|
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 |
| |
李锦华 唐皓 廖晓星 余学清 张仕光
【摘 要】 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，hIL-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pLX(hIL-10)SN，经PA317细胞包装，G418筛选，NIH3T3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(PCR)，反转录聚合酶链反应(RT-PCR)和ELISA检测hIL-10基因的整合和表达。结果 外源性hIL-10基因已整合到靶细胞染色体DNA并有效地表达。ELISA检测显示hIL-10基因在pLX(hIL-10)SN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hIL-10基因可以转移到大鼠关节滑膜细胞并稳定表达，为研究通过hIL-10基因转移治疗免疫性疾病的关节损害提供了物质保证。
【关键词】 基因转移 逆转录病毒载体 人白细胞介素10基因 大鼠关节滑膜细胞
Expression of human interleukin 10 in rat synoviocytes by retroviral vector
LI Jinhua,TANG Hao,LIAO Xiaoxing,et al.
(First Affiliated Hospital,Sun Yat-sen University of Medical Sciences,Guangzhou 510080)
【Abstract】 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 (6×108 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.10－6cells.24h－1.Conclusion Exogenous hIL-10 gene can be[1] [2] 下一页
上一个医学论文： 血清阴性脊柱关节病与致密性髂骨炎的鉴别
下一个医学论文： 单相酶扩散法测定SLE患者血清DNA酶的浓度
|
|
|
|
|
|
|
您现在的位置： 绿色健康网 >> 医学论文 >> 内科论文 >> 正文