本文選題：HBV　切入點：IFN-γ　出處：《華中科技大學》2013年博士論文

【摘要】：目的 本研究發(fā)現(xiàn),在高壓水注射HBV復制小鼠模型中,IFN-y能夠延長HBV持續(xù)時間,但其具體的作用機制尚不明確。因此,本研究從病毒學及免疫學角度來探討IFN-γ延長HBV持續(xù)時間的機制。 方法 1.采用高壓水注射的方法將pAAV/HBV1.2和pCMV2/IFN-null或pAAV/HBV1.2和pCMV2/IFN-y質(zhì)粒轉染到BALB/c小鼠肝細胞內(nèi),不同時間點采集小鼠血清和肝脾標本,ELISA方法檢測小鼠血清HBV抗原和抗體,免疫組化檢測肝內(nèi)HBcAg表達水平,Real-Time PCR檢測HBV復制水平及肝內(nèi)免疫相關分子的mRNA水平,ELISPOT檢測HBV特異性免疫應答,FACS檢測肝脾內(nèi)淋巴細胞PD-1和FoxP3的表達,HE染色觀察肝臟炎性浸潤,IFCC法測血清ALT水平。觀察IFN-y對HBV的作用并探討IFN-y是否通過調(diào)節(jié)HBV特異性免疫應答及免疫負調(diào)控來延長HBV持續(xù)時間。 2.將質(zhì)粒pcDNA3.1(+)/HBs采用肌肉注射聯(lián)合電擊免疫BALB/c小鼠,免疫后2周,高壓水注射pAAV/HBV1.2和pCMV2/IFN-γ質(zhì)粒,不同時間點采集小鼠血清,ELISA法檢測HBV抗原和抗體水平,觀察IFN-y對HBV的作用。 3.高壓水注射方法將HBeAg/core-null pAAV/HBV1.2和pCMV2/IFN-γ或pcDNA3.1(+)/HBs和pCMV2/IFN-γ分別轉染BALB/c小鼠,不同時間點采集小鼠血清,ELISA檢測血清HBV抗原和抗體水平,觀察IFN-y對HBsAg持續(xù)時間的影響。 結果 1.HBV平均持續(xù)時間為：pAAV/HBV1.2組為28天、pCMV2/IFN-null處理組為22.8天、pCMV2/IFN-γ處理組為44.6天。結果表明,IFN-y顯著延長HBV在BALB/c小鼠體內(nèi)持續(xù)時間(P=0.000)。 2. IFN-y具有抗HBV作用,早期抑制HBV復制及表達,晚期促進HBV持續(xù)。 3. IFN-y抑制HBsAb和HBcAb產(chǎn)生,抑制HBV體液免疫應答。pAAV/HBV1.2注射后第10天,IFN-y抑制HBcAg特異性T細胞免疫應答；第20天,3組間沒有統(tǒng)計學差異；第30天,IFN-y促進HBcAg特異性T細胞免疫應答持續(xù)。此外,在第10、20、30天,未檢測到HBsAg特異性T細胞免疫應答。 4. IFN-y在第注射后10、20、30天均未誘導肝脾淋巴細胞PD-1及FoxP3的表達,但誘導肝內(nèi)PD-L1、NOS2、IDO、SOCS等分子高表達。 5.在pAAV/HBV1.2小鼠模型中,注射低劑量pAAV/HBV1.2(2.5μg), IFN-y延長HBV持續(xù)時間(P=0.009)；注射高劑量質(zhì)粒(50μg), IFN-y延長HBV持續(xù)時間(P=0.015)。 6. pcDNA3.1(+)/HBs基因免疫小鼠成功后,再注射pAAV/HBV1.2和pCMV2/IFN-y, IFN-y促進HBsAb產(chǎn)生,促進HBV清除。 7.在HBeAg/core-null pAAV/HBV1.2小鼠模型中,IFN-y則促進HBsAg的清除(P=0.036)。在pcDNA3.1(+)/HBs小鼠模型中,IFN-y對HBsAg清除無影響。 結論 1.在BALB/c小鼠模型中IFN-y早期抑制HBV復制和表達,同時抑制HBV體液免疫應答,并且延長HBV持續(xù)時間。 2. HBcAg引發(fā)的特異性免疫應答與病毒清除無直接關系,IFN-y也未通過誘導免疫負調(diào)控來延長病毒的持續(xù)。 3. IFN-γ延長HBV持續(xù)時間與抑制HBcAg免疫應答及HBsAb產(chǎn)生有關。本研究的創(chuàng)新點 1.在HBV復制小鼠模型中發(fā)現(xiàn)IFN-y延長HBV持續(xù)時間。 2.闡明IFN-y延長HBV持續(xù)時間與抑制HBcAg免疫應答有關。 本研究的意義 IFN-γ是一個多功能細胞因子,其作用效果和機制與病毒和宿主的多種因素有關,本研究以高壓水注射HBV復制小鼠模型為基礎,探討IFN-γ延長HBV持續(xù)時間的作用機理,為揭示HBV持續(xù)感染的機制和其誘導的免疫學效應提供實驗依據(jù)。
[Abstract]:objective
In this study, we found that IFN-y can prolong HBV duration in high pressure water injection HBV replication mice, but its specific mechanism is not clear. Therefore, this study explored the mechanism of IFN- gamma prolonging HBV duration from the perspective of Virology and immunology.
Method
1. using the method of high-pressure water injection of pAAV/HBV1.2 and pCMV2/IFN-null or pAAV/HBV1.2 and pCMV2/IFN-y plasmid was transfected into BALB/c mouse liver cells at different time points were collected in serum and liver and spleen specimens, ELISA method for detection of serum HBV antigen and antibody, immunohistochemistry to detect the expression of HBcAg in liver, immune related molecules Real-Time PCR replication and HBV detection the level of mRNA in liver, ELISPOT detection of HBV specific immune response, the expression of PD-1 and FoxP3 FACS lymphocytes detected in the liver and spleen, observation of liver inflammatory infiltration of HE staining, measured the serum level of ALT IFCC. To observe the effects of IFN-y on HBV and explore whether IFN-y by adjusting the HBV specific immune response and immune negative regulation to extend HBV duration.
2., plasmid pcDNA3.1 (+) /HBs was immunized to BALB/c mice by intramuscular injection combined with electric shock. After 2 weeks, pAAV/HBV1.2 and pCMV2/IFN- pAAV/HBV1.2 plasmid were injected with high pressure water, serum was collected at different time points, HBV antigen and antibody level were detected by ELISA, and the effect of IFN-y on HBV was observed.
