IL2、ASGR1和Cav-1在乙型肝炎病毒入胞过程中的作用
发布时间：2018-09-07 08:08
【摘要】：背景：由于缺乏有效的体外乙肝病毒感染的模型,乙肝病毒入胞早期过程的研究进展十分缓慢,在一定程度上影响了乙型肝炎及其相关肝脏疾病的基础和临床研究。越来越多的研究表明受体在病毒入胞过程中发挥了一定的作用。 目的：建立一个I-IBV体外自然感染HepG2细胞的模型,探讨与HBV入胞相关的分子和受体。 方法：收集]3BeAg阳性、高拷贝(HBV-DNA1×108copies/ml)乙肝病人血清；未注射过乙肝疫苗的乙肝两对半全阴的正常人血清；以及2.2.15细胞上清。用PEG沉淀和蔗糖密度梯度离心得到去除病毒的高拷贝病人血清和2.2.15细胞病毒颗粒。将高拷贝血清和2.2.15细胞的病毒混合物作为实验组,正常人血清和2.2.15细胞的病毒混合物作为阴性对照组,和HepG2细胞共孵育,用DMSO处理六天的HepG2细胞加实验组血清作为阳性对照组。24h后去除旧培养基,PBS清洗3次,胰酶消化重悬继续孵育。收集去感染后细胞上清和细胞。ELISA检测细胞培养上清中的HBsAg, PCR方法检测细胞培养上清中的HBVDNA,电镜观察细胞上清中的病毒颗粒,组织化学、共聚焦和western blot观察细胞中的HBcAg。然后利用抗体中和方法中和血清中的IL-2, IL-12,定量PCR观察进入细胞的HBcAg的mRNA。用RNA干扰降低HepG2细胞表面的ASGR1或者CAV-1含量,PCR、western blot方法观察细胞内HBcAg含量。 结果：HBV高拷贝血清作为实验组自然感染的HepG2细胞上清中, ELISA检测到HBsAg并且持续到120h,PCR检测实验组细胞上清中的HBV DNA呈阳性,免疫电镜可以观察到细胞培养上清中Dane样颗粒和20nm左右的杆状颗粒和球形颗粒。共聚焦、组织化学、Western Blot观察到细胞内的HBsAg和HBcAg。用抗体中和血清中IL-2后RT-PCR方法检测发现细胞内HBcAg mRNA发达也减低。RNA干扰细胞膜上的ASGR1和cav-1后,用RT-PCR和WB检测细胞内HBcAg, mRNA水平和蛋白水平的核心蛋白的表达均有所下降。 结论：建立了HBV阳性血清直接感染HepG2细胞的模型,IL-2, ASGR1,CAV-1在HBV入胞时发挥作用。
[Abstract]:Background: due to the lack of an effective model of hepatitis B virus infection in vitro, the progress in the early stage of hepatitis B virus entry is very slow, which to some extent affects the basic and clinical study of hepatitis B and its related liver diseases. More and more studies have shown that receptors play a role in the process of virus entry. Aim: to establish a model of I-IBV naturally infecting HepG2 cells in vitro and to explore the molecules and receptors associated with HBV entry. Methods: 3BeAg positive, high copy (HBV-DNA1 脳 108copies/ml) hepatitis B patients' serum, two pairs and half negative normal serum without hepatitis B vaccine, and 2.2.15 cell supernatant were collected. High copy patient serum and 2.2.15 cell viral particles were obtained by PEG precipitation and sucrose density gradient centrifugation. The virus mixture of high-copy serum and 2.2.15 cells was used as experimental group, and that of normal human serum and 2.2.15 cell as negative control group, and incubated with HepG2 cells. The HepG2 cells were treated with DMSO for six days and the serum of the experimental group was used as the positive control group. 24 hours later, the old culture medium was cleaned for 3 times, and the trypsin digestion was incubated again. Detection of HBsAg, PCR in supernatant of cell culture by HBsAg, PCR electron microscope observation of virus particles in supernatant, histochemistry, confocal detection and western blot observation of HBcAg. in supernatant of cell culture Then the mRNA. of the HBcAg entering the cell was observed by using the IL-2, IL-12, quantitative PCR in the antibody neutralization method and in the serum. RNA interference was used to reduce the content of ASGR1 or CAV-1 on the surface of HepG2 cells. Results HBsAg was detected by ELISA in the supernatant of HepG2 cells infected naturally by the high copy serum of ELISA, and the HBV DNA in the supernatant of the experimental group was detected by polymerase chain reaction (HBV DNA) for 120 hours. The Dane-like particles and the 20nm-like and spherical particles in the supernatant of cell culture were observed by immunoelectron microscopy. Confocal and histochemical observation of HBsAg and HBcAg. in cells by Western Blot The results of RT-PCR assay after antibody neutralizing IL-2 in serum showed that HBcAg mRNA developed in cells also decreased ASGR1 and cav-1 on cell membrane, and the expression of HBcAg, mRNA and core protein in cells were decreased by RT-PCR and WB. Conclusion: the model of direct infection of HepG2 cells with HBV positive serum was established. ASGR1,CAV-1 plays a role in the entry of HBV.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.62
