本文關(guān)鍵詞：抗體修飾腫瘤細(xì)胞疫苗實(shí)驗(yàn)研究　出處：《中國(guó)協(xié)和醫(yī)科大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 腫瘤細(xì)胞疫苗 CD20 Fc受體 樹(shù)突狀細(xì)胞 美羅華

【摘要】： 腫瘤疫苗作為生物療法之一被人們廣泛研究,尤其在防止腫瘤復(fù)發(fā)轉(zhuǎn)移中具有重要價(jià)值。腫瘤細(xì)胞疫苗含有所有的腫瘤相關(guān)抗原,所以至今腫瘤細(xì)胞疫苗仍有不可替代的優(yōu)勢(shì)。為了提高腫瘤疫苗的免疫效果,該課題研究了新型的抗體修飾腫瘤細(xì)胞疫苗。為了進(jìn)行臨床前的體內(nèi)實(shí)驗(yàn)研究,我們建立了小鼠模型。通過(guò)文獻(xiàn)查閱和抗原抗體結(jié)合空間結(jié)構(gòu)分析,選擇克隆人CD20胞外區(qū)和部分跨膜區(qū)基因與次級(jí)淋巴組織趨化因子信號(hào)肽基因區(qū)域進(jìn)行拼接,構(gòu)建了融合基因真核表達(dá)質(zhì)粒(pcDNA3f-sig-CD20)。該質(zhì)粒導(dǎo)入B16F10腫瘤細(xì)胞后篩選出穩(wěn)定克隆株,經(jīng)流式細(xì)胞法和熒光顯微鏡驗(yàn)證該細(xì)胞株(B16/CD20)表達(dá)目的蛋白。通過(guò)與臨床抗CD20單克隆抗體藥物Rituximab(美羅華)結(jié)合后制備成疫苗,治療同系C57BL/6小鼠人工肺腫瘤轉(zhuǎn)移模型,并對(duì)其抗腫瘤效應(yīng)機(jī)制進(jìn)行了初步探討。此外,應(yīng)用Rituximab修飾人CD20~+腫瘤細(xì)胞制備疫苗誘導(dǎo)抗腫瘤特異性T細(xì)胞,并用抗HLA-A0201抗體封閉實(shí)驗(yàn)驗(yàn)證MHC限制性的殺傷腫瘤作用。 研究結(jié)果顯示,在人工肺轉(zhuǎn)移腫瘤實(shí)驗(yàn)動(dòng)物模型中,該疫苗能有效的干預(yù)腫瘤結(jié)節(jié)的生長(zhǎng),延長(zhǎng)小鼠生存期,獲得較好的抗腫瘤效果。LDH釋放法測(cè)定表明:該疫苗免疫小鼠后顯著提高了脾淋巴細(xì)胞特異性殺傷腫瘤的能力。CD8~+細(xì)胞和NK刪除實(shí)驗(yàn)進(jìn)一步驗(yàn)證CD8~+T淋巴細(xì)胞在抗腫瘤轉(zhuǎn)移過(guò)程中是主要的效應(yīng)細(xì)胞。實(shí)驗(yàn)中我們還觀察到抗體修飾瘤苗不僅能誘導(dǎo)針對(duì)轉(zhuǎn)染了CD20的腫瘤細(xì)胞殺傷反應(yīng),而且還提高對(duì)野生型黑色素瘤的殺傷效應(yīng)。共聚焦顯微鏡和流式細(xì)胞測(cè)定顯示:抗體修飾瘤苗與DC孵育后能明顯增強(qiáng)DC對(duì)腫瘤細(xì)胞的捕獲率,提示該疫苗通過(guò)抗原呈遞細(xì)胞Fc受體途徑增強(qiáng)對(duì)腫瘤抗原捕捉,從而提高了抗原呈遞效果和誘導(dǎo)了特異性抗腫瘤作用。人體外實(shí)驗(yàn)系統(tǒng)表明:Rituximab修飾人的腫瘤細(xì)胞疫苗誘導(dǎo)了抗腫瘤效應(yīng)細(xì)胞。HLA-A2抗體封閉實(shí)驗(yàn)進(jìn)一步證實(shí)了該效應(yīng)細(xì)胞是MHC限制性的細(xì)胞毒性T細(xì)胞。我們建立的抗體修飾瘤苗制備方法簡(jiǎn)單,體內(nèi)使用安全有效,抗體用量較少,因此制備成本低廉。此法為臨床抗腫瘤單克隆抗體藥物開(kāi)辟了新的應(yīng)用方法,有望成為臨床腫瘤免疫治療的新手段。
[Abstract]:Tumor vaccine has been widely studied as one of biotherapy, especially in preventing tumor recurrence and metastasis. Tumor cell vaccine contains all tumor related antigens. So up to now tumor cell vaccine still has irreplaceable advantage. In order to improve the immune effect of tumor vaccine, a new antibody modified tumor cell vaccine has been studied. The mouse model was established and the antigen-antibody binding spatial structure was analyzed by literature review. The cloned human CD20 extracellular and transmembrane region genes were spliced with the secondary lymphoid tissue chemokine signal peptide region. The eukaryotic expression plasmid of fusion gene, pcDNA3f-sig-CD20, was constructed, and the stable clone strain was screened after transfection into B16F10 tumor cells. Flow cytometry and fluorescence microscopy showed that the cell line B16 / CD20 expressed the target protein. Rituxima, a clinical monoclonal antibody against CD20, was detected by Rituximab. After binding, the vaccine was prepared. The model of artificial lung tumor metastasis in the same C57BL / 6 mice was treated and the mechanism of its antitumor effect was discussed. Anti-tumor specific T cells were induced by Rituximab modified human CD20 ~ tumor cells. The anti-HLA-A0201 antibody blocking test was used to verify the anti-tumor effect of MHC. The results showed that the vaccine could effectively interfere with the growth of tumor nodules and prolong the survival time of mice in the experimental animal model of artificial lung metastasis. The results of anti-tumor effect and LDH release assay showed that the vaccine could significantly improve the ability of spleen lymphocytes to kill tumor after immunizing mice. Further verification of CD8 ~ by cell and NK deletion assay. T-lymphocytes are the main effector cells in the process of anti-tumor metastasis. We also observed that the antibody-modified tumor vaccine can not only induce the killing effect of tumor cells transfected with CD20. Confocal microscopy and flow cytometry showed that DC could significantly enhance DC capture rate of tumor cells after incubating with DC. These results suggest that the vaccine enhances the capture of tumor antigen through the FC receptor pathway of antigen-presenting cells. Thus, the antigen presentation effect and the specific anti-tumor effect were improved. The human body external experiment system showed that:. Rituximab modified human tumor cell vaccine induced anti-tumor effector cell. HLA-A2 antibody blocking assay further confirmed that the effector cell was MHC restricted cytotoxic T cell. The preparation method of antibody modified tumor vaccine was simple. It is safe and effective to use in vivo, so the preparation cost is low. This method opens up a new method for clinical anti-tumor monoclonal antibody drugs, and is expected to become a new method of clinical tumor immunotherapy.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392;R730.5

【共引文獻(xiàn)】

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