本文關鍵詞： 牛乳頭狀瘤病毒 C127細胞 苦參堿 細胞凋亡　出處：《東北農(nóng)業(yè)大學》2015年碩士論文　論文類型：學位論文

【摘要】：牛乳頭狀瘤病毒(Bovine Papillomavirus,BPV)是牛乳頭狀瘤病的病原體,BPV能夠引起牛的皮膚、黏膜上皮增生性病變,目前BPV已呈全球內(nèi)流行趨勢,帶給了養(yǎng)牛業(yè)巨大的經(jīng)濟損失。當前防治牛乳頭狀瘤病的方法有外科手術治療法、中藥治療法及疫苗免疫法等。本研究利用BPV感染C127細胞,建立細胞感染模型,進行牛乳頭狀瘤病防治藥物的體外篩選,為牛乳頭狀瘤病的臨床治療提供理論基礎及試驗依據(jù)。試驗選取臨床典型的牛乳頭狀瘤組織,對其進行光鏡及電鏡觀察,通過間接ELISA法檢測腫瘤組織中BPV,并進行PCR擴增、測序確定其基因型;BPV野毒株感染C127細胞,利用光鏡對比觀察細胞形態(tài)學變化,透射電鏡觀察細胞內(nèi)病毒粒子分布;以利巴韋林作為陽性對照藥物,通過實時熒光定量PCR法檢測苦參堿及氧化苦參堿對BPV直接滅活作用、抑制BPV在細胞內(nèi)增殖作用及預防BPV對細胞的感染作用,評價苦參堿及氧化苦參堿對BPV的作用效果;利用細胞流式技術檢測藥物作用后,對染毒細胞的凋亡影響,探索藥物作用機制。結果表明:光鏡觀察可見腫瘤組織呈多個指狀突起,每個突起的中心為纖維組織和血管,突起外為多層鱗狀上皮細胞,異型性低,未見明顯角化,且無異常核分裂象。透射電鏡觀察可見腫瘤組織基底細胞、棘細胞及顆粒細胞均出現(xiàn)細胞形狀不規(guī)則、線粒體空泡狀等病理變化。間接ELISA法檢測腫瘤組織BPV呈陽性反應;經(jīng)PCR擴增、測序結果與分離自中國的BPV-2病毒SW株(Gen Bank:KC256805.1)L1基因的相似性高達99%。BPV野毒株感染C127細胞,細胞形狀變小、細長、生長密集、排列紊亂,PCR檢測在450bp左右出現(xiàn)特異條帶。細胞透射電鏡觀察發(fā)現(xiàn)病毒粒子,并伴隨高爾基體、粗面內(nèi)質(zhì)網(wǎng)、線粒體變形等病理變化。通過藥物作用后染毒細胞CPE及實時熒光定量PCR檢測結果表明具直接抗BPV作用的為:利巴韋林濃度為0.625μg/ml時抑制率最高,可達90%,苦參堿濃度為80μg/ml時抑制率最高,可達98%,與對照組相比差異極顯著(P㩳0.01);具預防BPV感染作用的為:苦參堿濃度為80μg/ml時抑制率最高,可達95%,與對照組相比差異極顯著(P㩳0.01);具抑制BPV在細胞內(nèi)增殖作用的為:利巴韋林濃度為0.625μg/ml時抑制率最高,可達93%,苦參堿濃度為80μg/ml時抑制率最高,可達95%以上,與對照組相比差異極顯著(P㩳0.01);而氧化苦參堿藥物最大無毒濃度的三種作用途徑對BPV的抑制率均低于30%,視為無效。BPV感染細胞24h時,己經(jīng)開始抑制細胞凋亡,且細胞主要處于晚期凋亡,同時苦參堿的三種給藥途徑處理BPV,均對細胞晚期凋亡具有一定促進作用。結論:苦參堿直接作用于BPV可使其活性喪失,苦參堿又具有抑制BPV在細胞內(nèi)增殖及預防BPV感染的作用。
[Abstract]:Bovine papillomavirus (BPV) is the pathogen of bovine papillomatosis. The current methods of prevention and treatment of bovine papillomatosis include surgical treatment, traditional Chinese medicine therapy and vaccine immunization. In this study, C127 cells were infected with BPV, and cell infection models were established. In order to provide theoretical basis and experimental basis for clinical treatment of bovine papillomatosis, the clinical typical bovine papillomatous tissue was selected and observed by light and electron microscope, and in vitro screening of drugs for the prevention and treatment of bovine papillomatosis was carried out in order to provide theoretical basis and experimental basis for clinical treatment of bovine papillomatosis. BPVs in tumor tissues were detected by indirect ELISA method, and PCR amplification was carried out. The genotypes of BPV-wild strains were determined by sequencing. The morphological changes of C127 cells were observed by light microscopy and the distribution of virus particles in the cells was observed by transmission electron microscope (TEM). Ribavirin was used as a positive control drug. The direct inactivation of BPV by matrine and oxymatrine was detected by real-time fluorescence quantitative PCR method, which inhibited the proliferation of BPV in cells and prevented the infection of BPV. To evaluate the effect of matrine and oxymatrine on BPV and to