在大鼠关节滑膜细胞表达人白细胞介素10的基因转移系统的建立

Expression of human interleukin 10 in rat synoviocytes by retroviral vector

目的 建立一个在大鼠关节滑膜细胞表达人白细胞介素１０（ｈｕｍａｎ ｉｎｔｅｒｌｅｕｋｉｎ １０ ，ｈ Ｉ Ｌ１０） 的重组逆转录病毒载体基因转移系统，为下一步的研究工作做准备。方法 构建表达人白细胞介素１０ 的逆转录病毒重组体ｐ Ｌ Ｘ（ｈ Ｉ Ｌ１０） Ｓ Ｎ，经 Ｐ Ａ３１７ 细胞包装， Ｇ４１８ 筛选， Ｎ Ｉ Ｈ３ Ｔ３ 细胞进行病毒滴度测定，选滴度最高的克隆（６ ×１０８ 集落形成单位／ Ｌ） 作为感染大鼠关节滑膜细胞的感染细胞；用重组逆转录病毒感染大鼠关节滑膜细胞，应用聚合酶链反应（ Ｐ Ｃ Ｒ） ，反转录聚合酶链反应（ Ｒ Ｔ Ｐ Ｃ Ｒ） 和 Ｅ Ｌ Ｉ Ｓ Ａ 检测ｈ Ｉ Ｌ１０ 基因的整合和表达。结果 外源性ｈ Ｉ Ｌ１０ 基因已整合到靶细胞染色体 Ｄ Ｎ Ａ 并有效地表达。 Ｅ Ｌ Ｉ Ｓ Ａ 检测显示ｈ Ｉ Ｌ１０ 基因在ｐ Ｌ Ｘ（ｈ Ｉ Ｌ１０） Ｓ Ｎ 转染大鼠关节滑膜细胞４８ 小时后开始表达，达７２０ ｎｇ·１０ － ６ｃｅｌｌｓ·２４ｈ － １ ，高峰在第３ 天，为１ ９８２ ｎｇ·１０ － ６ｃｅｌｌｓ·２４ｈ － １ 。第７ 、１４ 、２８天分别为１２ ７６１ 、１ ０５４ 、９４２ ｎｇ·１０ － ６ｃｅｌｌｓ·２４ｈ － １ 。结论 外源性ｈ Ｉ ?

Objective To establish a model expressing human interleukin 10 in rat synoviocytes (RSC) by retrovial vector for further study.Methods The constructed recombinant retroviral vector pLX (hIL 10) SN encoding human interleukin 10 gene was introduced into packaging cell line PA317,then PA317 was selected by G418.Retrovirus producer cells titre was determined by NIH3T3 for identification of producer clones making high titre virus.Exogenous gene hIL 10 was transferred into RSC by the highest titre proudcer cells clone (6×10 8 CFU/L).After gene transfer 72 hours,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 hIL 10 in transfected RSC was checked by ELISA.Results Exogenous hIL 10 gene has been integrated into the chromosal DNA of the transfected RSC.RT PCR and ELISA showed that exogenous hIL 10 gene was expressed in the transfected RSC after transfection 48 hours and the highest expression level was 1 982 ng·10 -6 cells·24h -1 .Conclusion Exogenous hIL 10 gene can be transferred into RSC and be expressed stably.