骨桥蛋白调节树突状细胞对乙肝病毒的反应

发布时间：2018-01-19 22:52

本文关键词： 树突状细胞骨桥蛋白乙型肝炎病毒 HBcAg HBsAg　出处：《浙江大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究背景及目的乙型肝炎病毒(hepatitis B virus, HBV)感染目前仍是严重影响中国乃至世界人类健康的重要公共卫生问题。但目前发病机制不明。病毒性肝炎的发生、发展过程中均与免疫细胞的活化及细胞因子的产生有密切的关系。树突状细胞(Dendriticcells,DC)在乙型肝炎病毒的识别及抗原呈递过程中起重要作用,影响病毒性肝炎病理生理过程中Thl、Th2的活化。研究发现DC既可诱导对肝炎病毒的免疫,又可诱导免疫耐受,取决于DC的活化状态。并有研究发现在HBV病毒未清除患者中DC的功能受损,并在抗病毒治疗后,病毒清除过程中DC的功能得到一定的恢复,同时伴随着T细胞功能的恢复。骨桥蛋白(Osteopontin, OPN)是一种分泌性磷酸化糖蛋白,作为免疫调节因子在DC的分化,成熟、存活方面发挥重要作用。本实验室之前的研究证明OPN在多种肝脏疾病中大量表达,与肝炎、肝癌、肝脏纤维化的发生发展及预后密切相天。本实验目的在于揭示在乙肝病毒感染过程中骨桥蛋白对树突状细胞的成熟以及分泌功能的影响,进而研究乙型肝炎中骨桥蛋白对树突状细胞作用的调节作用。材料及方法分别取野生鼠与骨桥蛋白基因敲除小鼠骨髓细胞进行培养,并给与IL-4和GM-CSF诱导向DC方向的分化。于第六天分别给予刺激：LPS, HepG2.2.15培养上清,HepG2培养上清,HBcAg,HBsAg,以及空白对照,共十二组。于48h后收集细胞及培养上清。通过流式细胞仪检测DC表面CD分子的表达,通过ELISA检测培养上清中细胞因子水平分别取野生鼠与骨桥蛋白基因敲除小鼠脾脏单个核细胞,并通过磁珠分选获得CD4+T细胞,野生鼠来源DC或骨桥蛋白基因敲除小鼠来源DC分别与野生鼠来源或骨桥蛋白基因敲除小鼠来源CD4+T细胞共培养,培养24h后收集培养上清。通过ELISA检测培养上清中细胞因子水平。收集健康成人抗凝血分离PBMC并进行培养,使用骨桥蛋白中和性抗体阻断骨桥蛋白作用。分别给予刺激LPS,HBcAg, HBsAg,于培养24h后收集细胞和上清。通过流式细胞仪检测DC表面CD分子的表达,通过ELISA检测培养上清中细胞因子水平。结果野生型小鼠来源DC与骨桥蛋白基因敲除小鼠来源DC,在无抗原刺激情况下表面CD80,CD86,MHC-II分子表达无明显差异,在接受HepG2.2.15培养上清或HBsAg刺激后,骨桥蛋白基因敲除小鼠骨髓来源DC表达CD80,CD86, MHC-II分子明显下降,其成熟受到抑制。但HBcAg刺激则使骨桥蛋白基因敲除小鼠来源DC的CD80,CD86,MHC-II分子表达有所升高。通过对DC分泌IFN-a,IL-12的分析,发现在无抗原刺激时骨桥蛋白缺陷的DC与野生鼠来源DC相比IFN-a分泌水平降低,在HBV抗原刺激后仍低于野生鼠来源的DC的分泌水平,IL-12的表达类似。还发现,虽然在HBcAg抗原刺激后骨桥蛋白基因敲除小鼠来源DC表面CD分子表达升高,但其分泌功能是下降的。通过对不同来源DC与相同来源CD4+T的共培养并检测Th1细胞因子以及Th2细胞因子,发现骨桥蛋白基因敲除小鼠来源DC的共培养与野生鼠来源DC的共培养相比,培养上清中Thl细胞因子TNF-a分泌减少,而Th2细胞因子IL-4,IL-6,IL-10的产生水平升高。同时通过对相同来源DC与不同来源CD4+T的共培养发现来源相同的DC和来源不同的CD4+T共培养对此实验中产生细胞因子无明显影响,更进一步证明在DC诱导的T细胞反应过程中DC是否产生骨桥蛋白对细胞因子的产生起决定的作用。通过人PBMC培养并给予骨桥蛋白中和性抗体封闭骨桥蛋白功能,发现骨桥蛋白抗体使DC表面CD80,CD86的水平降低,在HBV抗原刺激后保持同样的趋势,说明骨桥蛋白功能被阻断后,DC的成熟受到抑制。通过人PBMC培养并给予骨桥蛋白中和性抗体来封闭骨桥蛋白功能,发现骨桥蛋白功能被阻断后可导致DC分泌IL-12功能下降,同时Thl型细胞因子IFN-a分泌降低,而Th2型细胞因子分泌水平升高。结论 1.DC在发育和成熟过程中能够分泌大量骨桥蛋白,并且骨桥蛋白在DC发育以及成熟过程中发挥重要作用。 2.骨桥蛋白的缺乏或不足抑制HBV抗原刺激后DC表面CD80,CD86分子的表达,使DC的成熟受抑制。 3.骨桥蛋白的缺乏或不足导致HBV抗原刺激后DC分泌细胞因子水平下降,分泌功能受影响。 4.骨桥蛋白的缺乏或不足导致DC表型和功能的缺陷使DC在HBV抗原刺激后诱导Thl细胞活化这一过程受到抑制,导致Th1/Th2细胞因子的失衡,从而影响DC在乙肝病毒感染过程中发挥功能。
[Abstract]:Background and purpose of research
Hepatitis B virus (HBV) infection is still an important public health problem that seriously affects human health in China and the world. However, the pathogenesis is unknown.
