新型可復制型抗腫瘤DNA疫苗PSCK-2PFcGB的抑瘤活性及免疫學機制研究
發(fā)布時間：2018-08-19 20:32
【摘要】：隨著免疫學、分子生物學和細胞生物學理論與技術的不斷深入發(fā)展，更高效、更安全的疫苗形式也在不斷涌現。近年來，一種具有“自主復制”功能的、以RNA病毒復制元件為基礎的可復制型DNA疫苗的出現是DNA疫苗發(fā)展歷程中的一個重大突破。這種疫苗集合了傳統DNA疫苗、RNA疫苗以及RNA復制子疫苗的優(yōu)勢：(1)穩(wěn)定性好，便于生產、儲存和運輸；(2)具有自我復制能力，外源基因可以得到高效表達；(3)轉錄和翻譯過程均在宿主細胞的細胞質內進行，大大降低了外源基因與宿主細胞基因組整合的概率，提高了安全性；(4)由于其高效的復制和翻譯，占用了宿主細胞的大部分資源，最終會引起宿主細胞的凋亡而被機體清除，降低了機體的免疫耐受。綜上所述，可復制型DNA疫苗在治療性疫苗的開發(fā)中顯示出了廣闊的應用前景。 本研究的目的在于構建一種基于塞姆利基森林病毒(Sen1ijkj forest vjms，SFv)的新型可復制型抗腫瘤DNA疫苗并對其抑瘤活性及免疫學作用機制進行深入的探討。 首先，我們將含有紅色和綠色熒光蛋白報告基因的片段“DsRed．．IRES—EGFP”克隆入本實驗室前期構建保存的、經過卡那霉素(kfdl]a)抗性改造的基于SFv的PSCA(命名為PSCK)載體，以驗證其真核表達能力；然后將本室驗證具有良好抗腫瘤活性的DNA疫苗pwL)(1—2PFcGB中的多靶點融合抗原基因片段2PFcGB克隆入PSCK載體，構建成新型可復制型抗腫瘤DNA疫苗PSCK一2PFcGB，將該重組質粒瞬時轉染293T細胞檢測其在體外培養(yǎng)的細胞中的表達能力；通過肌肉注射加上電詠沖刺激的方法免疫小鼠，剝取免疫部位肌肉組織進行免疫組化分忻，對其活體內抗原的表達進行驗證。 接著，我們采用PCR方法分別擴增出人Survivin、hCGB全長基因的cDNA編碼區(qū)序列，利用DNA重組技術將其定向插入到真核表達載體pIRES一1]co中，，利用陽離子脂質體轉染和G418篩選技術，分別篩選出能夠穩(wěn)定表達人SuTvivin、hCGB的小鼠黑色素瘤(B16)細胞株；利用上述細胞株以不同濃度分別接種C57BL/6小鼠，觀察穩(wěn)定轉染外源基因后B16細胞的致瘤能力，建立人survivin、hcGBI的黑色素瘤荷瘤小鼠模型。 隨后，我們將編碼熒光素酶基因的pGL3一CMV質粒利用肌肉注射、基因槍、電詠沖三種方式遞送至BALB／c小鼠體內，在質粒遞送后24—144h，連續(xù)檢測小鼠體內的熒光素酶活性，通過熒光素酶活性的變化來比較經不同途徑遞送質粒DNA至BALB／c小鼠體內所誘導的表達效率。 最后，在上述研究結果的基礎上，我們通過兩種不同的免疫接種策略(治療模型、予頁防模型)來研究新型可復制型抗腫瘤DNA疫苗PSCK_2PFcGB的抗腫瘤效應。同時，從細胞免疫和體液免疫兩個方面，對該疫苗所誘導的免疫學效應機制進行深入探討。我們通過肌肉注射加電詠沖刺激的方式，將該疫苗遞送至荷瘤C57BL／6小鼠體內，通過觀察記錄各組小鼠腫瘤生長情況來研究可復制型DNA疫苗的抑瘤活性；ElJISA法檢測免疫小鼠血清中特異性抗體的滴度；LDH法體外檢測免疫小鼠細胞毒性T淋巴細胞(CTL)反應；Elispot法檢測免疫小鼠脾淋巴細胞IFN—v的分泌；流式細胞術和病理組織切片觀察的方法檢測免疫小鼠的離體腫瘤組織內的腫瘤浸潤淋巴細胞。 結果證實：(1)經過kfdl]a抗性改造后的PS(：K載體能夠高水平的表達紅色和綠色熒光蛋白；成功構建了可復制型抗腫瘤DNA疫苗PS(：K_2PFcGB，利用流式細胞術及免疫組化法對該疫苗的體內外表達進行了驗證。結果表明，IRES序列上游的融合抗原片段及下游的佐劑分子片段均獲得良好的表達。(2)分別篩選出了能夠穩(wěn)定表達幾SuTvivin、hCGB基因的B16細胞株，利用流式細胞儀、Weste：m blot、R_r_PCR及免疫熒光法檢測了抗原分子的表達，為研究新型抗腫瘤DNA疫苗PSCK_2PFcGB的抑瘤作用機制提供了細胞模型；同時我們還建立了SuTvivhlI、hCGB’腫瘤細胞的C57BL／6荷瘤小鼠模型，為研究該疫苗的抑瘤活性研究提供了動物模型。(3)與傳統的裸DNA質粒直接肌肉注射比較，電詠沖和基因槍均能有效的導入外源基因并提高外源基因在活體內的表達效率。