血红素加氧酶1在牛病毒性腹泻病毒感染MDBK细胞中的作用研究

发布时间：2018-01-01 01:19

  本文关键词：血红素加氧酶1在牛病毒性腹泻病毒感染MDBK细胞中的作用研究　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 血红素加氧酶1 牛病毒性腹泻病毒 MDBK细胞 病原-细胞相互作用

【摘要】：牛病毒性腹泻(Bovine viral diarrhea,BVD)是由牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)感染引起的一种以腹泻、急/慢性黏膜炎、繁殖障碍、持续性感染和免疫耐受为特征的传染病。BVDV为单股正链RNA病毒,它与猪瘟病毒、羊边界病病毒同属于黄病毒科瘟病毒属。该病毒具有广泛的宿主嗜性,除了感染牛,还可感染猪、羊、鹿、骆驼及其它多种动物并引起发病,使其在自然界中难以清除,给整个牲畜养殖业造成巨大的危害。BVDV感染怀孕母畜常引起免疫耐受,导致持续性感染(Persistent infection,PI)牛的产生。PI牛对疫苗免疫不产生相应的抗体,病毒得以在体内持续存在并不断向外排毒感染其它牛群,这使得PI牛成为BVD感染中最主要的传染源。现有的防控策略很难将BVDV从牛群中根除,因此,我们亟需寻找一种新的抗病毒方案以有效防控BVDV的感染。近期研究发现,血红素加氧酶1(Heme oxygenase-1,HO-1)作为细胞中的一种重要的抗氧化应激蛋白,不仅具有抗炎、抗氧化、抗凋亡等多种生物学功能,而且在病毒感染中也发挥着非常重要的调控作用。乙型肝炎病毒、丙型肝炎病毒、猪繁殖与呼吸综合征病毒等病毒感染可以抑制HO-1的表达,上调HO-1表达可有效抑制这些病毒的感染。但是猪瘟病毒感染却可促进HO-1的表达,降低HO-1的表达反而抑制了该病毒的感染,这说明不同病毒的感染,HO-1所发挥的作用并不相同。BVDV感染能否调控细胞中HO-1的表达?HO-1在BVDV感染细胞的过程中发挥什么作用?这一切都尚未清楚。因此本研究拟明确BVDV感染对宿主细胞HO-1表达的影响,进一步阐明HO-1在BVDV感染细胞过程中的作用,主要研究结果如下:1.BVDV感染降低MDBK细胞中HO-1的表达水平我们首先检测了BVDV感染后不同时间点MDBK细胞中BVDV、HO-1、HO-2、NQO1(NAD(P)H醌氧化还原酶1,与HO-1同属于抗氧化反应元件所调控)mRNA和蛋白的表达情况。结果表明,BVDV感染后MDBK细胞中HO-1及NQO1的表达水平显著下降,且下降的趋势与病毒感染时间呈正相关,而HO-2的表达无显著变化。这说明BVDV感染可抑制MDBK细胞中HO-1表达,其抑制作用初步证实与抗氧化通路相关,而HO-2的表达不受影响。2.上调HO-1表达显著抑制BVDV的感染为了研究HO-1在BVDV感染过程中的作用,我们首先应用HO-1的诱导剂CoPP处理MBDK细胞,然后检测CoPP诱导HO-1对BVDV复制的影响。结果表明,CoPP诱导显著上调了细胞中HO-1的表达而HO-2表达水平没有变化,并且细胞的活性不受影响。CoPP呈浓度梯度依赖性的降低BVDV mRNA和蛋白的表达水平及病毒滴度,而且不论病毒感染前或感染后用CoPP处理MDBK细胞,均可显著降低BVDV的复制。该结果表明,CoPP诱导HO-1上调表达可有效抑制BVDV的感染。为证明CoPP对BVDV感染的抑制作用是由HO-1介导的,我们构建了可表达HO-1的重组腺病毒,利用该病毒感染MDBK细胞,在细胞中特异性过表达HO-1,研究其对BVDV感染的影响。结果表明,感染重组腺病毒的细胞中HO-1的表达水平显著升高,而BVDV的NS5B蛋白含量及细胞上清中病毒RNA的含量和病毒滴度均呈显著降低。该结果证实了特异性上调HO-1的表达可有效抑制细胞中BVDV的感染。3.特异性降低HO-1基础表达水平促进BVDV的感染由于诱导或过表达HO-1均显著降低了BVDV的感染,我们想进一步了解降低细胞中HO-1的基础表达水平能否影响BVDV的复制。因此我们将特异性干扰HO-1表达的siRNA转入MDBK细胞以降低细胞中HO-1的基础表达水平,然后检测HO-1和BVDV mRNA和蛋白表达。结果表明,转染siRNA的细胞HO-1的表达水平显著降低,而细胞上清中病毒RNA的含量及病毒滴度却显著升高。说明特异性降低HO-1的基础表达水平可促进BVDV的感染。4.CoPP通过HO-1介导抑制BVDV的复制为了证明CoPP处理对BVDV感染的抑制作用是由HO-1特异性介导的,转染siRNA的MDBK细胞,接毒后用CoPP处理,然后检测BVDV的病毒滴度和蛋白表达情况。结果表明,转染siRNA可部分逆转CoPP诱导对BVDV感染的抑制作用,该结果证明CoPP是通过HO-1的介导抑制BVDV的复制。5.HO-1抑制BVDV复制的作用与其代谢产物有关为进一步了解HO-1抑制BVDV感染的原因,我们采用biliverdin、CORM-2及FeCl3(模拟HO-1的代谢产物biliverdin,CO和Fe2+)分别处理细胞,然后检测BVDV的感染情况。结果表明,采用biliverdin和CORM-2处理的细胞,BVDV的感染均显著降低,而采用FeCl3处理的细胞,BVDV感染无显著变化,这初步证明了HO-1抑制BVDV感染的作用与其代谢产物biliverdin和CO有关。综上所述,本研究证明了BVDV感染可以抑制MDBK细胞中HO-1的表达,而上调HO-1表达可显著抑制BVDV的感染,其抑制作用与HO-1的代谢产物biliverdin和CO相关。该研究为阐明BVDV的分子致病机制提供了有用的参考资料,并为抗BVDV感染的研究提供了新的思路及潜在的靶点。
[Abstract]:Bovine viral diarrhea (Bovine viral diarrhea, BVD) from bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) an infection caused by diarrhea, acute / chronic mucositis, reproductive disorders, persistent infection and immune tolerance of infectious disease.BVDV positive single strand RNA virus, it classical swine fever virus, border disease virus belonging to the Flaviviridae pestivirus genus. The virus has a wide host tropism, apart from infected cows can also infect pigs, sheep, deer, camels and other animal and cause disease, making it difficult to remove in nature, for the livestock breeding