本文選題：樹突狀細(xì)胞　切入點(diǎn)：膀胱腫瘤　出處：《復(fù)旦大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的樹突狀細(xì)胞(dendritic cell, DC)是迄今為止所知道的抗原遞呈功能最強(qiáng)的專職抗原遞呈細(xì)胞(antigen presenting cell, APC),也是唯一可以激活初始免疫反應(yīng)的APC,在對(duì)腫瘤產(chǎn)生主動(dòng)免疫上起著重要的作用。樹突狀細(xì)胞瘤苗的制備與應(yīng)用成為了腫瘤免疫治療的研究熱點(diǎn)。本實(shí)驗(yàn)研究輻射凋亡腫瘤細(xì)胞誘導(dǎo)的DC疫苗的制備及對(duì)C57BL/6小鼠負(fù)載膀胱腫瘤的免疫學(xué)效應(yīng)。方法采用輻射法獲取MB49細(xì)胞抗原并用其致敏骨髓來源的DC來制備DC疫苗。DC采用骨髓分離法從C57BL/6小鼠骨髓中分離細(xì)胞,接種于含10%胎牛血清的RPMI1640培養(yǎng)基中,加入重組鼠粒巨噬細(xì)胞集落刺激因子(rmGM-CSF)、重組鼠白細(xì)胞介素4(rmIL-4),體外誘導(dǎo)培養(yǎng)為未成熟DC,于第6天分別加入經(jīng)輻射獲取的凋亡的MB49小鼠膀胱癌細(xì)胞,刺激使其成熟,制備膀胱腫瘤疫苗。觀察其形態(tài)同時(shí)流式細(xì)胞儀檢測(cè)各組成熟DC表面分子CD80、CD86的表達(dá)情況。用MB49小鼠膀胱癌細(xì)胞建立荷瘤動(dòng)物模型,隨機(jī)分為實(shí)驗(yàn)組和實(shí)驗(yàn)對(duì)照組,于腫瘤細(xì)胞接種后第7、14天給予相應(yīng)DC疫苗治療或者PBS,每組分2亞組,分別用于測(cè)量瘤質(zhì)量、體積及用于觀察荷瘤小鼠存活情況。結(jié)果經(jīng)鑒定建立的以DC為基礎(chǔ)的膀胱腫瘤疫苗具有典型DC形態(tài)學(xué)和表型特征。DC疫苗擴(kuò)增到第5天,通過檢測(cè)表面抗體顯示CDllc高表達(dá),但是CD80,CD86以及I-A低表達(dá),提示為未成熟DC細(xì)胞。加入.LPS共同培養(yǎng)后第7天通過檢測(cè)CD80,CD86以及I-A提示DC細(xì)胞成熟。培養(yǎng)至第5天的小鼠骨髓來源的未成熟DC和經(jīng)照射獲得的凋亡小鼠膀胱癌細(xì)胞及無血清共培養(yǎng)1天后,經(jīng)過流式細(xì)胞儀測(cè)定顯示未成熟DC細(xì)胞成為成熟的DC細(xì)胞。經(jīng)過X射線照射三天后腫瘤細(xì)胞的凋亡達(dá)到最佳數(shù)目。負(fù)載輻射凋亡腫瘤細(xì)胞抗原的DC疫苗實(shí)驗(yàn)組荷瘤鼠腫瘤平均體積和平均瘤質(zhì)量均顯著低于對(duì)照組(P0.01),生存期長于對(duì)照組,并且有2只小鼠無瘤長期存活。經(jīng)DC疫苗實(shí)驗(yàn)組治愈的2只小鼠30天后無腫瘤生長,再次皮下接種MB49細(xì)胞觀察30天仍無腫瘤發(fā)生。結(jié)論DC是功能最強(qiáng)大的抗原提呈細(xì)胞,負(fù)載輻射凋亡腫瘤細(xì)胞的DC疫苗對(duì)膀胱腫瘤荷瘤小鼠具有抑瘤效應(yīng)和生存期延長作用。我們建立的DC疫苗誘導(dǎo)的特異性CTL對(duì)MB49小鼠膀胱癌細(xì)胞建立荷瘤動(dòng)物模型具有的很強(qiáng)的細(xì)胞毒活性。負(fù)載腫瘤抗原的DC疫苗可誘導(dǎo)機(jī)體產(chǎn)生并維持長期的腫瘤記憶性細(xì)胞免疫反應(yīng),對(duì)膀胱腫瘤細(xì)胞再次攻擊具有免疫保護(hù)作用,在膀胱腫瘤復(fù)發(fā)預(yù)防中具有良好應(yīng)用前景。充分說明以DC為中心的腫瘤生物治療有望提高膀胱癌綜合治療水平。
[Abstract]:Objective dendritic cells (DCs) are the most potent antigen-presenting cells known to date for antigen presenting presenting cells, and are the only ones that can activate the initial immune response. The preparation and application of dendritic cell tumor vaccine has become the focus of tumor immunotherapy. The preparation of DC vaccine induced by radiation apoptosis and the immunology of bladder tumor loaded with C57BL/6 mice. Methods MB49 cell antigen was obtained by radiometric method and DC from bone marrow was sensitized to prepare DC vaccine. DC cells were isolated from the bone marrow of C57BL/6 mice by bone marrow separation method. Inoculated in RPMI1640 medium containing 10% fetal bovine serum, The recombinant mouse granulocyte macrophage colony-stimulating factor (rmGM-CSFN) was added, and the recombinant mouse interleukin-4rmIL-4 was induced to