3., the HBeAg/core-null pAAV/HBV1.2 and pCMV2/IFN- gamma or pcDNA3.1 (+) /HBs and pCMV2/IFN- gamma were transfected into BALB/c mice by high pressure water injection. The serum of mice was collected at different time points, and the levels of serum HBV antigen and antibody were detected by ELISA. The effect of IFN-y on the duration of HBsAg was observed.
Result
The mean duration of 1.HBV was 28 days in group pAAV/HBV1.2, 22.8 days in pCMV2/IFN-null group, and 44.6 days in pCMV2/IFN- gamma treatment group. The results showed that IFN-y significantly prolonged the duration of HBV in BALB/c mice (P=0.000).
2. IFN-y has the effect of anti HBV, which inhibits the replication and expression of HBV in the early stage, and promotes the HBV in the late stage.
3. IFN-y inhibited HBsAb and HBcAb production, inhibit the humoral immune response to HBV tenth days after.PAAV/HBV1.2 injection, IFN-y inhibited HBcAg specific T cell immune response; twentieth days, no statistically significant difference between the 3 groups; thirtieth days, IFN-y promotes HBcAg specific T cell immune response sustained. In addition, in the first 10,20,30 days, HBsAg was not detected specific T cell immune response.
4. IFN-y did not induce the expression of PD-1 and FoxP3 in liver and spleen lymphocytes at 10,20,30 days after injection, but induced the high expression of PD-L1, NOS2, IDO, SOCS and other molecules in the liver.
5. in the pAAV/HBV1.2 mouse model, low dose pAAV/HBV1.2 (2.5 g) was injected, IFN-y prolonged the duration of HBV (P=0.009), and high dose plasmid (50 g) was injected, IFN-y prolonged HBV duration (P=0.015).
After 6. pcDNA3.1 (+) /HBs gene immunization mice were successfully immunized with pAAV/HBV1.2 and pCMV2/IFN-y, IFN-y promoted HBsAb production and promoted HBV clearance.
7. in the HBeAg/core-null pAAV/HBV1.2 mouse model, IFN-y promoted the clearance of HBsAg (P=0.036). In the pcDNA3.1 (+) /HBs mouse model, IFN-y had no effect on HBsAg clearance.
conclusion
1. in the BALB/c mouse model, the early inhibition of the replication and expression of HBV and the inhibition of the HBV humoral immune response and the prolongation of the duration of HBV in the early stage of IFN-y were inhibited.
The specific immune response caused by 2. HBcAg was not directly related to the virus clearance, and IFN-y did not prolong the virus by inducing negative immuno regulation.
3. IFN- gamma prolongs the duration of HBV and is related to the suppression of HBcAg immune response and HBsAb production. The innovation of this study
1. in the HBV replicating mouse model, IFN-y was found to prolong the duration of HBV.
2. clarifies that IFN-y prolongs the duration of HBV and inhibits the immune response to HBcAg.
The significance of this study
IFN- is a multifunctional cytokine that is related to various factors and the effect and mechanism of the virus and the host, in this study, high pressure water injection of HBV mice model based on IFN- gamma extension mechanism of HBV duration, provide experimental basis for revealing the mechanism of HBV persistent infection and its inducing immunological effect.

【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

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