重组牛干扰素τ抗马传染性贫血病毒效果的研究
摘要
干扰素(interferon, IFN)是由动物细胞产生并释放的一种具有高度生物活性、多种生物学功能的蛋白质,特别是在抵御感染、抗细胞增殖以及免疫调节方面具有重要的作用。目前已经有IFN类生物制品在临床上应用。IFN-τ是一种独特的IFN,属于I类IFN。最初发现IFN-τ是反刍动物妊娠过程中比较重要的细胞因子,除与动物维持妊娠有关外,近年来也证实了它的免疫学功能,特别是有显著抗慢病毒增殖效果。与其他IFN相比,IFN-τ具有抗病毒效果好、细胞毒性低和副作用小甚至无毒副作用等特点。IFN-τ在临床应用上的潜力已经引起科研工作者的注意。近年来,越来越多的实验室开展了针对IFN-τ抗病毒效果和免疫调节功能的研究,并且已经开始进行IFN-τ跨种间抗病毒效果及免疫功能的研究。值得注意的是,鉴于IFN-τ有显著抗慢病毒增殖的特点,已经开始设想用IFN-τ对人免疫缺陷病毒(human immunodeficiency virus, HIV )感染者进行治疗。
马传染性贫血病毒(Equine Infectious Anemia Virus,EIAV)作为逆转录病毒亚科慢病毒属中基因结构最简单的成员,是目前公认最理想的慢病毒研究模型。由于EIAV和HIV在病毒形态、基因组结构、细胞嗜性、免疫机制及病毒与宿主相互作用等方面都十分相似,深入研究EIAV感染与免疫机制,能够为HIV的相关研究提供借鉴,因此EIAV的感染与免疫机制的研究已受到国内外学者的高度重视。本研究选择EIAV作为靶病毒,尝试研究和评价重组牛干扰素τ(recombinant bovine interferon tau, rBoIFN-τ)的抗慢病毒效果,并与重组马干扰素(γrecombinant equine interferon gamma, rEIFN-γ)在抗病毒增殖、干扰素刺激基因(Interferon Stimulate Gene, ISG)节点因子介导水平等方面进行比较,分析IFN-τ与IFN-γ抗EIAV病毒的效果上的差异,进而揭示两者的免疫调节机理。本研究采用VSV-MDBK结晶紫染色法分别对rEIFN-γ和rBoIFN-τ进行滴定,并采用Cell Counting Kit-8对两种IFN在ED-EIAV上的细胞毒性进行检测和比较,然后在不产生细胞毒性的条件下,应用逆转录酶活性检测法和real-Time PCR检测和评价两种IFN在ED-EIAV细胞上的抗病毒效果。在上述研究的基础上,用Real-Time PCR检测经过两种IFN处理后不同时间点的ED-EIAV细胞中EIF2AK2、OAS1和Mx protein三种因子基因表达情况,并通过结合比较两种IFN处理后ISG基因的表达情况,探讨两种IFN抗病毒作用机理。
本研究证明,尽管其抗病毒效果低于rEIFN-γ,rBoIFN-τ在ED-EIAV细胞上确实表现出了一定的抗EIAV病毒的效果,大约10倍量的rBoIFN-τ抗EIAV效果与rEIFN-γ相当,但是其细胞毒性明显低于rEIFN-γ。另外,同rEIFN-γ一样,rBoIFN-τ也能刺激三种节点ISG基因的表达,而且表达上调的趋势也具有相似性,但rBoIFN-τ诱导这三种因子表达的能力显著低于rEIFN-γ,特别是在诱导OAS1基因和Mx protein基因表达的能力上两者差异更为显著。由此推测,IFN-τ的抗病毒机制可能与IFN-γ以及其他IFN的抗病毒机制不同,除了EIF2AK2、OAS1和Mx protein三种因子为代表的通路之外,很可能还存在其它的抗病毒通路,并且这一机制上的差异也可能与IFN-τ细胞毒性低有关。
Interferons are a type of protein produced by animal cells, which have high biological activity and a series of biological functions, especially in infection defense, anti-cell proliferation and immunoregulation. Till now, in-depth studies have been carried out to dissect the biological functions of interferon gamma and it has been applied in clinical cases as a biomedicine. Interferon tau belongs to the type I interferons, originally found as an important cytokine involved in maintaining the pregnancy of ruminant. Recently, efforts have been exerted to explore its roles in immunology, especially in the anti-multiplication of the lentivirus. Compared with other interferons, tau possesses some unique qualities such as high effectiveness of anti-virus, low toxicity to cells and minor or even no side-effects. These excellent characteristics draw great attention from various laboratories trying to expand its functions in effectiveness of anti-interspecies viruses and immunology. It is worth mentioning that some researchers even assume to use interferon tau in the treatment for HIV patients due to the excellent anti-lentivirus effect.
