白细胞介素-10基因转移抑制滑膜细胞产生炎性细胞因子

Effects of interleukin 10 gene transfer on secretion of inflammatory cytokines in rat synoviocytes induced by lipopolysaccharide in vitro

目的 通过建立人白细胞介素 (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)诱导的大鼠关节滑膜细胞 (RSC)中炎性细胞因子的产生及其基因表达的影响。方法 将构建的重组逆转录病毒载体pLX(IL 10 )SN经包装、筛选、病毒滴度测定 ,选滴度最高的克隆 (6×10 6 CFU/ml)感染RSC :(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RSC肿瘤坏死因子 α(TNF α)的mRNA表达的影响 ,以ELISA测定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RSC产生IL 1β、TNF α。 结论 外源性IL 10基因可以转移到RSC并稳定表达 ,它能抑制RSC炎性效应中细胞因子的产生及其基因表达 ,为今后研究通过IL 10基因转移治疗免疫性疾病的关节损伤提供了重要的理论和实验依据。

Objective To investigate the effects of interleukin 10(IL 10) gene transfer on secretion of inflammatory cytokines in rat synoviocytes (RSC) induced by lipopolysaccharide (LPS) in vitro. Methods Constructed retroviral vector pLX (IL 10) SN was introduced into packaging cell line PA317. PA317 was selected by G418 and then retrovirus produce cells titre was determined by NIH3T3 for identification of producer clones making high titre virus. Through the highest titre produce cells clone (6×10 5 CFU/ml)exogenous gene IL 10 was transferred into RSC. 72 hours after gene transfer, DNA and RNA were prepared from rat transfected RSC for the polymerase chain reaction (PCR) and the reverse transcription polymerase chain reaction (RT PCR) ;expression of IL 10 in transfected RSC was checked by ELISA. Effects of IL 10 gene transfer on secretion of inflammatory cytokines induced by LPS in rat RSC cells were detected by RT PCR and ELISA in vitro.Results Exogenous IL 10 gene was integrated into the chromosal DNA of the transfected RSC cells. RT PCR and ELISA showed that exogenous IL 10 gene was expressed in the transfected RSC 48 hours after transfection and the expression lasted for over 28 days. Exogenous IL 10 gene transfer can inhibit secretion of inflammatory cytokines, IL 1β and TNF α in the transfected RSC at mRNA and protein level induced by LPS. Conclusion Exogenous IL 10 gene transfer can inhibit secretion of inflammatory cytokines in RSC, it is suggested that exogenous IL 10 gene transferring into RSC can be used in the treatment of joint injury caused by immunological diseases.