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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34853
Title: | 聚乙二烯胺作為DNA疫苗佐劑之研究 Studies of polyethylenimine as a DNA vaccine adjuvant |
Authors: | Yi-Fan Ma 馬依帆 |
Advisor: | 楊雅雯 |
Keyword: | 聚乙二烯胺,DNA疫苗,疫苗佐劑,癌症免疫治療, polyethylenimine,DNA vaccine,adjuvant,cancer immunotherapy, |
Publication Year : | 2006 |
Degree: | 碩士 |
Abstract: | DNA疫苗雖然比傳統活菌減毒疫苗或死菌疫苗更為安全且穩定,但由於單獨接種DNA疫苗效力有限,因此必須在疫苗佐劑的輔助下才能引起長期有效的免疫反應。在本論文中,我們嘗試使用大家熟知具有良好基因遞送能力的陽離子聚合物-聚乙二烯胺 (polyethylenimine;PEI) 作為DNA疫苗佐劑並於試管中與活體中評估其發展潛能。
首先,我們以MTT (3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) 測定法得知樹突狀細胞對於PEI的毒性耐受度為8 μg/ml,並使用界面電位測定儀與粒徑分析儀分析PEI/DNA複合物在不同N/P比例之物化特性,發現PEI/DNA複合物在N/P=10時,不僅劑型最穩定,對樹突狀細胞的基因轉染效率也最好。我們亦使用流式細胞儀分析樹突狀細胞經8 μg/ml PEI刺激24小時後的成熟活化情形,結果顯示樹突狀細胞不僅大量表現CD40、CD80、CD86等共刺激分子,其抗原捕捉能力也顯著下降,符合樹突狀細胞成熟活化的表型。細胞螢光免疫染色法和西方墨點法的結果顯示轉錄因子NFκB會在PEI的刺激下活化且轉位到細胞核,可能就是造成樹突狀細胞活化的主因。同時,我們使用2’,7’-dichlorofluorescein diacetate (DCFH-DA) 和hydroethidine (HE) 等螢光探針經流式細胞儀分析,確認了PEI刺激樹突狀細胞產生H2O2和O2-等活性氧系 (reactive oxygen species;ROS) 的情形。使用抗氧化分子ebselen會抑制ROS在樹突狀細胞的生成並降低樹突狀細胞的抗原呈現能力,再次證明了ROS在活化樹突狀細胞中扮演了重要角色。 我們使用PEI將pAc-neo-OVA質體DNA遞送至樹突狀細胞內後,發現樹突狀細胞不僅可以經由MHC class I分子呈現OVA抗原,還可引發抗原專一性的第一型免疫反應,有效的活化CD8+且具OVA專一性T細胞受器 (OVA-specific TCR) 的B3Z細胞。有別於其他基因遞送載體,我們亦從抗氧化分子ebselen的實驗證明了PEI在遞送基因的同時,能夠活化樹突狀細胞的獨特性質是其能有效引起免疫反應的原因之一。接著,我們以反轉錄聚合酵素鏈鎖反應 (RT-PCR) 分析經PEI刺激後樹突狀細胞mRNA表現量的消長情形,結果顯示僅第二型環氧化酶 (COX-2) 有大量表現的情形,卻未能觀察到其他發炎前期細胞激素如IL-1β、IL-6、 IL-12和 TNF-α等基因表現的增加。這些結果顯示PEI引起樹突狀細胞成熟活化或許並非經由模式辨識受器 (pattern recognition receptors;PRRs) 所媒介,而是經由ROS直接引起發炎反應。 在動物實驗中,我們於小鼠腳掌經皮下/皮間的途徑接種疫苗,發現接種PEI/DNA複合物疫苗的小鼠,其接種位置的樹突狀細胞會呈現OVA抗原並移行到近端淋巴結。為了證實這些樹突狀細胞能有效活化T細胞,我們也以B3Z細胞進行小鼠體內OVA專一性免疫反應鑑定 (in vivo B3Z assay)。實驗結果顯示PEI/DNA複合物疫苗能有效活化第一型免疫反應,而這些活化的樹突狀細胞也只會滯留在近端淋巴結,並不會進一步移行到脾臟。同時我們亦使用terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL) assay確認了PEI在疫苗接種位置造成的組織傷害低於FDA核准可使用在人體上的疫苗佐劑-磷酸鋁 (aluminum phosphate;AlPi) ,彰顯其安全性。接著我們使用酵素連結免疫吸附法 (enzyme linked immunosorbant assay;ELISA) 分析小鼠接種疫苗後體內抗OVA抗體的生成情形與抗體亞型轉換的情形以確認免疫系統偏極化 (immunity polarization) 的現象。而活體內的細胞毒殺試驗 (in vivo CTL assay) 亦證實PEI/DNA複合物疫苗能在小鼠體內有效活化OVA專一性毒殺型T細胞,建立長期的OVA專一性細胞免疫反應。此外,無論我們將PEI/DNA複合物疫苗應用作為預防性或治療性的癌症疫苗,都可得到不錯的療效,達到有效延緩EG7-OVA腫瘤在小鼠體內的生長速度以及提高小鼠罹癌後的存活率之目的。 為了將質體DNA作進一步的改造以期能強化DNA疫苗的腫瘤專一性靶向能力,減少治療基因在正常細胞表現的機會以降低其副作用,我們以惡性黑色素細胞瘤B16F10小鼠腫瘤模式篩選出最具療效的共刺激分子基因CD86和自殺基因caspase 3後,再選用多數腫瘤細胞會大量表現的第二型環氧化酶之驅動子 (COX-2 promoter) 驅動上述兩個治療基因並構築在重組腺相關病毒 (rAAV) 的inverted terminal repeat (ITR) 之間,希望能在轉錄階層靶向表現在B16F10腫瘤細胞上。實驗結果顯示在PEI的輔助下,此DNA疫苗的確能有效抑制惡性黑色素細胞瘤B16F10在小鼠體內的生長。 總而言之,本研究不僅提供了PEI刺激樹突狀細胞成熟活化的作用機轉、在活體實驗中發現PEI可在低毒性範圍達到有效轉染治療基因並成功建立第一型與第二型抗原專一性免疫反應,也在小鼠腫瘤模式中證實了應用PEI作為預防性或治療性DNA癌症疫苗佐劑的可行性。 