【摘要】：背景：由于缺乏有效的體外乙肝病毒感染的模型,乙肝病毒入胞早期過程的研究進展十分緩慢,在一定程度上影響了乙型肝炎及其相關肝臟疾病的基礎和臨床研究。越來越多的研究表明受體在病毒入胞過程中發(fā)揮了一定的作用。 目的：建立一個I-IBV體外自然感染HepG2細胞的模型,探討與HBV入胞相關的分子和受體。 方法：收集]3BeAg陽性、高拷貝(HBV-DNA1×108copies/ml)乙肝病人血清；未注射過乙肝疫苗的乙肝兩對半全陰的正常人血清；以及2.2.15細胞上清。用PEG沉淀和蔗糖密度梯度離心得到去除病毒的高拷貝病人血清和2.2.15細胞病毒顆粒。將高拷貝血清和2.2.15細胞的病毒混合物作為實驗組,正常人血清和2.2.15細胞的病毒混合物作為陰性對照組,和HepG2細胞共孵育,用DMSO處理六天的HepG2細胞加實驗組血清作為陽性對照組。24h后去除舊培養(yǎng)基,PBS清洗3次,胰酶消化重懸繼續(xù)孵育。收集去感染后細胞上清和細胞。ELISA檢測細胞培養(yǎng)上清中的HBsAg, PCR方法檢測細胞培養(yǎng)上清中的HBVDNA,電鏡觀察細胞上清中的病毒顆粒,組織化學、共聚焦和western blot觀察細胞中的HBcAg。然后利用抗體中和方法中和血清中的IL-2, IL-12,定量PCR觀察進入細胞的HBcAg的mRNA。用RNA干擾降低HepG2細胞表面的ASGR1或者CAV-1含量,PCR、western blot方法觀察細胞內(nèi)HBcAg含量。 結果：HBV高拷貝血清作為實驗組自然感染的HepG2細胞上清中, ELISA檢測到HBsAg并且持續(xù)到120h,PCR檢測實驗組細胞上清中的HBV DNA呈陽性,免疫電鏡可以觀察到細胞培養(yǎng)上清中Dane樣顆粒和20nm左右的桿狀顆粒和球形顆粒。共聚焦、組織化學、Western Blot觀察到細胞內(nèi)的HBsAg和HBcAg。用抗體中和血清中IL-2后RT-PCR方法檢測發(fā)現(xiàn)細胞內(nèi)HBcAg mRNA發(fā)達也減低。RNA干擾細胞膜上的ASGR1和cav-1后,用RT-PCR和WB檢測細胞內(nèi)HBcAg, mRNA水平和蛋白水平的核心蛋白的表達均有所下降。 結論：建立了HBV陽性血清直接感染HepG2細胞的模型,IL-2, ASGR1,CAV-1在HBV入胞時發(fā)揮作用。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R512.62

【參考文獻】

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