As a simplest member of the retrovirus family, by now, EIAV was generally acknowledged to be a perfect model for retrovirus researching. In this study, EIAV was used as the target virus to analyze the role of tau in anti-lent virus and compare with interferon gamma on antiviral effects and ISG cytokines gene level, and thereby revealing the different immunological mechanisms between interferon tau and gamma. Here, VSV-MDBK detecting system was established based on the titer of vesicular stomatitis virus (VSV), then titration was perform for interferon gamma and tau with VSV-MDBK Dye takes method. Cytotoxicity of interferon gamma and tau was detected using ED-EIAV cell with Cell Counting Kit-8 (DOJINDO). And then based on the condition without cytotoxicity, anti-EIAV effects of them were detected and compared with Reverse transcriptase activity detection and One Step Taqman absolutely quantitive Real-Time PCR. Two methods, Reverse Transcriptase Assay Colorimetric Kit and One Step Taqman absolutely quantitive Real-Time PCR, were used to confirm virus changes in the EIAV cells treated by interferon gamma and tau respectively. At last, three cytokines gene in the ED-EIAV cell, namely EIF2AK2, OAS1 and Mx protein, were detected with One Step relative quantitive Real-Time PCR at different time point after interferon gamma and interferon tau treatment. Then immunologic mechanism of the two IFNs was discussed after cytokines expression assay.
The results demonstrated that interferon tau indeed has anti EIAV effects in ED-EIAV cell, authough 10 times weaker than interferon gamma. While the cytotoxicity of interferon tau is significantly lower than that of interferon gamma. They both can stimulate the expression of EIF2AK2, OAS1 and Mx protein in similar tendency, but the stimulating capability of interferon tau is significantly lower than that of interferon gamma, especially to OAS1 and Mx protein. All these data showed that interferon tau has an anti-virus mechanism different from gamma and other interferons. Our hypothesis is that interferon tau may have other signaling pathways except the pathways through EIF2AK2, OAS1 and Mx protein, and this mechanism may involved in its low cytotoxicity to cells.
马传染性贫血病毒(Equine Infectious Anemia Virus,EIAV)作为逆转录病毒亚科慢病毒属中基因结构最简单的成员,是目前公认最理想的慢病毒研究模型。由于EIAV和HIV在病毒形态、基因组结构、细胞嗜性、免疫机制及病毒与宿主相互作用等方面都十分相似,深入研究EIAV感染与免疫机制,能够为HIV的相关研究提供借鉴,因此EIAV的感染与免疫机制的研究已受到国内外学者的高度重视。本研究选择EIAV作为靶病毒,尝试研究和评价重组牛干扰素τ(recombinant bovine interferon tau, rBoIFN-τ)的抗慢病毒效果,并与重组马干扰素(γrecombinant equine interferon gamma, rEIFN-γ)在抗病毒增殖、干扰素刺激基因(Interferon Stimulate Gene, ISG)节点因子介导水平等方面进行比较,分析IFN-τ与IFN-γ抗EIAV病毒的效果上的差异,进而揭示两者的免疫调节机理。本研究采用VSV-MDBK结晶紫染色法分别对rEIFN-γ和rBoIFN-τ进行滴定,并采用Cell Counting Kit-8对两种IFN在ED-EIAV上的细胞毒性进行检测和比较,然后在不产生细胞毒性的条件下,应用逆转录酶活性检测法和real-Time PCR检测和评价两种IFN在ED-EIAV细胞上的抗病毒效果。在上述研究的基础上,用Real-Time PCR检测经过两种IFN处理后不同时间点的ED-EIAV细胞中EIF2AK2、OAS1和Mx protein三种因子基因表达情况,并通过结合比较两种IFN处理后ISG基因的表达情况,探讨两种IFN抗病毒作用机理。
本研究证明,尽管其抗病毒效果低于rEIFN-γ,rBoIFN-τ在ED-EIAV细胞上确实表现出了一定的抗EIAV病毒的效果,大约10倍量的rBoIFN-τ抗EIAV效果与rEIFN-γ相当,但是其细胞毒性明显低于rEIFN-γ。另外,同rEIFN-γ一样,rBoIFN-τ也能刺激三种节点ISG基因的表达,而且表达上调的趋势也具有相似性,但rBoIFN-τ诱导这三种因子表达的能力显著低于rEIFN-γ,特别是在诱导OAS1基因和Mx protein基因表达的能力上两者差异更为显著。由此推测,IFN-τ的抗病毒机制可能与IFN-γ以及其他IFN的抗病毒机制不同,除了EIF2AK2、OAS1和Mx protein三种因子为代表的通路之外,很可能还存在其它的抗病毒通路,并且这一机制上的差异也可能与IFN-τ细胞毒性低有关。