相比較而言，電詠沖技術在肌肉注射DNA質粒后，加上電詠沖刺激即可完成，此方法可直接遞送裸DNA質粒，操作簡便、遞送效率高、DNA的制備成本低廉。此外，DNA的儲存和運輸亦更加方便。(4)通過對免疫小鼠移植瘤生長趨勢的觀察和免疫學機制的研究，證明了新型可復制型抗腫瘤DNA疫苗PSCK_2PFcGB在兩種免疫策略下均可有效地抑制腫瘤生長，并能誘導出較高水平的特異性細胞免疫和體液免疫應答。 本文主要以提高抗腫瘤DNA疫苗的免疫效力及增強疫苗安全性作為研究對象，在疫苗構建策略上，我們應用了基于SFV的可復制型DNA疫苗載體PSCK，該載體可以最大限度的提高外源基因的表達量且相對安全；在抗原選擇上，將人Survivin的主要T細胞抗原表位區(qū)域和人、猴的hCGB—CTP37基因利用連接臂進行連接，構成異種化復合抗原2PAG，同時引入了人IgG Fc、GM—CSF和B7．1等免疫黏附分子、細胞因子及共刺激分子以增強免疫效力，含有該融合抗原片段的DNA疫苗pwL)(1—2PFcGB在動物實驗中取得了較好的抗腫瘤效應。在靶細胞選擇上，為了更好的評價疫苗抑瘤效果，我們建立了能穩(wěn)定表達人Survivin、hCGB基因的小鼠黑色素瘤細胞模型及SurvivilI、hCGB’腫瘤細胞的荷瘤小鼠模型；在DNA疫苗遞送方式上，我們對常用遞送方式的遞送效率進行了比較，選擇了一種高效、穩(wěn)定、便捷的疫苗遞送途徑；在免疫策略上，根據DNA疫苗的特性設計了兩種不同的免疫方案，以此全面評估DNA疫苗的抑瘤效果。 綜上所述，本研究在疫苗載體的應用、DNA遞送方式和免疫策略的制定等多方面進行了新的嘗試，證實所構建的新型可復制型抗腫瘤DNA疫苗PSCK一2PFcGB可有效地誘導免疫小鼠體內特異性的細胞免疫及體液免疫反應，能夠有效地抑制移植瘤生長。這些結果為該疫苗進一步的藥效學及藥理學實驗研究提供了依據、為該疫苗將來可能的臨床應用奠定了良好的研究基礎、為惡性腫瘤的予頁防和治療提供了新的方法和思路。
[Abstract]:With the development of immunology, molecular biology and cell biology, more efficient and safer forms of vaccines have emerged. In recent years, the emergence of a replicable DNA vaccine based on RNA viral replication elements, which has the function of self-replication, is an important step in the development of DNA vaccine. Breakthroughs. This vaccine combines the advantages of traditional DNA vaccines, RNA vaccines and RNA replicon vaccines: (1) good stability, easy production, storage and transportation; (2) self-replicating ability, foreign genes can be highly expressed; (3) transcription and translation processes are carried out in the cytoplasm of the host cells, greatly reducing the number of foreign genes and The probability of genome integration of host cells improves safety; (4) Because of its efficient replication and translation, it occupies most of the resources of host cells, and eventually leads to apoptosis of host cells and is eliminated by the body, reducing the immune tolerance of the body. Broad application prospects.
