DCs来源的Exosomes用于抗HBV免疫治疗的实验研究

发布时间：2018-01-06 05:40

  本文关键词：DCs来源的Exosomes用于抗HBV免疫治疗的实验研究　出处：《福建医科大学》2009年硕士论文　论文类型：学位论文

  更多相关文章： Exosomes 树突状细胞 HBV 免疫治疗

【摘要】： 目的 初步探索DEXs体外抗HBV的免疫活性。 方法 以细胞因子GM-CSF、IL-4诱导培养慢性HBV携带者PBMCs来源的DCs,以HBcAg刺激,并加入TNF-α促进其成熟,通过流式细胞仪分析细胞表型,判断所收获的mDCs成熟度;采用多步骤离心法分离纯化DCs培养上清中的Exosomes,负染电镜观察并摄片,western blotting分析蛋白质性质。通过体外试验来评价mDEXs的抗HBV免疫活性,包括:ELISPOT法检测其刺激自体PBMCs产IFN-γ的能力、CCK-8法检测其刺激自体PBMCs增殖的能力、用流式细胞仪检测其刺激CD3+CD8+T细胞增殖的能力,并将它与mDCs和HBcAg的免疫活性进行比较。 结果 培养8天后镜下观察mDCs呈类圆形,表面可见许多树突状突触。流式检测所培养的HBcAg特异性mDCs ,高表达HLA-DR、CD80和CD86等分子(分别为63%、92.8%和95%)。电镜下观察成熟DCs分泌的Exosomes,直径约30-100nm,为类圆形或类椭圆形小体,western blotting证实其表达HLA-DR和CD86分子。ELISPOT结果显示:HBcAg特异性mDCs和mDEXs刺激产生的斑点数多于imDCs和imDEXs(分别为1122 vs 749和897 vs 398);HBcAg特异性mDEXs组刺激产生的斑点数明显多于HBcAg刺激组(897 vs 22);少量HBcAg特异性mDEXs与抗原非特异性imDC共同培养可显著增强imDC的刺激能力(斑点数从749上升到1394)。淋巴细胞增殖试验也显示相同趋势的结果:HBcAg特异性mDEXs体外刺激PBMCs增殖的能力大于HBcAg,但小于mDCs(吸光值分别为1.591、1.542和1.700)。mDEXs、HBcAg和mDCs与PBMCs共培养7天后,CD3+CD8+T细胞的比例由基础值的28.6%分别上升到37.3%、34%和39.4%。 结论 负载HBcAg的mDEXs在体外的抗病毒免疫活性明显强于HBcAg,但是不如负载HBcAg的mDCs;少量负载HBcAg的mDEXs在与抗原非特异性imDCs共育后能显著提高后者的免疫活性。
[Abstract]:objective
The immunological activity of DEXs in vitro against HBV was preliminarily explored.
Method
The cytokines GM-CSF, IL-4 induced by chronic HBV carriers from PBMCs DCs to HBcAg stimulation, and adding TNF- alpha promote its mature, through the analysis of the cell phenotype by flow cytometry, determine the harvest maturity of mDCs; separation and purification of DCs Exosomes in the culture supernatant by multi step centrifugation, negative staining electron microscopy and radiography, Western blotting analysis of protein properties. Including by in vitro test to evaluate the anti HBV immune activity, mDEXs: ELISPOT method was used to detect the ability to stimulate autologous PBMCs gamma producing IFN-, detect the proliferation ability of PBMCs stimulated by CCK-8 method, detected by the ability to stimulate the proliferation of CD3+CD8+T cells by flow cytometry, and the compared with mDCs and HBcAg immune activity.
Result
After 8 days of culture under the microscope mDCs were round, visible on the surface of dendritic synapses. Many specific HBcAg mDCs trained by flow cytometry, the high expression of HLA-DR, CD80 and CD86 molecules (respectively 63%, 92.8% and 95%). Exosomes observation of mature DCs secretion under electron microscope, a diameter of about 30-100nm, for the round or oval body, Western blotting confirmed that the expression of HLA-DR and CD86 molecular.ELISPOT results showed that HBcAg specific mDCs and mDEXs stimulated imDCs and imDEXs more than the number of spots (respectively 1122 and 897 vs 749 vs 398); the number of dot HBcAg specific mDEXs group stimulated significantly more than HBcAg group (897 vs 22) a small amount of HBcAg; mDEXs specific and nonspecific antigen imDC in co culture can significantly enhance the stimulatory capacity of imDC (the number of spots increased from 749 to 1394). Lymphocyte proliferation test also showed the same trend of the results: HBcAg specific mDEXs in vitro. The ability of stimulating PBMCs proliferation is greater than HBcAg, but less than mDCs (absorbance value is 1.591,1.542 and 1.700).MDEXs, HBcAg and mDCs co cultured with PBMCs for 7 days, the proportion of CD3+CD8+T cells increased from 28.6% to 37.3%, 34% and 39.4%. respectively.
conclusion
The antiviral immunity activity of mDEXs loaded with HBcAg in vitro is much stronger than that of HBcAg, but it is not as good as that of HBcAg loaded with HBcAg. A small amount of mDEXs loaded with HBcAg can significantly improve the latter's immune activity after CO incubation with antigen specific imDCs.

【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【参考文献】

相关期刊论文 前2条

1 赵岐刚,田小侠,张书广;乙肝疫苗负载的树突状细胞诱导慢性乙型肝炎患者特异性免疫应答[J];齐鲁医学检验;2004年03期

2 王健,史玉,王如美,苏安英,柴锡庆,门金娥;HBsAg致敏树突状细胞治疗原发性肝癌合并HBV感染临床研究[J];中国综合临床;2005年05期

，

本文编号：1386558