本文關(guān)鍵詞：DCs來(lái)源的Exosomes用于抗HBV免疫治療的實(shí)驗(yàn)研究　出處：《福建醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

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【摘要】： 目的 初步探索DEXs體外抗HBV的免疫活性。 方法 以細(xì)胞因子GM-CSF、IL-4誘導(dǎo)培養(yǎng)慢性HBV攜帶者PBMCs來(lái)源的DCs,以HBcAg刺激,并加入TNF-α促進(jìn)其成熟,通過(guò)流式細(xì)胞儀分析細(xì)胞表型,判斷所收獲的mDCs成熟度;采用多步驟離心法分離純化DCs培養(yǎng)上清中的Exosomes,負(fù)染電鏡觀察并攝片,western blotting分析蛋白質(zhì)性質(zhì)。通過(guò)體外試驗(yàn)來(lái)評(píng)價(jià)mDEXs的抗HBV免疫活性,包括:ELISPOT法檢測(cè)其刺激自體PBMCs產(chǎn)IFN-γ的能力、CCK-8法檢測(cè)其刺激自體PBMCs增殖的能力、用流式細(xì)胞儀檢測(cè)其刺激CD3+CD8+T細(xì)胞增殖的能力,并將它與mDCs和HBcAg的免疫活性進(jìn)行比較。 結(jié)果 培養(yǎng)8天后鏡下觀察mDCs呈類圓形,表面可見許多樹突狀突觸。流式檢測(cè)所培養(yǎng)的HBcAg特異性mDCs ,高表達(dá)HLA-DR、CD80和CD86等分子(分別為63%、92.8%和95%)。電鏡下觀察成熟DCs分泌的Exosomes,直徑約30-100nm,為類圓形或類橢圓形小體,western blotting證實(shí)其表達(dá)HLA-DR和CD86分子。ELISPOT結(jié)果顯示:HBcAg特異性mDCs和mDEXs刺激產(chǎn)生的斑點(diǎn)數(shù)多于imDCs和imDEXs(分別為1122 vs 749和897 vs 398);HBcAg特異性mDEXs組刺激產(chǎn)生的斑點(diǎn)數(shù)明顯多于HBcAg刺激組(897 vs 22);少量HBcAg特異性mDEXs與抗原非特異性imDC共同培養(yǎng)可顯著增強(qiáng)imDC的刺激能力(斑點(diǎn)數(shù)從749上升到1394)。淋巴細(xì)胞增殖試驗(yàn)也顯示相同趨勢(shì)的結(jié)果:HBcAg特異性mDEXs體外刺激PBMCs增殖的能力大于HBcAg,但小于mDCs(吸光值分別為1.591、1.542和1.700)。mDEXs、HBcAg和mDCs與PBMCs共培養(yǎng)7天后,CD3+CD8+T細(xì)胞的比例由基礎(chǔ)值的28.6%分別上升到37.3%、34%和39.4%。 結(jié)論 負(fù)載HBcAg的mDEXs在體外的抗病毒免疫活性明顯強(qiáng)于HBcAg,但是不如負(fù)載HBcAg的mDCs;少量負(fù)載HBcAg的mDEXs在與抗原非特異性imDCs共育后能顯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.

【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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本文編號(hào)：1386558