The aim of this study was to construct a novel replicable antitumor DNA vaccine based on Sen1ijkj forest vjms (SFv) and to investigate its antitumor activity and immunological mechanism.
Firstly, we cloned the fragment "DsRed. IRES - EGFP" containing red and green fluorescent protein reporter gene into the SFv-based PSCA (named PSCK) vector which was constructed and preserved in our laboratory and modified by kanamycin (kfdl] a) to verify its eukaryotic expression ability; then we verified its good anti-tumor activity in our laboratory. A novel replicable antitumor DNA vaccine PSCK-2PFcGB was constructed. The recombinant plasmid was transfected into 293T cells to detect its expression in vitro. Immunohistochemical staining was performed on the immune muscles of infected mice to verify the antigen expression in vivo.
Then, we amplified the full-length gene of human Survivin and hCGB by PCR, and inserted them into the eukaryotic expression vector pIRES-1] CO by DNA recombination technology. The fine murine melanoma (B16) which could stably express human SuTvivin and hCGB was screened by cationic liposome transfection and G418 screening respectively. C57BL/6 mice were inoculated with the above cell lines at different concentrations to observe the tumorigenicity of B16 cells stably transfected with exogenous genes and establish the melanoma-bearing mice model of human survivin and hcGBI.
Subsequently, the plasmid pGL3-CMV encoding luciferase gene was delivered to BALB/c mice by intramuscular injection, gene gun and electrophoresis. The activity of luciferase in mice was detected 24-144 hours after plasmid delivery. The activity of luciferase in mice was compared by the change of luciferase activity. The expression efficiency induced by mice.
Finally, on the basis of the above results, we studied the antitumor effect of a novel replicable DNA vaccine PSCK_2PFcGB by using two different immunization strategies (treatment model and anti-tumor model). The antitumor activity of replicable DNA vaccine was studied by observing the growth of tumor in C57BL/6 mice. The titer of specific antibody in serum of immunized mice was detected by ElJISA method. The titer of specific antibody in serum of immunized mice was detected by LDH method. Cytotoxic T lymphocyte (CTL) reaction; Elispot method was used to detect the secretion of IFN-v in splenic lymphocytes of immunized mice; flow cytometry and histopathological observation were used to detect tumor infiltrating lymphocytes in tumor tissues of immunized mice.
The results showed that: (1) PS (: K vector) could express high level of red and green fluorescent protein after resistance modification of kfdl] a, and the replicable DNA vaccine PS (: K_2PFcGB) was successfully constructed. The expression of PS (: K_2PFcGB) in vitro and in vivo was verified by flow cytometry and immunohistochemistry. Antigen fragments and downstream adjuvant fragments were well expressed. (2) B16 cell lines stably expressing several SuTvivin and hCGB genes were screened out. The expression of antigen molecules was detected by flow cytometry, Weste:m blot, R_r_PCR and immunofluorescence assay to study the anti-tumor mechanism of a novel anti-tumor DNA vaccine PSCK_2PFcGB. We also established C57BL/6 tumor-bearing mice model of SuTvivhlI, hCGB'tumor cells, which provided an animal model for studying the antitumor activity of the vaccine. (3) Comparing with direct intramuscular injection of naked DNA plasmids, both electro-Yongchong and gene gun can effectively introduce foreign genes and enhance foreign genes. Comparatively speaking, this technique can be accomplished by intramuscular injection of DNA plasmid and stimulation of DNA. This method can deliver naked DNA plasmid directly. It is easy to operate, high delivery efficiency and low cost of DNA preparation. In addition, DNA storage and transportation are more convenient. (4) The growth trend of transplanted tumor in immune mice is also more convenient. Potential observation and immunological mechanism study showed that the novel replicable DNA vaccine PSCK_2PFcGB could effectively inhibit tumor growth and induce a higher level of specific cellular and humoral immune responses.
