IFN-γ延长HBV持续时间及机制研究

发布时间：2018-04-08 16:48

  本文选题：HBV　切入点：IFN-γ　出处：《华中科技大学》2013年博士论文

【摘要】：目的 本研究发现,在高压水注射HBV复制小鼠模型中,IFN-y能够延长HBV持续时间,但其具体的作用机制尚不明确。因此,本研究从病毒学及免疫学角度来探讨IFN-γ延长HBV持续时间的机制。 方法 1.采用高压水注射的方法将pAAV/HBV1.2和pCMV2/IFN-null或pAAV/HBV1.2和pCMV2/IFN-y质粒转染到BALB/c小鼠肝细胞内,不同时间点采集小鼠血清和肝脾标本,ELISA方法检测小鼠血清HBV抗原和抗体,免疫组化检测肝内HBcAg表达水平,Real-Time PCR检测HBV复制水平及肝内免疫相关分子的mRNA水平,ELISPOT检测HBV特异性免疫应答,FACS检测肝脾内淋巴细胞PD-1和FoxP3的表达,HE染色观察肝脏炎性浸润,IFCC法测血清ALT水平。观察IFN-y对HBV的作用并探讨IFN-y是否通过调节HBV特异性免疫应答及免疫负调控来延长HBV持续时间。 2.将质粒pcDNA3.1(+)/HBs采用肌肉注射联合电击免疫BALB/c小鼠,免疫后2周,高压水注射pAAV/HBV1.2和pCMV2/IFN-γ质粒,不同时间点采集小鼠血清,ELISA法检测HBV抗原和抗体水平,观察IFN-y对HBV的作用。 3.高压水注射方法将HBeAg/core-null pAAV/HBV1.2和pCMV2/IFN-γ或pcDNA3.1(+)/HBs和pCMV2/IFN-γ分别转染BALB/c小鼠,不同时间点采集小鼠血清,ELISA检测血清HBV抗原和抗体水平,观察IFN-y对HBsAg持续时间的影响。 结果 1.HBV平均持续时间为：pAAV/HBV1.2组为28天、pCMV2/IFN-null处理组为22.8天、pCMV2/IFN-γ处理组为44.6天。结果表明,IFN-y显著延长HBV在BALB/c小鼠体内持续时间(P=0.000)。 2. IFN-y具有抗HBV作用,早期抑制HBV复制及表达,晚期促进HBV持续。 3. IFN-y抑制HBsAb和HBcAb产生,抑制HBV体液免疫应答。pAAV/HBV1.2注射后第10天,IFN-y抑制HBcAg特异性T细胞免疫应答；第20天,3组间没有统计学差异；第30天,IFN-y促进HBcAg特异性T细胞免疫应答持续。此外,在第10、20、30天,未检测到HBsAg特异性T细胞免疫应答。 4. IFN-y在第注射后10、20、30天均未诱导肝脾淋巴细胞PD-1及FoxP3的表达,但诱导肝内PD-L1、NOS2、IDO、SOCS等分子高表达。 5.在pAAV/HBV1.2小鼠模型中,注射低剂量pAAV/HBV1.2(2.5μg), IFN-y延长HBV持续时间(P=0.009)；注射高剂量质粒(50μg), IFN-y延长HBV持续时间(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促进HBsAb产生,促进HBV清除。 7.在HBeAg/core-null pAAV/HBV1.2小鼠模型中,IFN-y则促进HBsAg的清除(P=0.036)。在pcDNA3.1(+)/HBs小鼠模型中,IFN-y对HBsAg清除无影响。 结论 1.在BALB/c小鼠模型中IFN-y早期抑制HBV复制和表达,同时抑制HBV体液免疫应答,并且延长HBV持续时间。 2. HBcAg引发的特异性免疫应答与病毒清除无直接关系,IFN-y也未通过诱导免疫负调控来延长病毒的持续。 3. IFN-γ延长HBV持续时间与抑制HBcAg免疫应答及HBsAb产生有关。本研究的创新点 1.在HBV复制小鼠模型中发现IFN-y延长HBV持续时间。 2.阐明IFN-y延长HBV持续时间与抑制HBcAg免疫应答有关。 本研究的意义 IFN-γ是一个多功能细胞因子,其作用效果和机制与病毒和宿主的多种因素有关,本研究以高压水注射HBV复制小鼠模型为基础,探讨IFN-γ延长HBV持续时间的作用机理,为揭示HBV持续感染的机制和其诱导的免疫学效应提供实验依据。
[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.

【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

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