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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 investigate the expression of human interleukin - 10 (hIL 10) in human bronchial epithelial cell line H2 92 (H2 92) and its biological activity. Methods The pcDSRα plasmid containing hIL 10 gene (pcDSRα IL 10) was transfected into H2 92 cells via liposome Fuge 6. The expression of IL-10 mRNA was detected by reverse transcriptase-polymerase chain reaction (RT) and polymerase chain reaction (PCR) in 92 strains of H22 at 48 h after transfection. The expression of hIL 10 in H2 92 culture supernatant was detected by ELISA ; The inhibitory effect of hIL 10 on LPS-induced IFN [gamma] secretion by T lymphocytes was analyzed. Results RT-PCR showed that hIL 10 mRNA was expressed in H2-transfected cells, while untransfected H2 92 failed to detect its mRNA expression. The expression of hIL 10 in H 2 92 supernatants after 24 h of transfection was detected by ELISA and peaked at 48 h after transfection, and then decreased gradually after transfection. The supernatant of untransfected cells showed no expression of IL 10. The inhibitory effects of hIL 10 and hIL 10 on LPS-induced IFNγ secretion were not significantly different (P> 0.05), which were significantly higher than those of untransfected cells (P <0.01). Conclusion The recombinant hIL 10pcDSRα plasmid can successfully express hIL 10 in H2 92. The expressed hIL 10 has ideal biological activity and can be used in transgenic study in vivo.