP70 fusion protein was expressed when transformed into E.coli.BL21(DE3). ②SDS-PAGE indicated that the fusion protein was large expressed at the molecular weight of Mr22 000, but there were no band in the total lysate of bacteria harboring pET-28a-OPG-HSP70 without IPTG induction group. ③Western-blotting indicated that the OPG-HSP70 fusion protein could specifically react with anti-human OPG monoclonal antibodies. ④The osteoclast inhibition test demonstrated that the fusion protein could reduce the number of osteoclast, and had the ability to inhibit bone absorptions in vitro. ⑤The experiment of restraining inflammation showed that the fusion protein could significantly reduce the inflammation of delayed type hypersensitivity (DTH) mice, which explained that HSP70 in fusion protein had the inflammation inhibitory bioactivity. CONCLUSION: OPG-HSP70 fusion protein is expressed in E.coli.BL21(DE3),and function study in vitro illustrates the bioactivity of fusion protein.
ABSTRACT
AIM:This study was designed to use RNA interference technique to down-regulate the expression of survivin gene in human Burkitt's lymphoma cell line Daudi and to explore the effect on sensitivity of Daudi cells to adriamycin.METHODS:The survivin-shRNA expression vector was constructed and transfected into Daudi cells.Expression of survivin mRNA and protein were assessed by RT-PCR and Western blotting analysis,respectively.Apoptosis index of transfected Daudi cells was quantified by flow cytometry.The sensitivity of Daudi cells to adriamycin(ADR) before and after transfection was detected by MTT test.RESULTS:The mRNA and protein levels of survivin were down-regulated by 62.32% and 61.88%,respectively,compared to those in control-shRNA treated group and PBS treated group(P