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Zike病毒感染肾周管状上皮细胞后的持续性细胞反应相关研究
人阅读 发布时间：2018-04-25 11:23
Zika病毒是一种虫媒病毒，通过蚊子传播。该病毒和登革热病毒相似，感染后有类似的症状：发热、头痛、睑结膜充血、皮疹、肌肉疼痛和关节疼痛，临床表现相对温和，病程持续约4-7天。Zike病毒感染成人以后，能够引起胎儿畸形以及吉兰-巴利综合征，虽然对Zike病毒感染与小头畸形之间的关系已经有较多的研究，并且取得了不错的进展，但是针对该病毒病理机制方面的研究，尤其是寄生在人体内的Zika病毒，仍然知之甚少。既往的一些研究表明，被感染的患者尿液中的病毒存在时间比血液中存在的时间要长，这说明在人类的泌尿系统中很有可能存在着独立的病毒复制库。而且在临床实验中，人的泌尿系统中该病毒的寄生细胞很少被发现。上海复旦大学公共卫生临床中心的Chen Jian等研究证明Zika病毒不但能够感染免疫缺陷小鼠的肾周管状上皮细胞（RPTEpiCs），而且可以感染人的永生化及幼稚肾周管状上皮细胞（hRPTEpiCs）。此外，已有实验证明了Zika病毒感染小鼠肾脏以后会引起caspase-3介导的肾脏细胞凋亡。Zika病毒感染永生化及幼稚hRPTEpiCs也会导致类似的细胞反应。结合临床观察，研究者发现Zika病毒感染hRPTEpiCs后持续存在。RNA-Seq测序结果也显示被感染的hRPTEpiCs中存在较长的一段转录基因的变化，包括I型干扰素信号基因和抗病毒反应基因。这些都表明在人类的泌尿系统中，hRPTEpiCs是Zika病毒的潜在病毒复制库，为Zika病毒在患者尿液中长期存在提供了一种可能性的解释。
本实验主要通过免疫荧光、免疫组化、RNA-Seq测序分析等方法作为实验手段，主要实验结果如下：
Figure 1 Renal infection of Zika virus (ZIKV) in AG6 mice. (A) Components of the nephron. (B) Detection of ZIKV RNA in mouse kidneys. Left panel, in situ hybridization (ISH) using a ZIKV-specific probe. Blue and red arrows indicate renal glomerular and tubular cells, respectively. Hybridization signals of ZIKV RNA were developed by a chromogenic reaction and are observed as black particles. Right panel, hematoxylin and eosin (H&E) staining. Scale bar, 50 μm. (C) Immunofluorescence staining of caspase-3 in ZIKV-infected renal tissues of AG6 mice. Left panel, representative images. Scale bar, 200 μm. Right panel, statistical analyses of caspase-3-positive cells. **Significant difference between mock and infected cells (Po0.01; Student’s t-test).
Figure 2 ZIKV infection in the HK2 cell line. (A) Immunofluorescence staining of HK2 cells infected with SZ01 or MR766 ZIKV using an anti-ZIKV envelope protein antibody. Upper panel, representative images. Scale bar, 20 μm. Lower panel, quantification of infected cells. (B) Light microscopy images showing SZ01 ZIKV-induced CPEs. (C) Production of infectious SZ01 and MR766 ZIKV in HK2 cells. Cytopathogenic effects, CPEs.
Figure 3 ZIKV infection in primary hRPTEpiCs. (A) Immunofluorescence staining of primary hRPTEpiCs infected with SZ01 ZIKV at a MOI of 2.5 for 96 h. Scale bar, 30 μm. (B) Long-term persistence of SZ01 ZIKV in primary hRPTEpiCs. Viral copies in the supernatant of medium were determined by RT-qPCR using ZIKV standard samples. Values are shown as the mean±standard deviation (sd; n=5). (C) MR766 ZIKV-induced apoptosis in primary hRPTEpiCs. Upper panel, representative immunofluorescence images. Scale bar, 200 μm. Lower panel, quantification of ZIKV-positive and caspase-3-positive cells. ***Significant difference between mock and infected cells (Po0.001; Student’s t-test). (D) Production of infectious SZ01 and MR766 ZIKV particles in primary hRPTEpiCs.
Figure 4 RNA-Seq analysis of mock and ZIKV-infected primary hRPTEpiCs at 48 h post infection. (A) Volcano plot of genes differentially expressed in mock and ZIKV-infected hRPTEpiCs. (B) Genes with differential expression between mock and infected hRPTEpiCs were subjected to gene ontology (GO) analyses. The top 10 most significantly upregulated and downregulated biological processes are shown in red and blue, respectively.