In order to improve the immune efficacy and safety of anti-tumor DNA vaccine, we applied replicable DNA vaccine vector PSCK based on SFV to construct the vaccine, which can maximize the expression of foreign genes and is relatively safe; in antigen selection, the host of human Survivin was used. For T cell epitope regions and humans, the monkey hCGB-CTP37 gene is linked by a connecting arm to form a heterologous complex antigen 2PAG. Immune adhesion molecules such as human IgG Fc, GM-CSF and B7.1, cytokines and costimulatory molecules are introduced to enhance immune efficacy. DNA vaccine pwL containing the fusion antigen fragment (1-2PFcGB) is used in animals. In order to evaluate the anti-tumor effect of the vaccine, we established a mouse melanoma cell model stably expressing human Survivin, hCGB gene and a tumor-bearing mouse model of SurvivilI, hCGB'tumor cells. In the delivery mode of DNA vaccine, we used the commonly used delivery methods. A high efficient, stable and convenient route of vaccine delivery was selected, and two different immunization schemes were designed according to the characteristics of DNA vaccine in order to evaluate the anti-tumor effect of DNA vaccine.
In summary, the new replicable DNA vaccine PSCK-2PFcGB can effectively induce specific cellular and humoral immune responses in immunized mice and inhibit transplanted tumor effectively. These results provide a basis for further pharmacodynamic and pharmacological experimental studies of the vaccine, lay a good foundation for the future clinical application of the vaccine, and provide a new method and ideas for the prevention and treatment of malignant tumors.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392
本文編號：2192753
[Abstract]:With the development of immunology, molecular biology and cell biology, more efficient and safer forms of vaccines have emerged. In recent years, the emergence of a replicable DNA vaccine based on RNA viral replication elements, which has the function of self-replication, is an important step in the development of DNA vaccine. Breakthroughs. This vaccine combines the advantages of traditional DNA vaccines, RNA vaccines and RNA replicon vaccines: (1) good stability, easy production, storage and transportation; (2) self-replicating ability, foreign genes can be highly expressed; (3) transcription and translation processes are carried out in the cytoplasm of the host cells, greatly reducing the number of foreign genes and The probability of genome integration of host cells improves safety; (4) Because of its efficient replication and translation, it occupies most of the resources of host cells, and eventually leads to apoptosis of host cells and is eliminated by the body, reducing the immune tolerance of the body. Broad application prospects.
The aim of this study was to construct a novel replicable antitumor DNA vaccine based on Sen1ijkj forest vjms (SFv) and to investigate its antitumor activity and immunological mechanism.
Firstly, we cloned the fragment "DsRed. IRES - EGFP" containing red and green fluorescent protein reporter gene into the SFv-based PSCA (named PSCK) vector which was constructed and preserved in our laboratory and modified by kanamycin (kfdl] a) to verify its eukaryotic expression ability; then we verified its good anti-tumor activity in our laboratory. A novel replicable antitumor DNA vaccine PSCK-2PFcGB was constructed. The recombinant plasmid was transfected into 293T cells to detect its expression in vitro. Immunohistochemical staining was performed on the immune muscles of infected mice to verify the antigen expression in vivo.
Then, we amplified the full-length gene of human Survivin and hCGB by PCR, and inserted them into the eukaryotic expression vector pIRES-1] CO by DNA recombination technology. The fine murine melanoma (B16) which could stably express human SuTvivin and hCGB was screened by cationic liposome transfection and G418 screening respectively. C57BL/6 mice were inoculated with the above cell lines at different concentrations to observe the tumorigenicity of B16 cells stably transfected with exogenous genes and establish the melanoma-bearing mice model of human survivin and hcGBI.
