| Proinflammatory cytokines appear to be of special importance in the creation of neuropathic pain. Investigators have addressed the potential therapeutic role of regulatory cytokines and several studies have shown that IL-10 protein administered before or at the time of onset of the symptoms of neuropathic pain resulted in significant amelioration of the pain. However, IL-10 is rapidly cleared from the circulation after in vivo administration, limiting its availability to target tissues. To overcome the problem of limited bioavailability of IL-10, we used gene transfer technology to achieve overproduction of IL-10 in vivo in rats CCI models. The experiment is designed to carry out at three steps:Part one: Preparation of Eukaryotic Expression Plasmid Of IL-10Objective: The purpose of this study is to construct the eukaryotic expression plasmid of IL~10. Methods: IL-10cDNA with signal peptide gene was obtained from pUC-19-IL-10 by PCR (twice) through designing a 5' primer containing the signal peptide gene of human monocyte chemoattractant protein I (MCP I). Then, it was cloned to pUC-T vector via T4DNA ligase enzymes. IL-10-cDNA fragment was obtained through cleaving pUC-T-IL-10cDNA by reconstriction enzymes. After that , IL IOcDNA was cloned to pcDNA3.1 which is also cleaved by reconstriction enzymess.pcDNA3. 1-IL-10 was constructed and confirmed by restriction enzyme mapping and sequencing. Results: Restriction enzyme mapping shows that one fragment about 560bp was inserted into pcDNA3. 1. Sequencing shows that sequence was consistent with IL-10 gene, and contained MCP I gene. In addition, the gene contained two terminal position of restriction enzymes Hind III and EcoR I , initiator condon ( ATG ) and terminator condon (TAA). Conclusion: We succesfu] ly construct eukaryotic expression plasmid of IL-10 which contains signal peptide gene of MCP I .Part two: The Transfection and expression of IL-10gene in extra cultural celles.Objective:To investigate the biological activities of the prepared Eukaryotic expression plasmid of pcDNA3. 1-IL-10 by transgenic expression researches in vitro in several cultured cell lines. Methods: Firstly, we research the ability of pcDNA3. l-IL-10 to transfer cells of ECV304> RAW264. 7 and astroglia. These cells were grown in DMEM supplemented with 10% fetal bovine serum in 6-well tissue culture plates.When cells were at about 60% conflence, they were co-transfected with lug/well pcDNA3. l-IL-10 or pcDNA3. 1 plasmid respectively mediated by SA. The cells and cell culture supernatant was collected at the second day to detect IL-10 and IL-10mRNA. Secondly, we research the express course of pcDNA3. l-IL-10. Astroglial cells in 6-well tissue culture plates at about 60% conflence were cotransfected lug/well pcDNA3. l-IL-10 or pcDNA3.1 plasmid respectively mediated by SA. The cell culture supernatant was collected daily for 10 days after transfection. Contents of IL-10 were analyzed by ELISA kit. Thirdly, we research the function of pcDNA3. l-IL-10 in inhibiting proinflammatory cytokines. Astroglial cells were grown in DMEM supplemented with 10% fetal bovine serum and LPSlOmg/L. Astroglial cells in 6-well tissue culture plates at about 60% conflence were cotransfected with lug/well pcDNA3. l-IL-10 or pcDNA3. 1 plasmid respectively mediated by SA. The cell culture supernatant was collected daily for 3 days after transfaction.Contents of IL-10 and TNFa were analyzed by ELISA kit and cell lysis of L929. Results: (1) IL-10 could be detected at the second day in all the supernatant of the three kinds of cells after pcDNA3. l-IL-10 transfection, whereas there was no IL-10 production in the culture supernatant after pcDNA3. 1 transfection. (2) IL-10 could be detected from day 1 to day 8 in the culture supernatant of astroglia after pcDNA3. l-IL-10 transfection, which reached peak at day 2, whereas there was no IL-10 production in the culture supernatant after pcDNA3. 1 transfection. (3) LPS significantly induce astroglia to release TNF a . pcDNA3. l-IL-10 markedlyattenuated release o...
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