IFN-γ诱导鹦鹉热嗜衣原体持续性感染及相关基因的表达

发布时间：2018-01-17 02:06

  本文关键词：IFN-γ诱导鹦鹉热嗜衣原体持续性感染及相关基因的表达　出处：《南华大学》2014年硕士论文　论文类型：学位论文

  更多相关文章： 鹦鹉热嗜衣原体 持续性感染 人重组干扰素-γ 基因表达

【摘要】：目的：建立IFN-γ诱导的鹦鹉热嗜衣原体（Chlamydophila psittaci，Cps）持续性感染的细胞模型，检测与Cps6BC持续性感染相关基因的表达变化。 方法： 体外培养HeLa单层细胞感染Cps6BC标准菌株，分别用5、15、25、35、45、55ng/mL的人重组干扰素-γ（recombinant hunan IFN-γ，rhIFN-γ）诱导Cps6BC持续性感染30h和用35ng/mL rhIFN-γ诱导12、24、36、48、60h，利用间接免疫荧光染色法（indirect immunofluorescence assay，IFA）检测Cps6BC感染能力的变化。用IFA和透射电镜检测rhIFN-γ诱导Cps6BC持续性感染后包涵体形态的变化。rhIFN-γ诱导Cps6BC持续性感染后，去除诱导剂，补充足量的L-色氨酸，利用IFA和透射电镜检测Cps6BC感染能力和包涵体形态的恢复情况。用Realtime-PCR检测Cps6BC持续性感染状态下目的基因的mRNA表达水平的变化以及Western blot检测CPSIT_0844、CPSIT_0594蛋白表达水平的变化。 结果： 用不同浓度的rhIFN-γ诱导Cps6BC标准株持续性感染30h后，Cps6BC的感染能力均降低，且呈明显的剂量依赖关系。35ng/mL rhIFN-γ诱导不同时间后，Cps6BC的感染能力也出现不同程度的下降，尤其是36、48、60h后感染能力显著降低。IFA和透射电镜观察显示，rhIFN-γ诱导后，Cps6BC包涵体体积变小，结构疏松，并可见异形网状体，成熟子代原体数目减少，呈现典型的持续性感染特征。去除诱导剂，，补充足量的L-色氨酸后，Cps6BC的感染能力及包涵体形态均可恢复到急性感染状态。 rhIFN-γ诱导Cps6BC持续性感染后，CPSIT_0844、CPSIT_0846、CPSIT_0959、CPSIT_0594、CPSIT_0310、CPSIT_0870、CPSIT_0057、CPSIT_0208在mRNA水平的表达均发生了一定的改变。核糖体蛋白CPSIT_0870在2～36h显著上调，而半胱氨酸脱硫酶CPSIT_0959在36h、48h显著下调。Ⅲ型分泌系统效应蛋白CPSIT_0594、CPSIT_0844均在2～48h上调，且分别在36h和48h显著上调，而CPSIT_0846在2～24h显著上调，36h后显著下调。膜蛋白CPSIT_0057、CPSIT_0310在2～48h上调，而CPSIT_0208在48h后下调。Westernblot结果显示，rhIFN-γ诱导Cps6BC持续性感染后，CPSIT_0844、CPSIT_0594蛋白表达的改变与其mRNA水平的表达变化一致。 结论： 1.建立了rhIFN-γ诱导的Cps6BC持续性感染细胞模型； 2. CPSIT_0844、 CPSIT_0846、 CPSIT_0959、 CPSIT_0594、 CPSIT_0310、CPSIT_0870、CPSIT_0057、CPSIT_0208在rhIFN-γ诱导Cps6BC持续性感染过程中的表达出现一定的变化，这些基因可能与衣原体持续性感染有关。
[Abstract]:Objective: to establish a cell model of persistent infection of Chlamydophila psittacius (Cpss) induced by IFN- 纬. The expression changes of genes associated with persistent infection of Cps6BC were detected. Methods: In vitro, HeLa monolayer cells were infected with Cps6BC standard strain. Recombinant hunan IFN- 纬 was obtained with 55 ng / mL of recombinant human interferon- 纬. RhIFN- 纬) induced Cps6BC persistent infection for 30 h and 35 ng / mL rhIFN- 纬 for 48 h. Indirect immunofluorescence staining was used to detect indirect immunofluorescence assay. IFA). To detect the change of Cps6BC infection ability. To detect the changes of inclusion body morphology after Cps6BC persistent infection induced by rhIFN- 纬 by IFA and transmission electron microscopy. RhIFN- 纬 induced Cps6. After persistent BC infection. Remove the inducer and supplement a sufficient amount of L-tryptophan. IFA and transmission electron microscopy (TEM) were used to detect the ability of Cps6BC infection and the shape recovery of inclusion body. Realtime-PCR was used to detect the mR of the target gene under the condition of persistent Cps6BC infection. Changes of na expression level and Western. CPSIT_0844 was detected by blot. Changes of CPSIT_0594 protein expression level. Results: The ability of Cps6BC infection of Cps6BC standard strain was decreased after 30 hours of persistent infection induced by different concentrations of rhIFN- 纬. In a dose-dependent manner, the infection ability of Cps6BC was decreased in different time after rhIFN- 纬 was induced by 35 ng / mL rhIFN- 纬, especially 36ng / mL rhIFN- 纬. After 60 hours, the ability of infection decreased significantly. IFA and TEM observation showed that the volume of Cps6BC inclusion body decreased after rhIFN- 纬 induction, the structure was loose, and the heteromorphic reticular bodies could be seen. The number of mature progenies decreased, showing a typical persistent infection. After removing the inducer and replenishing sufficient amounts of L-tryptophan. The infection ability and inclusion body morphology of Cps6BC can be restored to acute infection state. After Cps6BC persistent infection induced by rhIFN- 纬, CPSITT: 0844 / CPSITT: 0846 / CPSIT0959 / CPSIT0594. CPSIT_0310,CPSIT_0870,CPSIT_0057. The expression of CPSIT_0208 in mRNA was changed to some extent, and ribosomal protein CPSIT_0870 was upregulated significantly at 2 ~ 36 h. The cysteine desulfurization enzyme CPSIT_0959 was significantly down-regulated at 36h and 48h. Type 鈪

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