胆固醇对鸡传染性支气管炎病毒感染鸡胚肾细胞的影响

发布时间：2018-04-23 11:48

本文选题：鸡传染性支气管炎病毒 + 鸡胚肾细胞　；参考：《东北农业大学》2017年硕士论文

【摘要】：鸡传染性支气管炎(Infectious bronchitis,IB)是由鸡传染性支气管炎病毒(Infectious bronchitis virus,IBV)引起的一种急性、高度接触性传染病,被认为是危害全球养禽业的主要病毒性传染病之一。鸡胚肾(Chicken embryo kidney,CEK)细胞源于IBV易感宿主,相较于其他细胞系更贴近于IBV的感染环境,且对IBV具有极高的敏感性,接种后能观察到明显的细胞病变,CEK已成为研究IBV感染细胞致病过程及其机制的适宜宿主细胞模型。脂筏是质膜中富含胆固醇和鞘脂的微结构域,具有介导病毒感染、信号转导、蛋白分选等生物学功能。胆固醇作为脂筏的重要组分,可维持脂筏的稳定结构并参与脂筏功能的发挥。研究表明,胆固醇在某些病毒感染细胞时扮演着非常重要的作用,包括病毒的进入、吸附和释放过程,但胆固醇在IBV感染过程中的作用还并未有报道。本实验分别研究了细胞膜和病毒囊膜胆固醇在IBV M41株感染CEK细胞过程中的作用。首先检测了胆固醇萃取剂甲基-β-环糊精(MβCD)对CEK细胞活性的影响,结果发现,当MβCD浓度小于10 m M时,不会对CEK细胞活性造成明显影响,所以本实验中所用的MβCD最大浓度为10 m M。同时进行胆固醇含量测定,当MβCD浓度为10 m M时,细胞膜胆固醇含量下降至37%,补充外源胆固醇后,胆固醇浓度会得到相应恢复,当充补胆固醇浓度为200μM时,胆固醇含量恢复至接近MβCD处理前。MβCD还能有效去除IBV囊膜胆固醇,用10m M MβCD处理病毒后,病毒囊膜胆固醇含量下降至约66%。本实验利用Real Time PCR、病毒滴度试验和Western Blot方法检测了细胞膜胆固醇在IBV M41株感染CEK细胞不同过程中的作用。结果表明,加入不同浓度MβCD去除细胞膜胆固醇,会以剂量依赖性的方式抑制IBV的进入,病毒感染力和IBV N蛋白的表达量也会梯度下降。为进一步确定该抑制作用是由于去除胆固醇引起的,用10 mM MβCD去除细胞膜胆固醇后再补充外源胆固醇,结果显示,随着补充胆固醇浓度的增加,IBV感染力和蛋白表达水平均得到相应恢复,说明去除胆固醇可抑制IBV进入CEK细胞,且该抑制作用是可逆的。本实验还研究了胆固醇在IBV吸附到CEK细胞和从CEK细胞释放过程中的作用,结果显示,随着MβCD浓度增大,IBV的吸附和释放过程均受到剂量依赖性的抑制。同时,将CEK细胞转染GPI-pEGFP后感染IBV,结果显示IBV与GPI锚定蛋白可共定位在同一个CEK细胞膜上,说明IBV可定位到细胞膜脂筏。病毒囊膜胆固醇在很多病毒进入细胞的过程中也扮演重要的作用。本实验将不同浓度MβCD处理后的IBV M41株感染CEK细胞,结果发现IBV感染力无明显趋势性变化,IBV N蛋白表达量也无显著性变化,证明病毒囊膜胆固醇并不是IBV进入CEK细胞所必须的。综上所述,IBV感染CEK细胞过程中需要细胞膜胆固醇参与,而不需要病毒囊膜胆固醇。
[Abstract]:Infectious bronchitis (IBV) is an acute and highly contagious disease caused by infectious bronchitis virus (IBV). It is considered to be one of the major viral infectious diseases in poultry industry worldwide. Chicken embryo kidney1 cells originate from susceptible hosts of IBV, and are more close to IBV infection environment than other cell lines, and highly sensitive to IBV. After inoculation, it was observed that the obvious cytopathic changes had become a suitable host cell model for studying the pathogenetic process and mechanism of IBV infection. Lipid rafts are microdomains rich in cholesterol and sphingolipid in plasma membrane, which can mediate virus infection, signal transduction, protein sorting and so on. Cholesterol, as an important component of lipid raft, can maintain the stable structure of lipid raft and play an important role in the function of lipid raft. Studies have shown that cholesterol plays a very important role in the process of virus infection, including virus entry, adsorption and release, but the role of cholesterol in the process of IBV infection has not been reported. The effects of membrane and viral envelope cholesterol on the infection of IBV M41 strain CEK cells were studied in this experiment. The effect of cholesterol extractant methyl 尾 -cyclodextrin M 尾 CD on the activity of CEK cells was investigated. The results showed that when the concentration of M 尾 CD was less than 10 mm, the activity of CEK cells would not be significantly affected. Therefore, the maximum concentration of M 尾 CD in this experiment was 10 mm. At the same time, cholesterol content was measured. When the concentration of M 尾 CD was 10 mm, the cholesterol content of cell membrane decreased to 37. After supplementation of exogenous cholesterol, the cholesterol concentration recovered accordingly, and when the filling cholesterol concentration was 200 渭 M, the cholesterol content of the cell membrane decreased to 37 渭 M. Cholesterol content returned to close to that before treatment with M 尾 CD, and the cholesterol of IBV capsule membrane was removed effectively. After treated with 10 mm M 尾 CD, the cholesterol content of virus capsule membrane decreased to about 66. In this study, Real Time PCR, virus titer test and Western Blot were used to detect the effect of cell membrane cholesterol on the different processes of IBV M41 cell infection in CEK cells. The results showed that adding different concentrations of M 尾 CD to remove membrane cholesterol inhibited the entry of IBV in a dose-dependent manner, and the viral infectivity and the expression of IBV N protein decreased in a gradient. In order to further confirm that the inhibition was caused by cholesterol removal, 10 mm M 尾 CD was used to remove membrane cholesterol and then replenish exogenous cholesterol. With the increase of cholesterol supplementation, the infectivity and protein expression level of IBV recovered accordingly, which indicated that removal of cholesterol could inhibit the entry of IBV into CEK cells, and the inhibition effect was reversible. The effect of cholesterol on the adsorption of IBV to CEK cells and its release from CEK cells was also studied. The results showed that the adsorption and release of IBV were inhibited in a dose-dependent manner with the increase of M 尾 CD concentration. At the same time, CEK cells were transfected with GPI-pEGFP. The results showed that IBV and GPI anchoring proteins could be co-located on the same CEK cell membrane, indicating that IBV could be located on the membrane lipid raft. Viral envelope cholesterol also plays an important role in the entry of many viruses into cells. In this study, CEK cells were infected with IBV M41 strain treated with different concentrations of M 尾 CD. The results showed that there was no obvious trend change in IBV infectivity and no significant change in the expression of IBV N protein, which proved that viral envelope cholesterol was not necessary for the entry of IBV into CEK cells. In conclusion, membrane cholesterol is required in the process of CEK cell infection, but not the viral envelope cholesterol.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

【参考文献】