DNA-based vaccines are safer and more stable than conventional protein vaccines containing live-attenuated or killed bacteria/viruses. However, the immunogenicity of this new generation vaccine is often poor when administered alone, and it often requires incorporation with the adjuvants to enhance the immune response. In the present study, polyethylenimine (PEI), a cationic polymer commonly used in gene delivery, was examined in vitro and in vivo for the potential of being a candidate adjuvant for DNA vaccines. The 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the direct cytotoxicity of PEI in the bone marrow-derived dendritic cells (BMDCs), the most potent antigen presenting cells (APCs), from C57BL/6J mice. The physicochemical properties, including size and zeta potential, and the transfection efficiency of the PEI-DNA complexes were measured at various N/P ratios. Maturation of dendritic cells is a crucial step in the initiation of an adaptive immune response. To examine the direct immuno-stimulatory effect of PEI on the APCs, the BMDCs at Day 6 were treated with PEI (8 μg/ml) for 24 hrs, followed by flow cytometric analysis of co-stimulatory molecules expression. Treatment of BMDCs with PEI for 24 hrs increased the expression of maturation markers (CD40, CD80, and CD86) of CD11c+ dendritic cells and decreased their antigen-capturing capability. Fluorescence microscopic examination and Western blot analysis demonstrated that treatment of BMDCs with PEI resuled in translocation of transcription factor NFκB (Nuclear Factor kappa B) to the nucleus. These results illustrated the involvement of NFκB signaling in immunogenicity elicited by PEI. The possible roles of oxidative stress on immunogenicity after treatment of PEI were examined in BMDCs by measuring the production of reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and superoxide (O2‾), using 2’,7’-dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE). Treatment of BMDCs with PEI exhibited a time- and dose-dependent increase in the fluorescent products 2’,7’-dichlorofluorescein (DCF) and ethidium bromide (EB), peaked at 32 μg/ml PEI. Addition of ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one), a substrate for thioredoxin reductase, suppressed the increased ROS production induced by PEI with concomitant decreased expression of co-stimulatory molecules in the BMDCs. Transfection of BMDCs with pAc-neo-OVA plasmid DNA-PEI complexes resulted in MHC-I restricted OVA presentation, as assayed by B3Z cells, a CD8+ murine T cell hybridoma expresses OVA-specific T cell receptors and transfected with LacZ reporter gene under the control of NFAT (Nuclear Factor of Activated T cells) promoter. Pretreatment of BMDCs with ebselen decreased B3Z activation without changes in MHC-I restricted antigen presentation, suggesting the roles of the ROS in adjuvant-mediated DC maturation. Analysis of the pro-inflammatory effect of PEI by reverse transcription-polymerase chain reaction (RT-PCR) illustrated that treatment of BMDCs with PEI induced significant up-regulation of COX-2 mRNA but not other pro-inflammatory cytokines tested, including IL-1β, IL-6, IL-12, and TNF-α, etc.. These results suggested that signaling mediated by toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs) may