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目的:通过建立白细胞介素10(IL-10)的重组逆转录病毒载体基因转移系统,观察IL-10对脂多糖(LPS)诱导的大鼠肾小球系膜细胞(glomerularmesengialcel,GMC)中细胞因子的产生及其基因表达的影响。方法:通过构建的重组逆转录病毒载体pLX(IL-10)SN将外源基因IL-10转移至大鼠GMC:(1)应用聚合酶链反应(PCR),反转录聚合酶链反应(RT-PCR)和ELISA检测IL-10基因的整合和表达;(2)以RT-PCR观察IL-10基因转移对LPS诱导的GMC肿瘤坏死因子-α(TNF-α)的mRNA表达的影响,以ELISA测定白细胞介素-1β(IL-1β)和TNF-α的蛋白质表达。结果:外源性IL-10基因已整合到靶细胞染色体DNA并有效地表达,它能抑制LPS诱生GMC过度产生IL-1β,TNF-α。结论:外源性IL-10基因可以转移到GMC并稳定表达,它能抑制GMC炎症效应中细胞因子的产生及其基因表达。
OBJECTIVE: To investigate the effect of IL-10 on glomerular mesangial cells (GMC) induced by lipopolysaccharide (LPS) in rats by establishing recombinant retroviral vector gene transfer system of interleukin 10 (IL-10) The production of factors and their effects on gene expression. Methods: The exogenous gene IL-10 was transferred to rat GMC via the constructed retroviral vector pLX (IL-10) SN. (1) The expression of IL-10 was detected by polymerase chain reaction (2) The effect of IL-10 gene transfer on mRNA expression of tumor necrosis factor-α (TNF-α) induced by LPS in GMCs was observed by RT-PCR, The protein expression of interleukin-1β (IL-1β) and TNF-α was measured by ELISA. Results: The exogenous IL-10 gene has been integrated into the chromosomal DNA of target cells and effectively expressed. It can inhibit LPS-induced GMC overproduction of IL-1β and TNF-α. CONCLUSION: Exogenous IL-10 gene can be transferred to GMC and stably expressed. It can inhibit the production of cytokines and its gene expression in GMC inflammatory effect.