become immature DCin in vitro. On the 6th day, the apoptotic MB49 mouse bladder cancer cells were added separately to stimulate them to mature. The bladder tumor vaccine was prepared and the expression of CD80 CD86 on the surface of mature DC in each group was detected by flow cytometry. The tumor bearing animal model was established by using MB49 mouse bladder cancer cells and was randomly divided into experimental group and experimental control group. The tumor cells were treated with corresponding DC vaccine or PBS14 days after tumor cell inoculation. Each group was divided into 2 subgroups, which were used to measure tumor quality. Results the DC based bladder tumor vaccine had typical DC morphologic and phenotypic characteristics. The DC vaccine was amplified to 5 days later, and the surface antibody was detected to show the high expression of CDllc. But CD80, CD86 and I-A are low expressed, On the 7th day after co-culture with LPS, CD80 CD86 and I-A indicated that DC cells matured. Immature DC derived from bone marrow of mice cultured to the 5th day and apoptotic mouse bladder cancer were obtained by irradiation. Cell and serum-free co-culture for 1 day, Flow cytometry analysis showed that immature DC cells became mature DC cells. After three days of X-ray irradiation, the apoptosis of tumor cells reached the best number. DC vaccine loaded with radiation apoptosis tumor cell antigen. The mean tumor volume and mean tumor mass of the tumor mice were significantly lower than that of the control group (P 0.01), and the survival time was longer than that of the control group. Two mice survived without tumor for a long time. 2 mice cured by DC vaccine had no tumor growth after 30 days, and no tumor occurred after subcutaneously inoculating MB49 cells again for 30 days. Conclusion DC is the most powerful antigen presenting cell. DC vaccine loaded with radiation apoptosis tumor cells can inhibit tumor and prolong the survival of bladder tumor bearing mice. A tumor bearing animal model of MB49 mice was established by specific CTL induced by DC vaccine. DC vaccine loaded with tumor antigen can induce the body to produce and maintain long-term tumor memory cellular immune response. It has a good application prospect in the prevention of bladder cancer recurrence, which indicates that the tumor biotherapy with DC as the center is expected to improve the comprehensive treatment level of bladder cancer.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.14

【共引文獻(xiàn)】

相關(guān)期刊論文 前4條

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本文編號(hào)：1646604