抗猪繁殖与呼吸综合征病毒（PRRSV）中药高效成分的体外筛选及其抗病毒机制的研究

发布时间：2018-12-27 20:20

【摘要】：目的：猪繁殖与呼吸综合征(porcine reproductive and respiration syndrome, PRRS),又称为蓝耳病,是一种传染性较强的常见猪病,病毒具有极易变异的特点,爆发后会给养猪业造成极大的经济损失。目前针对该病主要采用注射疫苗进行预防的手段,可以起到一定作用,但尚没有针对PRRS的有效治疗方法。本试验选取从常见抗病毒中草药金银花、板蓝根和淫羊藿中提取的天然化合物绿原酸、表告依春和淫羊藿苷,以Marc-145细胞为体外模型,研究其是否具有体外抗猪繁殖与呼吸综合征病毒作用,是否能够有效抑制由PRRSV引起的细胞凋亡,并初步探讨这些中药主要成分的抗病毒机制。方法：(1)通过采用细胞病变法(cytopathologic effect, CPE)以及噻唑蓝比色法(3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT)来测定中药提取物在Marc-145细胞体外模型上的最大安全浓度(maximum no-cytotoxic concentration, MNTC)。(2)运用RT-PCR方法测定PRRSV对Marc-145细胞的侵染作用,通过免疫组化的方法观察PRRSV对Marc-145细胞的侵染部位,并研究中药化合物在其安全浓度下对病毒侵染Marc-145细胞的抑制作用效果。(3)运用细胞凋亡检测的方法研究中药化合物在其安全浓度下对PRRSV诱导细胞凋亡所起到的作用效果。结果：中药提取的天然化合物绿原酸、表告依春和淫羊藿苷均具有抗PRRSV的作用,以绿原酸最为明显。绿原酸的最大安全浓度为：500μg/mL,表告依春的最大安全浓度为：100μg/mL,淫羊藿苷的最大安全浓度为：31.25μg/mL。在最大安全浓度下添加绿原酸和表告依春,可以使得Marc-145细胞免受PRRSV的侵染,同时细胞的凋亡数量明显低于未添加药物的病毒对照组,而淫羊藿苷不能完全防止PRRSV侵染Marc-145细胞,但可以减少受病毒侵染的细胞数目,并起到一定抑制细胞凋亡的作用。结论：中药提取的天然化合物绿原酸、表告依春具有较强的体外抗PRRSV的作用,而淫羊藿苷的体外抗PRRSV的作用并不理想,推测绿原酸与表告依春的抗病毒机制可能是直接灭活PRRSV并抑制由PRRSV引起的细胞凋亡来实现,而淫羊藿苷则可以一定程度的抑制由PRRSV引起的细胞凋亡。
[Abstract]:Objective: porcine reproductive and respiratory syndrome (porcine reproductive and respiration syndrome, PRRS), also known as blue ear disease, is a common infectious disease in pigs. At present, injection vaccine can be used to prevent the disease, but there is no effective treatment for PRRS. In this experiment, the natural compounds Lv Yuan acid extracted from common antiviral herbs Flos Lonicerae, Radix Isatidis and Herba Epimedii were selected, and the Marc-145 cells were used as the model in vitro. To study whether it has anti-porcine reproductive and respiratory syndrome virus effect in vitro, whether it can effectively inhibit apoptosis induced by PRRSV, and to explore the antiviral mechanism of the main components of these traditional Chinese medicines. Methods: (1) tetrazolium bromide, was obtained by using cytopathic method (cytopathologic effect, CPE) and thiazolium blue colorimetry (3- (4- (4) -dimeththiazol-2-yl) -2- (5-diphenyl) tetrazolium bromide,). MTT) was used to determine the maximum safe concentration (maximum no-cytotoxic concentration, MNTC). (2) of Chinese traditional medicine extract on Marc-145 cell model in vitro. The infection of PRRSV on Marc-145 cells was determined by RT-PCR method. The site of PRRSV infection on Marc-145 cells was observed by immunohistochemical method. The inhibitory effect of traditional Chinese medicine compounds on virus infection of Marc-145 cells at safe concentration was also studied. (3) apoptosis induced by traditional Chinese medicine compounds on PRRSV cells was studied by the method of cell apoptosis detection. The effect. Results: the natural compound Lv Yuan acid, epiguuicine and icariin extracted from Chinese medicine all had the effect of anti PRRSV. The maximum safe concentration of Lv Yuan was 500 渭 g / mL, the maximum safe concentration of epigulium was 100 渭 g / mL, and the maximum safe concentration of icariin was 31.25 渭 g / mL. At the maximum safe concentration, the addition of Lv Yuan acid and epiglumic acid could protect Marc-145 cells from PRRSV infection, and the number of apoptotic cells was significantly lower than that of the control group. Icariin could not completely prevent PRRSV from infecting Marc-145 cells, but could reduce the number of cells infected by virus and inhibit apoptosis. Conclusion: the natural compound Lv Yuan acid extracted from traditional Chinese medicine has a strong effect on PRRSV in vitro, but icariin has no effect on PRRSV in vitro. It is speculated that the antiviral mechanism of Lv Yuan and epiguicun may be the direct inactivation of PRRSV and the inhibition of apoptosis induced by PRRSV, while icariin can inhibit the apoptosis induced by PRRSV to some extent.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28

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