人白细胞介素10在支气管上皮细胞株H292中的表达及生物活性鉴定

Expression and biological activity of human interleukin-10 in human bronchial epithelial cell line H292

目的 探讨在支气管上皮细胞H2 92株 (H2 92 )中表达人白细胞介素 10 (hIL 10 ) ,并鉴定其生物活性。方法 将载有hIL 10基因的pcDSRα质粒 (pcDSRα IL 10 ) ,经脂质体Fuge 6介导转染H2 92株。应用逆转录聚合酶反应 (RT)及聚合酶链式反应 (PCR) ,检测转染后 48hH2 92株中IL 10mRNA的表达 ;用ELISA法测定hIL 10在不同时段H2 92培养上清液中的表达的量 ;分析hIL 10对LPS诱导T淋巴细胞IFN γ分泌的抑制作用。结果 RT PCR检测显示转染后的H2 92中有hIL 10mRNA表达 ,未转染的H2 92则未能检测到其mRNA表达。ELISA检测转染后H2 92上清液中 2 4h已有hIL 10表达 ,48h达高峰 ,以后逐渐下降 ,未转染组上清液则无IL 10表达。表达的hIL 10与标准hIL 10对LPS诱导的IFN γ分泌抑制作用差别不显著 (P >0 .0 5 ) ,均显著强于未转染组 (P <0 .0 1)。结论 重组的hIL 10pcDSRα质粒可成功在H2 92中表达hIL 10 ,表达的hIL 10有理想的生物活性 ,可用于体内转基因研究。

Objective To explore the expression and biological epithelial activity of human interleukn 10 (hIL 10) in human bronchial epithelial cell line H292. Methods Plasmid pcDSR α containing hIL 10 gene (pcPSR α IL 10) was transfected into H292 with lipofectin Fuge 6. The expression of IL 10 mRNA in H292 cells was identified with reverse transcription polymerase chain reaction (RT PCR) 48 h after the transfection and its quantity of expression was detected with ELISA in the supernatant of H292 culture medium at different time points. Inhibitory effect of expressed IL 10 on the secretion of IFN γ by LPS induced T cells was compared with that of expressed standard hIL 10 (shIL 10) of the supernatant of untransfected H292. Results hIL 10 mRNA was detected by PCR in the transfected H292 but not in untransfected H292. With ELISA, the expression of hIL 10 in the supernatant of transfected H292 began at 24 h, reached to the peak at 48 h, and then slowly went down, but no was in the supernatant of the untransfected H292. No difference of inhibitory effect on IFN γ secretion was found between hIL 10 and shIL 10, which were more effective than the supernatant of untransfected H292 (P<0.01). Conclusion hIL 10 can be successfully expressed in H292 by transferring with pcDSR γ hIL 10 with same biological activity as shIL 10 and can be used in transgenic application.