detect the apoptosis of exposed cells by flow cytometry. The results showed that the tumor tissue showed multiple digital processes, the center of each process was fibrous tissue and blood vessel, and the outer process was multilayer squamous epithelial cells, with low heterogeneity and no obvious keratosis. There were no abnormal mitotic signs. The basal cells of tumor tissues were observed by transmission electron microscope, the cells of spinous cells and granulosa cells were irregular, and the mitochondria were vacuolated. Indirect ELISA method was used to detect the positive reaction of BPV in tumor tissues. The results of PCR amplification showed that the similarity of the gene of Genin Bank:KC256805.1)L1 of BPV-2 virus SW strain isolated from China was as high as 99k.BPV wild strain infected C127 cells. The cells became smaller, longer and more dense in growth. The specific bands were detected by PCR at about 450 BP. The virus particles were observed by transmission electron microscope and accompanied by Golgi body and rough endoplasmic reticulum. The results of CPE and real-time fluorescence quantitative PCR analysis showed that the inhibition rate of ribavirin was the highest when the concentration of ribavirin was 0.625 渭 g / ml, and the results of real-time fluorescence quantitative PCR analysis showed that the inhibition rate of ribavirin was the highest when the concentration of ribavirin was 0.625 渭 g / ml. Up to 90%, matrine concentration is 80 渭 g / ml, the inhibition rate is the highest, up to 98, compared with the control group, the difference is very significant. The inhibitory rate of matrine was the highest when the concentration of matrine was 80 渭 g / ml, up to 95%, and the difference was significant compared with the control group. The inhibition rate of ribavirin was the highest when the concentration of ribavirin was 0.625 渭 g / ml, and that of matrine was 80 渭 g / ml, which was more than 95%. However, the inhibitory rate of the three pathways of maximal nontoxic concentration of oxymatrine on BPV was lower than that of 30%, which was regarded as ineffective. After 24 hours of infection, the apoptosis of the cells was inhibited, and the cells were mainly in the late stage of apoptosis. At the same time, the three ways of administration of matrine can promote the late apoptosis of BPV.Conclusion: matrine can inhibit the proliferation of BPV in cells and prevent BPV infection by directly acting on BPV.Conclusion: matrine has the function of inhibiting the proliferation of BPV in cells and preventing the infection of BPV.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S853.7

【參考文獻】

相關期刊論文 前2條

1 李孝楠;穆傳軍;;牛纖維乳頭狀瘤的癥狀及防治方法[J];養(yǎng)殖技術顧問;2008年11期

2 梁克勤;王云龍;郭建平;呂尋玉;陳紅香;;燒烙術治療牛乳頭狀瘤[J];山西農(nóng)業(yè)(畜牧獸醫(yī));2008年12期

，

本文編號：1512782