industry great harm.BVDV infection of pregnant female often cause immune tolerance, cause persistent infection (Persistent infection, PI.PI) cattle cattle do not produce antibodies to the vaccine, the virus can persist in vivo and continue to infect other cattle from the pigs, the The PI become the main source of infectious bovine infection in BVD. The existing prevention and control strategy is very difficult to eradicate BVDV from the herd, therefore, we need to find a new antivirus solutions to effective prevention and control of BVDV infection. A recent study found that heme oxygenase 1 (Heme oxygenase-1 HO-1) is a kind of important oxidative stress proteins in the cell, not only has the anti-inflammatory, antioxidant, anti apoptosis of various biological functions, but also in the viral infection also plays a very important role in the regulation of hepatitis B virus, hepatitis C virus, porcine reproductive and respiratory syndrome virus can inhibit the expression of HO-1 infection, can effectively inhibit the upregulation of the expression of HO-1 the virus infection. But the swine fever virus infection can promote the expression of HO-1, decreased the expression of HO-1 may suppress the virus infection, indicating that different virus infection, HO-1 plays the role of Is not the same as the.BVDV infection in HO-1 cells can express HO-1? What is the role of BVDV in the infected cells? All this is not clear. Therefore, this study intends to clear effect of BVDV infection on the expression of host cell HO-1, to further elucidate the role of HO-1 in BVDV infected cells in the process, the main results are as follows: 1.BVDV the infection decreased the expression of HO-1 in MDBK cells, we first detected the infection of BVDV BVDV at different time points after MDBK cells in HO-1, HO-2, NQO1 (NAD (P) H quinone oxidoreductase 1, and HO-1 belong to the antioxidant response element regulated) expression of mRNA and protein. The results showed that the expression level of BVDV infection after MDBK and HO-1 in NQO1 cells decreased significantly, and decreased with the infection time was positively related to the expression of HO-2 showed no significant changes. This shows that BVDV infection can inhibit the expression of HO-1 in MDBK cells and its inhibition Preliminary confirmed with antioxidant pathway, whereas the expression of HO-2 was not affected by the expression of.2. up-regulated HO-1 significantly inhibited BVDV infection in order to study the HO-1 during BVDV infection, the induction of CoPP MBDK cells we applied HO-1, then detect the effect of HO-1 on CoPP induced BVDV replication. The results showed that CoPP induced significant upregulation of the expression of HO-1 in the cells and the expression level of HO-2 did not change, and the activity of the cells was not affected by.CoPP concentration gradient dependent and virus titer of BVDV decreased the expression of mRNA and protein, and no virus infection before or after infection with CoPP MDBK cells, BVDV could significantly decrease the replication of