[Abstract]:Background: due to the lack of an effective model of hepatitis B virus infection in vitro, the progress in the early stage of hepatitis B virus entry is very slow, which to some extent affects the basic and clinical study of hepatitis B and its related liver diseases. More and more studies have shown that receptors play a role in the process of virus entry. Aim: to establish a model of I-IBV naturally infecting HepG2 cells in vitro and to explore the molecules and receptors associated with HBV entry. Methods: 3BeAg positive, high copy (HBV-DNA1 脳 108copies/ml) hepatitis B patients' serum, two pairs and half negative normal serum without hepatitis B vaccine, and 2.2.15 cell supernatant were collected. High copy patient serum and 2.2.15 cell viral particles were obtained by PEG precipitation and sucrose density gradient centrifugation. The virus mixture of high-copy serum and 2.2.15 cells was used as experimental group, and that of normal human serum and 2.2.15 cell as negative control group, and incubated with HepG2 cells. The HepG2 cells were treated with DMSO for six days and the serum of the experimental group was used as the positive control group. 24 hours later, the old culture medium was cleaned for 3 times, and the trypsin digestion was incubated again. Detection of HBsAg, PCR in supernatant of cell culture by HBsAg, PCR electron microscope observation of virus particles in supernatant, histochemistry, confocal detection and western blot observation of HBcAg. in supernatant of cell culture Then the mRNA. of the HBcAg entering the cell was observed by using the IL-2, IL-12, quantitative PCR in the antibody neutralization method and in the serum. RNA interference was used to reduce the content of ASGR1 or CAV-1 on the surface of HepG2 cells. Results HBsAg was detected by ELISA in the supernatant of HepG2 cells infected naturally by the high copy serum of ELISA, and the HBV DNA in the supernatant of the experimental group was detected by polymerase chain reaction (HBV DNA) for 120 hours. The Dane-like particles and the 20nm-like and spherical particles in the supernatant of cell culture were observed by immunoelectron microscopy. Confocal and histochemical observation of HBsAg and HBcAg. in cells by Western Blot The results of RT-PCR assay after antibody neutralizing IL-2 in serum showed that HBcAg mRNA developed in cells also decreased ASGR1 and cav-1 on cell membrane, and the expression of HBcAg, mRNA and core protein in cells were decreased by RT-PCR and WB. Conclusion: the model of direct infection of HepG2 cells with HBV positive serum was established. ASGR1,CAV-1 plays a role in the entry of HBV.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.62
【参考文献】
相关期刊论文 前3条
1 李瑗;苏建家;杨春;曹骥;欧超;梁亮;杨芳;王琦;;乙型肝炎病毒树，
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