其中，RNA的ISH和HE染色、组织的IF和IHC染色结果均由奥地利TG公司的Tissue FAXS200系统采图并分析，通过以上实验，最终得出以下结论：1.对I型和II型IFN缺陷的小鼠接种Zika病毒会导致其肾小管及肾小球中病毒大量复制增殖，且肾小球中的病毒量更高。2.Zika病毒在幼稚hRPTEpiCs中长期存在。3.Zika病毒的2种菌株（SZ01和MR766）均能在永生化及幼稚hRPTEpiCs中复制增殖。综上，肾脏的hRPTEpiCs很可能是Zika病毒持续复制存在的病毒库，除此以外，本研究还发现了美洲和亚洲的Zika病毒菌株具有不同的感染方式。其结果可以为CDC(疾病防治中心)提供理论实验数据支持
本实验主要通过免疫荧光、免疫组化、RNA-Seq测序分析等方法作为实验手段，主要实验结果如下：
Figure 1 Renal infection of Zika virus (ZIKV) in AG6 mice. (A) Components of the nephron. (B) Detection of ZIKV RNA in mouse kidneys. Left panel, in situ hybridization (ISH) using a ZIKV-specific probe. Blue and red arrows indicate renal glomerular and tubular cells, respectively. Hybridization signals of ZIKV RNA were developed by a chromogenic reaction and are observed as black particles. Right panel, hematoxylin and eosin (H&E) staining. Scale bar, 50 μm. (C) Immunofluorescence staining of caspase-3 in ZIKV-infected renal tissues of AG6 mice. Left panel, representative images. Scale bar, 200 μm. Right panel, statistical analyses of caspase-3-positive cells. **Significant difference between mock and infected cells (Po0.01; Student’s t-test).
Figure 2 ZIKV infection in the HK2 cell line. (A) Immunofluorescence staining of HK2 cells infected with SZ01 or MR766 ZIKV using an anti-ZIKV envelope protein antibody. Upper panel, representative images. Scale bar, 20 μm. Lower panel, quantification of infected cells. (B) Light microscopy images showing SZ01 ZIKV-induced CPEs. (C) Production of infectious SZ01 and MR766 ZIKV in HK2 cells. Cytopathogenic effects, CPEs.
Figure 3 ZIKV infection in primary hRPTEpiCs. (A) Immunofluorescence staining of primary hRPTEpiCs infected with SZ01 ZIKV at a MOI of 2.5 for 96 h. Scale bar, 30 μm. (B) Long-term persistence of SZ01 ZIKV in primary hRPTEpiCs. Viral copies in the supernatant of medium were determined by RT-qPCR using ZIKV standard samples. Values are shown as the mean±standard deviation (sd; n=5). (C) MR766 ZIKV-induced apoptosis in primary hRPTEpiCs. Upper panel, representative immunofluorescence images. Scale bar, 200 μm. Lower panel, quantification of ZIKV-positive and caspase-3-positive cells. ***Significant difference between mock and infected cells (Po0.001; Student’s t-test). (D) Production of infectious SZ01 and MR766 ZIKV particles in primary hRPTEpiCs.
Figure 4 RNA-Seq analysis of mock and ZIKV-infected primary hRPTEpiCs at 48 h post infection. (A) Volcano plot of genes differentially expressed in mock and ZIKV-infected hRPTEpiCs. (B) Genes with differential expression between mock and infected hRPTEpiCs were subjected to gene ontology (GO) analyses. The top 10 most significantly upregulated and downregulated biological processes are shown in red and blue, respectively.
其中，RNA的ISH和HE染色、组织的IF和IHC染色结果均由奥地利TG公司的Tissue FAXS200系统采图并分析，通过以上实验，最终得出以下结论：1.对I型和II型IFN缺陷的小鼠接种Zika病毒会导致其肾小管及肾小球中病毒大量复制增殖，且肾小球中的病毒量更高。2.Zika病毒在幼稚hRPTEpiCs中长期存在。3.Zika病毒的2种菌株（SZ01和MR766）均能在永生化及幼稚hRPTEpiCs中复制增殖。综上，肾脏的hRPTEpiCs很可能是Zika病毒持续复制存在的病毒库，除此以外，本研究还发现了美洲和亚洲的Zika病毒菌株具有不同的感染方式。其结果可以为CDC(疾病防治中心)提供理论实验数据支持
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