Subsequently, the plasmid pGL3-CMV encoding luciferase gene was delivered to BALB/c mice by intramuscular injection, gene gun and electrophoresis. The activity of luciferase in mice was detected 24-144 hours after plasmid delivery. The activity of luciferase in mice was compared by the change of luciferase activity. The expression efficiency induced by mice.
Finally, on the basis of the above results, we studied the antitumor effect of a novel replicable DNA vaccine PSCK_2PFcGB by using two different immunization strategies (treatment model and anti-tumor model). The antitumor activity of replicable DNA vaccine was studied by observing the growth of tumor in C57BL/6 mice. The titer of specific antibody in serum of immunized mice was detected by ElJISA method. The titer of specific antibody in serum of immunized mice was detected by LDH method. Cytotoxic T lymphocyte (CTL) reaction; Elispot method was used to detect the secretion of IFN-v in splenic lymphocytes of immunized mice; flow cytometry and histopathological observation were used to detect tumor infiltrating lymphocytes in tumor tissues of immunized mice.
The results showed that: (1) PS (: K vector) could express high level of red and green fluorescent protein after resistance modification of kfdl] a, and the replicable DNA vaccine PS (: K_2PFcGB) was successfully constructed. The expression of PS (: K_2PFcGB) in vitro and in vivo was verified by flow cytometry and immunohistochemistry. Antigen fragments and downstream adjuvant fragments were well expressed. (2) B16 cell lines stably expressing several SuTvivin and hCGB genes were screened out. The expression of antigen molecules was detected by flow cytometry, Weste:m blot, R_r_PCR and immunofluorescence assay to study the anti-tumor mechanism of a novel anti-tumor DNA vaccine PSCK_2PFcGB. We also established C57BL/6 tumor-bearing mice model of SuTvivhlI, hCGB'tumor cells, which provided an animal model for studying the antitumor activity of the vaccine. (3) Comparing with direct intramuscular injection of naked DNA plasmids, both electro-Yongchong and gene gun can effectively introduce foreign genes and enhance foreign genes. Comparatively speaking, this technique can be accomplished by intramuscular injection of DNA plasmid and stimulation of DNA. This method can deliver naked DNA plasmid directly. It is easy to operate, high delivery efficiency and low cost of DNA preparation. In addition, DNA storage and transportation are more convenient. (4) The growth trend of transplanted tumor in immune mice is also more convenient. Potential observation and immunological mechanism study showed that the novel replicable DNA vaccine PSCK_2PFcGB could effectively inhibit tumor growth and induce a higher level of specific cellular and humoral immune responses.
In order to improve the immune efficacy and safety of anti-tumor DNA vaccine, we applied replicable DNA vaccine vector PSCK based on SFV to construct the vaccine, which can maximize the expression of foreign genes and is relatively safe; in antigen selection, the host of human Survivin was used. For T cell epitope regions and humans, the monkey hCGB-CTP37 gene is linked by a connecting arm to form a heterologous complex antigen 2PAG. Immune adhesion molecules such as human IgG Fc, GM-CSF and B7.1, cytokines and costimulatory molecules are introduced to enhance immune efficacy. DNA vaccine pwL containing the fusion antigen fragment (1-2PFcGB) is used in animals. In order to evaluate the anti-tumor effect of the vaccine, we established a mouse melanoma cell model stably expressing human Survivin, hCGB gene and a tumor-bearing mouse model of SurvivilI, hCGB'tumor cells. In the delivery mode of DNA vaccine, we used the commonly used delivery methods. A high efficient, stable and convenient route of vaccine delivery was selected, and two different immunization schemes were designed according to the characteristics of DNA vaccine in order to evaluate the anti-tumor effect of DNA vaccine.
In summary, the new replicable DNA vaccine PSCK-2PFcGB can effectively induce specific cellular and humoral immune responses in immunized mice and inhibit transplanted tumor effectively. These results provide a basis for further pharmacodynamic and pharmacological experimental studies of the vaccine, lay a good foundation for the future clinical application of the vaccine, and provide a new method and ideas for the prevention and treatment of malignant tumors.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392
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