【作者】 胡静；

【导师】 姚云清；

【作者基本信息】 重庆医科大学 ， 内科学， 2008， 硕士

【摘要】 目的在体外模型研究肝细胞能否被诱导产生干扰素等细胞因子,探讨肝细胞在乙型肝炎病毒清除过程中可能发挥的作用。方法选用聚肌胞(polyI:C)、植物血凝素(PHA)与结核菌素纯蛋白衍生物(PPD)为细胞因子诱生剂,以诱生剂最大无毒浓度刺激HepG2细胞及2.2.15细胞,分别以无细胞因子诱生剂作用的HepG2细胞及2.2.15细胞为对照组,用双抗体夹心ELlSA法检测细胞上清液及细胞裂解液中24小时、48小时IFN-α、TNF-α与IFN-γ的含量,并检测了2.2.15细胞被细胞因子诱生剂刺激后24小时、48小时细胞上清液及细胞裂解液中的HBV病毒标志物sAg、eAg的含量。结果1. PPD、polyI:C、PHA以最大无毒浓度分别是10μg/ml、20μg/ml、30μg/ml;刺激HepG2后24hr,48 hr时点HepG2培养上清液及裂解液中均能产生IFN-α,与对照组比较有显著性差异,其含量48hr时点大于24hr时点,胞内、胞外含量有显著性差异; PPD、polyI:C与PHA刺激HepG2后,细胞上清液及裂解液均检测出TNF-α,但与对照组比较无显著性差异,胞内胞外含量无显著性差异,不同时点间亦无显著性差异; PPD、PHA、PoLyI:C刺激后上清液中均检出IFN-γ,但与对照组比较无显著性差异,PPD、PHA刺激后细胞培养裂解液中检测出IFN-γ,与对照组比较有显著性差异,胞内、胞外含量有显著性差异;PoLyI:C刺激后培养细胞裂解液中检出IFN-γ,但与对照组比较无显著性差异,不同时点间无显著性差异;2. PPD、polyI:C、PHA以最大无毒浓度,刺激2.2.15细胞后,细胞裂解液、上清液均检测出IFN-α,与对照组比较有显著性差异,胞内含量比胞外含量多,有显著性差异,不同时点间无显著性差异;细胞裂解液、上清液中均检出IFN-γ、TFN-α;但与对照组比较无显著性差异,胞内含量与胞外含量比,无显著性差异,不同时点间无显著性差异;3. PPD、polyI:C、PHA以最大无毒浓度,刺激2.2.15细胞,细胞裂解液、上清液清除sAg、eAg的效果与对照组相比无显著性差异。结论1. PPD、polyI:C、PHA能够刺激HepG2细胞产生IFN-α并分泌至胞外,且IFN-α的量与诱生剂作用时间成正比。PPD、PHA能刺激HepG2细胞产生IFN-γ,但未能或未及分泌至胞外,IFN-γ的量与诱生剂作用时间长短无关。polyI:C刺激HepG2细胞产生IFN-γ作用不明显;另外,三种诱生剂刺激HepG2产生TNF-α作用也不明显。2. PPD、polyI:C、PHA,刺激2.2.15细胞后,能产生IFN-α,并分泌至胞外,但IFN-α的量与诱生剂作用时间长短无关;三种诱生剂刺激2.2.15细胞产生IFN-γ、TNF-α作用均不明显。3. PPD、polyI:C、PHA,刺激2.2.15细胞,清除sAg、eAg的效果不明显。更多还原

【Abstract】 ObjectiveTo investgate if cytokines can be Induced by cytokine Inducer in hepatocytes in vitro.MethodsHepG2 and 2.2.15 Cells were treated with maximal tolerance dosage of polyI:C ,PHA, and PPD. After 24h and 48h ,the contents of IFN-α、TNF-αand IFN-γboth in the supernatant and cell were determined by ELISA, and the contents of HBV virus marker sAg、eAg in 2.2.15 Cells in the supernatant and cell were also determined by ELISA.Results1、IFN-αwere detected in the supernatant and lysate in 24h and 48h,IFN-αlevels were significantly higher than that of control groups,IFN-αlevels in 24h were significantly higher than in 48h,IFN-αlevels in intra-cell and ecto-cell were significant difference。HepG2 were treated with PPD、polyI:C and PHA, TNF-αwere detected in the supernatant and lysate, The difference of TNF-αlevels were not significant with control groups,The difference of IFN-αlevels in intra-cell and ecto-cell were not significant. The difference of IFN-αlevels in 24h and 48h were not significant. HepG2 were treated with PPD、polyI:C and PHA , IFN-γwere detected in the supernatant ,The difference of IFN-γlevels were not significant with control groups,HepG2 were treated with PPD and PHA,IFN-γwere detected in the lysate,The difference of IFN-γlevels were not significant with control groups,The difference of IFN-γlevels in 24h and 48h were not significant.2. 2.2.15cells were treated with PPD、polyI:C and PHA with the maximal tolerance dosage,IFN-αwere detected in the supernatant and lysate,The difference of IFN-αlevels were significant with control groups,The content of IFN-αin intra-cell were higher than in ecto-cell,the difference was significant. The difference in 24h and 48h was not significant .IFN-γ、TFN-αwere detected in the supernatant and lysate,The difference was not significant with control groups,The difference in ecto-cell and intra-cell was not significant . The difference in 24h and 48hwas not significant.3.2.2.15cells were treated with PPD、polyI:C and PHA with the maximal tolerance dosage,the effect of cleaning HbsAg and HBeAg was not significant with control groups.Conclusion1. IFN-αcan be Induced by PPD、polyI:C and PHA in HepG2 cell and then be secreted out;The content of IFN-αand action time were direct ratio. IFN-γcan be induced by PPD and PHA and then were not secreted out; The content of IFN-γand action time were independence. IFN-γcan be induced by polyI:C ,but the effect was not obviously. TNF-αcan be Induced byPPD、polyI:C and PHA but the effect was not obviously.2. IFN-αcan be Induced by PPD、polyI:C and PHA in2.2.15 cell and then be secreted out ; The content of IFN-αand action time were independence. IFN-γ、TNF-αcan be induced by PPD、polyI:C and PHA but the effect was not obviously3. 2.2.15 cells were treated with PPD、polyI:C and PHA,the effect of cleaning sAg and eAg was not significant.更多还原