the results. Show that the expression of BVDV can effectively inhibit the infection of HO-1 induced upregulation of CoPP. In order to prove the inhibitory effect of CoPP on BVDV infection is mediated by HO-1, we constructed the recombinant adenovirus expressing HO-1 , the virus infected MDBK cells, the cell specific expression of HO-1, to study its effect on BVDV infection. The results showed that the HO-1 expression level of recombinant adenovirus infected cells significantly increased, while the content of RNA and the virus titer of virus NS5B protein and BVDV in the cell supernatant were significantly reduced. The results confirm the specificity of the expression regulation of HO-1 infected.3. specific cells can effectively inhibit the decrease of BVDV in HO-1 based BVDV expression level to promote infection due to induction or overexpression of HO-1 decreased significantly in BVDV infection, we want to know more about reducing the expression level of HO-1 in cells can influence the replication of BVDV. So we the specificity of siRNA expression by HO-1 interference into MDBK cells to reduce the expression level of HO-1 in cells, and the detection of HO-1 and BVDV mRNA and protein expression. The results showed that the transfection of siRNA fine The expression level of HO-1 cells decreased significantly, while the content of virus and virus titer in the supernatant of RNA cells was significantly increased. Lower specificity HO-1 based expression can promote the infection of.4.CoPP BVDV through HO-1 mediated inhibition of BVDV replication in order to prove that CoPP treatment of BVDV infection is inhibited by HO-1 mediated by specific the siRNA transfected MDBK cells after inoculation with CoPP, then the virus titer of BVDV and protein expression. The results showed that the transfection of siRNA could reverse CoPP induced inhibition of BVDV infection, the results show that CoPP inhibition of BVDV replication and its metabolites related to further understand the inhibition of HO-1 BVDV infection by HO-1 mediated inhibition of BVDV.5.HO-1 replication, we use biliverdin, CORM-2 and FeCl3 (analog HO-1 metabolites of biliverdin, CO and Fe2+) were treated cells, then detected The infection of BVDV. The results showed that the biliverdin and CORM-2 treated cells, BVDV infection were significantly decreased, while the use of FeCl3 treated cells, no significant changes of BVDV infection, which demonstrated that HO-1 inhibited the role of BVDV infection and its metabolites biliverdin and CO. In conclusion, this study demonstrated that HO-1 can inhibit the expression of MDBK cells in BVDV infection, and overexpression of HO-1 can inhibit BVDV infection, and the inhibitory effect of HO-1 metabolites biliverdin and CO related. This study provides a useful reference for the molecular pathogenesis of BVDV is clarified, and provides a new idea and a potential target for the study of anti BVDV infection.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S855.3
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本文编号：1362306