姜黄素抗猪繁殖与呼吸综合征病毒感染的作用机制

发布时间：2018-09-11 10:42

【摘要】：猪繁殖与呼吸综合征给养猪业造成了巨大的经济损失。该病的临床症状为怀孕母猪流产和各年龄阶段猪的呼吸系统疾病。猪繁殖与呼吸综合征的病原是猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV),该病毒属动脉炎病毒科的单股正链RNA病毒,自1991年发现至今已有20多年之久,主要分为两种类型:欧洲型(Ⅰ型)和美洲型(Ⅱ型)。中国发现并分离的高致病性毒株属于PRRSVⅡ型。虽然这两种毒株几乎同时被发现,但两者核苷酸序列的同源性只有70%,并且具有不同的地理分布。自1994年起就已经有商品化的PRRSV疫苗问世,然而疫苗免疫并缺乏全面的交叉保护效果,而且PRRSV感染后引起的抗感染免疫机制目前仍不甚清晰。鉴于此,亟待需要安全高效的疫苗,新型抗病毒药物和免疫佐剂的出现以有效控制PRRSV的感染。姜黄素是一种从姜黄根茎提取的具有生物活性的天然多酚类化合物。据报道,姜黄素能够影响细胞代谢,具有抗肿瘤、抗氧化、抗炎症、抗病毒和免疫调节等活性。有研究显示,姜黄素可抑制水疱性口炎病毒(vesicular stomatitis virus,VSV),丙型肝炎病毒(hepatitis C virus,HCV)和基孔肯雅病毒(chikungunya virus,CHIKV)进入细胞。但目前还没有关于姜黄素在PRRSV中的研究报道。所以本研究主要探讨姜黄素在PRRSV感染MARC-145细胞和猪肺泡巨噬细胞中对PRRSV感染的作用。主要结果如下:1.MARC-145细胞与不同浓度的姜黄素(0μM,5μM,10μM和15μM)按以下三种方法孵育:(1)姜黄素与PRRSV的混合物与细胞共孵育1h;(2)PRRSV感染细胞后与姜黄素共孵育1h;(3)姜黄素预处理细胞1h后接入病毒。细胞样品和细胞上清分别用western blot和TCID50进行分析。结果表明姜黄素和病毒的混合物同时接种入MARC-145细胞时,姜黄素抑制PRRSV感染,并具有浓度梯度依赖性。无PRRSV毒株依赖性。2.为了证明姜黄素的抑制作用是否是由于其细胞毒性以及是否是由于对细胞表面PRRSV受体的影响,实验中选择CCK-8试剂盒检测姜黄素对MARC-145细胞的毒性作用,并用western blot方法检测细胞表面受体CD163,非肌肉肌球蛋白重链9(non-muscle myosin heavy chain 9,MYH9)蛋白和波形蛋白(vimentin)的表达情况,结果表明姜黄素对MARC-145细胞无细胞毒性,MARC-145细胞表面PRRSV受体表达无抑制作用。3.为了研究姜黄素对PRRSV感染MARC-145细胞每个步骤的作用,包括病毒吸附,内化和内化后感染过程。RT-PCR检测病毒吸附实验中ORF7 mRNA的表达。Western blot和TCID50分别检测病毒内化实验和内化后实验中的N蛋白的表达和细胞上清中的子代病毒滴度。进一步通过低pH依赖性细胞融合实验来验证姜黄素是否阻止病毒G蛋白介导的细胞融合。结果表明:姜黄素对PRRSV吸附无抑制作用,而是抑制病毒内化以及通过阻止G蛋白介导的细胞融合抑制病毒脱衣壳。4.猪肺泡巨噬细胞PAM(porcine alveolar macrophages,PAM)实验中,将病毒和姜黄素的混合物与PAM细胞共同孵育1h。RT-PCR方法检测细胞中病毒ORF7 mRNA的表达,并检测细胞上清中子代病毒滴度。结果表明,姜黄素抑制PRRSV感染PAMs。
[Abstract]:Porcine reproductive and respiratory syndrome (porcine * * *) has caused enormous economic losses to the pig industry. The clinical symptoms of this disease are the miscarriage of pregnant sows and respiratory diseases of pigs at all ages. The pathogen of porcine reproductive and respiratory syndrome is the reproductive and respiratory syndrome virus (PRRSV), which belongs to the genus. The single-stranded positive-stranded RNA viruses of the family Arteritis Viridae have been discovered for more than 20 years since 1991 and are mainly divided into two types: European (type I) and American (type II). Since 1994, commercial PRRSV vaccines have been available. However, vaccines do not have comprehensive cross-protective effects and the mechanism of anti-infective immunity after PRRSV infection is still unclear. In view of this, safe and efficient vaccines, new antiviral drugs and immune adjuvants are urgently needed. Curcumin is a natural polyphenolic compound with biological activity extracted from the rhizome of Curcuma longa. It has been reported that curcumin can affect cell metabolism, has anti-tumor, anti-oxidation, anti-inflammatory, anti-virus and immune regulation activities. Studies have shown that curcumin can inhibit Vesic stomatitis virus (vesic stomatitis virus). Ular stomatitis virus (VSV), hepatitis C virus, HCV, and Chikungunya virus CHIKV have entered the cell. However, there is no research report on Curcumin in the process of killing. Therefore, this study mainly focused on the effects of curcumin on infection of * cells and porcine alveolar macrophages. The main results were as follows: 1. MARC-145 cells were incubated with curcumin of different concentrations (0, 5, 10 and 15 mu M) in three ways: (1) the mixture of curcumin and PRRSV was co-incubated with cells for 1 hour; (2) PRRSV infected cells were co-incubated with curcumin for 1 hour; (3) curcumin pretreated cells were inoculated with virus after 1 hour. The results showed that curcumin inhibited PRRSV infection in a concentration gradient dependent manner when the mixture of curcumin and virus was inoculated into MARC-145 cells at the same time. The effect of RRSV receptor was tested by CCK-8 kit. The expression of CD163, MYH9 and vimentin on MARC-145 cells was detected by Western blot. The results showed that curcumin had a fine effect on MARC-145 cells. The expression of PRRSV receptor on the surface of MARC-145 cells was not inhibited. 3. To investigate the effect of curcumin on each step of PRRSV-infected MARC-145 cells, including virus adsorption, internalization and post-internalization infection, the expression of ORF7 mRNA was detected by RT-PCR. Viral internalization was detected by Western blot and TCID50, respectively. The results showed that curcumin had no inhibitory effect on PRRSV adsorption, but inhibited virus internalization and G protein mediated cell fusion. Cell fusion inhibits virus (.4.) PAM porcine (alveolar * macrophages, PAM) experiment. The expression of virus ORF7 mRNA was detected by CO incubation of the mixture of virus and curcumin with PAM cells, and the titer of progeny virus in cell supernatants was detected. The results showed that curcumin inhibited the sense of PRRSV. Dyed PAMs.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.28

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