【摘要】：目的：豬繁殖與呼吸綜合征(porcine reproductive and respiration syndrome, PRRS),又稱為藍(lán)耳病,是一種傳染性較強(qiáng)的常見(jiàn)豬病,病毒具有極易變異的特點(diǎn),爆發(fā)后會(huì)給養(yǎng)豬業(yè)造成極大的經(jīng)濟(jì)損失。目前針對(duì)該病主要采用注射疫苗進(jìn)行預(yù)防的手段,可以起到一定作用,但尚沒(méi)有針對(duì)PRRS的有效治療方法。本試驗(yàn)選取從常見(jiàn)抗病毒中草藥金銀花、板藍(lán)根和淫羊藿中提取的天然化合物綠原酸、表告依春和淫羊藿苷,以Marc-145細(xì)胞為體外模型,研究其是否具有體外抗豬繁殖與呼吸綜合征病毒作用,是否能夠有效抑制由PRRSV引起的細(xì)胞凋亡,并初步探討這些中藥主要成分的抗病毒機(jī)制。方法：(1)通過(guò)采用細(xì)胞病變法(cytopathologic effect, CPE)以及噻唑藍(lán)比色法(3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT)來(lái)測(cè)定中藥提取物在Marc-145細(xì)胞體外模型上的最大安全濃度(maximum no-cytotoxic concentration, MNTC)。(2)運(yùn)用RT-PCR方法測(cè)定PRRSV對(duì)Marc-145細(xì)胞的侵染作用,通過(guò)免疫組化的方法觀察PRRSV對(duì)Marc-145細(xì)胞的侵染部位,并研究中藥化合物在其安全濃度下對(duì)病毒侵染Marc-145細(xì)胞的抑制作用效果。(3)運(yùn)用細(xì)胞凋亡檢測(cè)的方法研究中藥化合物在其安全濃度下對(duì)PRRSV誘導(dǎo)細(xì)胞凋亡所起到的作用效果。結(jié)果：中藥提取的天然化合物綠原酸、表告依春和淫羊藿苷均具有抗PRRSV的作用,以綠原酸最為明顯。綠原酸的最大安全濃度為：500μg/mL,表告依春的最大安全濃度為：100μg/mL,淫羊藿苷的最大安全濃度為：31.25μg/mL。在最大安全濃度下添加綠原酸和表告依春,可以使得Marc-145細(xì)胞免受PRRSV的侵染,同時(shí)細(xì)胞的凋亡數(shù)量明顯低于未添加藥物的病毒對(duì)照組,而淫羊藿苷不能完全防止PRRSV侵染Marc-145細(xì)胞,但可以減少受病毒侵染的細(xì)胞數(shù)目,并起到一定抑制細(xì)胞凋亡的作用。結(jié)論：中藥提取的天然化合物綠原酸、表告依春具有較強(qiáng)的體外抗PRRSV的作用,而淫羊藿苷的體外抗PRRSV的作用并不理想,推測(cè)綠原酸與表告依春的抗病毒機(jī)制可能是直接滅活PRRSV并抑制由PRRSV引起的細(xì)胞凋亡來(lái)實(shí)現(xiàn),而淫羊藿苷則可以一定程度的抑制由PRRSV引起的細(xì)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.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28

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