新型可复制型抗肿瘤DNA疫苗PSCK-2PFcGB的抑瘤活性及免疫学机制研究

发布时间：2018-08-19 20:32

【摘要】：随着免疫学、分子生物学和细胞生物学理论与技术的不断深入发展，更高效、更安全的疫苗形式也在不断涌现。近年来，一种具有“自主复制”功能的、以RNA病毒复制元件为基础的可复制型DNA疫苗的出现是DNA疫苗发展历程中的一个重大突破。这种疫苗集合了传统DNA疫苗、RNA疫苗以及RNA复制子疫苗的优势：(1)稳定性好，便于生产、储存和运输；(2)具有自我复制能力，外源基因可以得到高效表达；(3)转录和翻译过程均在宿主细胞的细胞质内进行，大大降低了外源基因与宿主细胞基因组整合的概率，提高了安全性；(4)由于其高效的复制和翻译，占用了宿主细胞的大部分资源，最终会引起宿主细胞的凋亡而被机体清除，降低了机体的免疫耐受。综上所述，可复制型DNA疫苗在治疗性疫苗的开发中显示出了广阔的应用前景。本研究的目的在于构建一种基于塞姆利基森林病毒(Sen1ijkj forest vjms，SFv)的新型可复制型抗肿瘤DNA疫苗并对其抑瘤活性及免疫学作用机制进行深入的探讨。首先，我们将含有红色和绿色荧光蛋白报告基因的片段“DsRed．．IRES—EGFP”克隆入本实验室前期构建保存的、经过卡那霉素(kfdl]a)抗性改造的基于SFv的PSCA(命名为PSCK)载体，以验证其真核表达能力；然后将本室验证具有良好抗肿瘤活性的DNA疫苗pwL)(1—2PFcGB中的多靶点融合抗原基因片段2PFcGB克隆入PSCK载体，构建成新型可复制型抗肿瘤DNA疫苗PSCK一2PFcGB，将该重组质粒瞬时转染293T细胞检测其在体外培养的细胞中的表达能力；通过肌肉注射加上电咏冲刺激的方法免疫小鼠，剥取免疫部位肌肉组织进行免疫组化分忻，对其活体内抗原的表达进行验证。接着，我们采用PCR方法分别扩增出人Survivin、hCGB全长基因的cDNA编码区序列，利用DNA重组技术将其定向插入到真核表达载体pIRES一1]co中，，利用阳离子脂质体转染和G418筛选技术，分别筛选出能够稳定表达人SuTvivin、hCGB的小鼠黑色素瘤(B16)细胞株；利用上述细胞株以不同浓度分别接种C57BL/6小鼠，观察稳定转染外源基因后B16细胞的致瘤能力，建立人survivin、hcGBI的黑色素瘤荷瘤小鼠模型。随后，我们将编码荧光素酶基因的pGL3一CMV质粒利用肌肉注射、基因枪、电咏冲三种方式递送至BALB／c小鼠体内，在质粒递送后24—144h，连续检测小鼠体内的荧光素酶活性，通过荧光素酶活性的变化来比较经不同途径递送质粒DNA至BALB／c小鼠体内所诱导的表达效率。最后，在上述研究结果的基础上，我们通过两种不同的免疫接种策略(治疗模型、予页防模型)来研究新型可复制型抗肿瘤DNA疫苗PSCK_2PFcGB的抗肿瘤效应。同时，从细胞免疫和体液免疫两个方面，对该疫苗所诱导的免疫学效应机制进行深入探讨。我们通过肌肉注射加电咏冲刺激的方式，将该疫苗递送至荷瘤C57BL／6小鼠体内，通过观察记录各组小鼠肿瘤生长情况来研究可复制型DNA疫苗的抑瘤活性；ElJISA法检测免疫小鼠血清中特异性抗体的滴度；LDH法体外检测免疫小鼠细胞毒性T淋巴细胞(CTL)反应；Elispot法检测免疫小鼠脾淋巴细胞IFN—v的分泌；流式细胞术和病理组织切片观察的方法检测免疫小鼠的离体肿瘤组织内的肿瘤浸润淋巴细胞。结果证实：(1)经过kfdl]a抗性改造后的PS(：K载体能够高水平的表达红色和绿色荧光蛋白；成功构建了可复制型抗肿瘤DNA疫苗PS(：K_2PFcGB，利用流式细胞术及免疫组化法对该疫苗的体内外表达进行了验证。结果表明，IRES序列上游的融合抗原片段及下游的佐剂分子片段均获得良好的表达。(2)分别筛选出了能够稳定表达几SuTvivin、hCGB基因的B16细胞株，利用流式细胞仪、Weste：m blot、R_r_PCR及免疫荧光法检测了抗原分子的表达，为研究新型抗肿瘤DNA疫苗PSCK_2PFcGB的抑瘤作用机制提供了细胞模型；同时我们还建立了SuTvivhlI、hCGB’肿瘤细胞的C57BL／6荷瘤小鼠模型，为研究该疫苗的抑瘤活性研究提供了动物模型。(3)与传统的裸DNA质粒直接肌肉注射比较，电咏冲和基因枪均能有效的导入外源基因并提高外源基因在活体内的表达效率。相比较而言，电咏冲技术在肌肉注射DNA质粒后，加上电咏冲刺激即可完成，此方法可直接递送裸DNA质粒，操作简便、递送效率高、DNA的制备成本低廉。此外，DNA的储存和运输亦更加方便。(4)通过对免疫小鼠移植瘤生长趋势的观察和免疫学机制的研究，证明了新型可复制型抗肿瘤DNA疫苗PSCK_2PFcGB在两种免疫策略下均可有效地抑制肿瘤生长，并能诱导出较高水平的特异性细胞免疫和体液免疫应答。本文主要以提高抗肿瘤DNA疫苗的免疫效力及增强疫苗安全性作为研究对象，在疫苗构建策略上，我们应用了基于SFV的可复制型DNA疫苗载体PSCK，该载体可以最大限度的提高外源基因的表达量且相对安全；在抗原选择上，将人Survivin的主要T细胞抗原表位区域和人、猴的hCGB—CTP37基因利用连接臂进行连接，构成异种化复合抗原2PAG，同时引入了人IgG Fc、GM—CSF和B7．1等免疫黏附分子、细胞因子及共刺激分子以增强免疫效力，含有该融合抗原片段的DNA疫苗pwL)(1—2PFcGB在动物实验中取得了较好的抗肿瘤效应。在靶细胞选择上，为了更好的评价疫苗抑瘤效果，我们建立了能稳定表达人Survivin、hCGB基因的小鼠黑色素瘤细胞模型及SurvivilI、hCGB’肿瘤细胞的荷瘤小鼠模型；在DNA疫苗递送方式上，我们对常用递送方式的递送效率进行了比较，选择了一种高效、稳定、便捷的疫苗递送途径；在免疫策略上，根据DNA疫苗的特性设计了两种不同的免疫方案，以此全面评估DNA疫苗的抑瘤效果。综上所述，本研究在疫苗载体的应用、DNA递送方式和免疫策略的制定等多方面进行了新的尝试，证实所构建的新型可复制型抗肿瘤DNA疫苗PSCK一2PFcGB可有效地诱导免疫小鼠体内特异性的细胞免疫及体液免疫反应，能够有效地抑制移植瘤生长。这些结果为该疫苗进一步的药效学及药理学实验研究提供了依据、为该疫苗将来可能的临床应用奠定了良好的研究基础、为恶性肿瘤的予页防和治疗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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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