not be essential for PEI-mediated adjuvanticity. The in vivo adjuvant effect of PEI for DNA vaccines was examined in EL4/EG7-OVA syngeneic mouse lymphoma model. Immunization was carried out in C57BL/6J mice by subcutaneous/intradermal inoculation of pAc-neo-OVA plasmid DNA-PEI complexes into the footpads of the animals, followed by periodic measurements of tumor sizes, enzyme linked immunosorbant assay (ELISA) for the antibody response, and determination of immunity polarization. Our results showed that DNA vaccination when incorporated with PEI significantly reduced tumor growth, increased the survival rate of the animals, and elevated the anti-OVA antibody titers. Flow cytometric analysis of lymphoid organs after vaccination showed increased numbers of OVA-specific CD11c+ DCs migrating to draining lymph nodes in those animals injected with DNA-PEI complexes. The terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL) assay of tissue sections examined at vaccination sites also demonstrated the safety in using PEI as an adjuvant with lower toxicity compared to FDA approved adjuvant-aluminum phosphate (AlPi). The in vivo B3Z assay demonstrated that immunization of mice with DNA-PEI complexes resulted in MHC class I-restricted antigen presentation predominantly in the draining lymph nodes. The in vivo adjuvant potency of PEI was further confirmed by the significant cytotoxic effect of OVA-specific cytotoxic T lymphocytes (CTLs) in the animals immunized with pAc-neo-OVA DNA-PEI complexes, exhibiting higher protection from EG7-OVA tumor challenge. To further examine the in vivo adjuvant effect of PEI for immuno-modulating DNA vaccines, studies were extended to examine the therapeutic effect of DNA vaccination in B16F10 melanoma-bearing animals. Significant anti-tumor effect was observed after immunization of tumor-bearing mice with the DNA-PEI complexes containing the mIL-2, h caspase3, mCD80, or mCD86 genes. In an attempt to transcriptionally target tumors overexpressing COX-2, plasmids were constructed by inserting the mCD86 and C2P-caspase3△9 genes, under the control of COX-2 promoter, into the recombinant adeno-associated virus (rAAV) vectors, followed by immunization in the melanoma-bearing mice. Complexation of these constructs with PEI was shown to significantly inhibit tumor growth in the B16F10 melanoma-bearing mice. In conclusion, strategies were developed in the present study employing PEI as an adjuvant for generation of effective DNA vaccination. Our results demonstrated that PEI activated BMDCs, triggered NFκB signaling cascade via ROS production, stimulated antigen-specific type I and type II immune responses, leading to effective tumor protection in tumor-bearing animals, both for prophylactic and therapeutic DNA vaccines. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34853 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 藥學系 |
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