Viral hepatitis occurred in the process of the development and activation of immune cells and cytokines are closely related. Dendritic cells (Dendriticcells, DC) play an important role in the recognition and antigen presentation of hepatitis B virus in viral hepatitis, influence of pathological physiology process in Thl, the activation of Th2. The study found DC can induce immunity to hepatitis B virus, and can induce immune tolerance and activation depends on the state of DC. And studies have found that damaged in the HBV virus is not clear in patients with DC, and after antiviral therapy, viral clearance during DC function was recovered to a certain extent, with the function of T cells recovery.
Osteopontin (Osteopontin, OPN) is a secreted phosphorylated glycoprotein, as immune regulator in mature DC differentiation, and play an important role in survival. Our previous research proved that high expression of OPN in various liver diseases and hepatitis, liver cancer, liver fibrosis occurrence development and prognosis was closely related to the day.
The purpose of this experiment is to reveal the effect of osteopontin on the maturation and secretion function of dendritic cells in the process of HBV infection, so as to further investigate the regulatory role of osteopontin in dendritic cells in hepatitis B virus infection.
Materials and methods
Take wild mouse gene and osteopontin knockout mice bone marrow cells were cultured in differentiation and give IL-4 and induced by GM-CSF to DC direction. On the sixth day were given stimulus: LPS, supernatant of HepG2.2.15 culture supernatant, HepG2, HBcAg, HBsAg, and blank control, a total of twelve groups. After 48h cells and culture supernatants were collected. Expression using flow cytometry DC surface CD molecules, cytokines levels of culture supernatant was detected by ELISA
Were collected from the wild rats and osteopontin gene knockout mice spleen mononuclear cells and CD4+T cells by immunomagnetic sorting, gene DC or wild mouse osteopontin knockout mice derived respectively with DC gene sources or wild mouse osteopontin knockout mice CD4+T cells were co cultured, 24h culture supernatant was collected. Cultivation of cytokines in the supernatant was detected by ELISA.
Collect healthy adult anticoagulant PBMC isolated and cultured, using osteopontin neutralizing antibody blocking osteopontin. Stimulation of LPS were given HBcAg, HBsAg, and 24h cells were collected and cultured in the supernatant. The expression was detected by flow cytometry DC surface CD molecules, cytokines levels of culture supernatant was detected by ELISA.
Result
Wild type mice from DC and OPN knock-out mice. DC, CD86 in the absence of surface antigen stimulation CD80 cases, the expression of MHC-II had no significant difference in the HepG2.2.15 culture supernatant or after HBsAg stimulation, osteopontin gene knockout mouse bone marrow-derived DC expression of CD80, CD86, MHC-II molecules decreased, the inhibition of mature but the HBcAg. Stimulation of OPN knock-out mice. DC, CD80, CD86, MHC-II expression increased.
By IFN-a on the secretion of DC, IL-12 analysis, found in the absence of antigen stimulation of osteopontin deficient DC and DC from wild type mice compared to IFN-a secretion levels decreased in HBV after antigen stimulation is still lower than the level of DC secretion from wild type mice, the expression of IL-12. Also found that although the osteopontin gene on HBcAg after antigen stimulation mice derived DC surface expression of CD molecules increased, but its secretion was decreased.
According to the different sources of DC and CD4+T were co cultured with the same source and detection of Th1 cytokines and Th2 cytokines, found that osteopontin knockout mice co cultured DC were co cultured with DC from wild type mice compared to the culture supernatant of Thl cytokine TNF-a secretion decreased, while Th2 cytokines IL-4, IL-6, elevated level IL-10.
At the same time through the co culture of the same source of DC and CD4+T from different sources found the source of the same DC and different sources of CD4+T co culture in this experiment to produce cytokines had no significant effect, further proof of DC whether OPN secreted plays a crucial role in the response of T cells induced by DC process.
Through human PBMC culture and osteopontin neutralization antibody blocking osteopontin function, we found that osteopontin antibody reduced the level of CD80 and CD86 on DC surface, and maintained the same trend after HBV antigen stimulation, indicating that the maturation of DC was inhibited after the function of osteopontin was blocked.
Through human PBMC culture and osteopontin neutralization antibody to block the function of osteopontin, it is found that after blocking the function of osteopontin, it can cause DC to secrete IL-12 function decline, while Thl type cytokine IFN-a secretion decreases, while Th2 type cytokine secretion level rises.
conclusion
1.DC can secrete a large number of Osteopontin during development and maturation, and osteopontin plays an important role in the development and maturation of DC.
The lack or deficiency of 2. osteopontin inhibits the expression of CD80 and CD86 molecules on the surface of DC after HBV antigen stimulation, which inhibits the maturation of DC.
The lack or deficiency of 3. osteopontin leads to a decrease in the level of DC secretory cytokines after HBV antigen stimulation, and the secretory function is affected.
4., deficiency or deficiency of osteopontin leads to the defect of DC phenotype and function, which inhibits DC activation induced by HBV antigen and induces Thl cell activation, resulting in imbalance of Th1/Th2 cytokines, thereby affecting DC in the process of HBV infection.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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