Interferons are a type of protein produced by animal cells, which have high biological activity and a series of biological functions, especially in infection defense, anti-cell proliferation and immunoregulation. Till now, in-depth studies have been carried out to dissect the biological functions of interferon gamma and it has been applied in clinical cases as a biomedicine. Interferon tau belongs to the type I interferons, originally found as an important cytokine involved in maintaining the pregnancy of ruminant. Recently, efforts have been exerted to explore its roles in immunology, especially in the anti-multiplication of the lentivirus. Compared with other interferons, tau possesses some unique qualities such as high effectiveness of anti-virus, low toxicity to cells and minor or even no side-effects. These excellent characteristics draw great attention from various laboratories trying to expand its functions in effectiveness of anti-interspecies viruses and immunology. It is worth mentioning that some researchers even assume to use interferon tau in the treatment for HIV patients due to the excellent anti-lentivirus effect.
As a simplest member of the retrovirus family, by now, EIAV was generally acknowledged to be a perfect model for retrovirus researching. In this study, EIAV was used as the target virus to analyze the role of tau in anti-lent virus and compare with interferon gamma on antiviral effects and ISG cytokines gene level, and thereby revealing the different immunological mechanisms between interferon tau and gamma. Here, VSV-MDBK detecting system was established based on the titer of vesicular stomatitis virus (VSV), then titration was perform for interferon gamma and tau with VSV-MDBK Dye takes method. Cytotoxicity of interferon gamma and tau was detected using ED-EIAV cell with Cell Counting Kit-8 (DOJINDO). And then based on the condition without cytotoxicity, anti-EIAV effects of them were detected and compared with Reverse transcriptase activity detection and One Step Taqman absolutely quantitive Real-Time PCR. Two methods, Reverse Transcriptase Assay Colorimetric Kit and One Step Taqman absolutely quantitive Real-Time PCR, were used to confirm virus changes in the EIAV cells treated by interferon gamma and tau respectively. At last, three cytokines gene in the ED-EIAV cell, namely EIF2AK2, OAS1 and Mx protein, were detected with One Step relative quantitive Real-Time PCR at different time point after interferon gamma and interferon tau treatment. Then immunologic mechanism of the two IFNs was discussed after cytokines expression assay.
The results demonstrated that interferon tau indeed has anti EIAV effects in ED-EIAV cell, authough 10 times weaker than interferon gamma. While the cytotoxicity of interferon tau is significantly lower than that of interferon gamma. They both can stimulate the expression of EIF2AK2, OAS1 and Mx protein in similar tendency, but the stimulating capability of interferon tau is significantly lower than that of interferon gamma, especially to OAS1 and Mx protein. All these data showed that interferon tau has an anti-virus mechanism different from gamma and other interferons. Our hypothesis is that interferon tau may have other signaling pathways except the pathways through EIF2AK2, OAS1 and Mx protein, and this mechanism may involved in its low cytotoxicity to cells.
引文
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