负载dsNKG2D-IL-21重组基因的壳聚糖纳米颗粒抑制小鼠结肠癌的研究

发布时间：2018-04-17 21:55

本文选题：IL-21 + NKG2D　；参考：《扬州大学》2017年硕士论文

【摘要】：IL-21是2000年发现的新型Ⅰ型细胞因子,属于IL-2家族一员,且共用一条γc链。IL-21主要由活化的CD4+T细胞(包括Th17细胞,滤泡性辅助性T细胞以及NKT细胞)产生,而同时IL-21也能诱导细胞活化,促使细胞增殖或者刺激细胞发挥细胞毒作用等等。IL-21的受体(IL-21R)是由IL-21Rα和普通γ链组成的异源二聚体,主要由淋巴细胞(比如NK细胞,T细胞,B细胞和NKT细胞)及骨髓细胞(比如巨噬细胞和树突状细胞)表达,而非免疫细胞(比如肠成纤维细胞和角质形成细胞)也能表达。NKG2D是一种C型凝集素样活化受体,由NK,NKT,CD8 + T和γδT细胞广泛表达。小鼠NKG2D配体由视黄酸早期转录因子(RAE-1),H60和鼠UL-16结合蛋白白样转录物1(MULT1)组成,人类NKG2D配体是主要组织相容性复合物Ⅰ类相关蛋白A和B(MICA和MICB)以及ULBP6-1。在大多数正常组织中NKG2D配体通常不表达,但病毒感染的细胞或肿瘤细胞能够诱导NKG2D配体的表达,因此,NKG2D的配体在免疫治疗中能够作为肿瘤靶向抗原。基于NKG2D配体能够靶向肿瘤的特点,本课题利用NKG2D的胞外区与IL-21基因片段重组形成dsNKG2D-IL-21重组基因,并插入到pcDNA3.1(一)质粒中构成重组质粒,研究dsNKG2D-IL-21目的蛋白的抗肿瘤活性。要将重组基因递送进细胞内必须借助生物载体,因此利用经过化学方法修饰过的强离子型化合物——壳聚糖(HTCC)来包裹重组质粒pcDNA3.1-dsNKG2D-IL21形成壳聚糖纳米颗粒(NP),大小200-400nm,表面带正电荷。MTS/PMS法发现纳米颗粒对CT-26细胞、NIH-3T3细胞以及RAW264.7细胞的毒性很小,且经纳米颗粒转染过的CT-26细胞能表达目的蛋白dsNKG2D-IL-21,而注射纳米颗粒的正常Balb/c小鼠其血清中也检测到了 IL-21的表达,证明本课题组制备的纳米颗粒能够在细胞水平或者宿主体内表达目的蛋白dsNKG2D-IL-21。注射纳米颗粒的荷瘤小鼠,其肿瘤体积明显比未注射的小鼠小,且肿瘤生长更缓慢,其脾脏及肿瘤组织中CD4+、CD8+、DX5+亚群的频率也明显更高,而且CD4+CD44+、CD8+CD44+细胞频率的升高表明T细胞处于活化的状态,另外肿瘤组织中DX5+CD69+、DX5+CD44+、DX5+CD107a+频率比未注射组高,表明纳米颗粒能够激活NK和T细胞,抑制肿瘤的生长。给小鼠二次荷瘤发现,注射纳米颗粒组的小鼠二次荷瘤后并未长出明显的肿瘤,暗示dsNKG2D-IL21 目的蛋白诱导小鼠发挥了记忆免疫应答,并产生了肿瘤相关抗体。综上所述,本研究中融合的NKG2D和IL-21基因片段通过壳聚糖递送纳米颗粒,纳米颗粒转染细胞后能够分泌dsNKG2D-IL-21蛋白并激活CD4+T,CD8+T和NK细胞,在荷瘤部位能大量累积,并通过小鼠T细胞和NK细胞抑制肿瘤生长,因此本课题组制备的NKG2D-IL-21基因纳米颗粒能够发挥有效的抗肿瘤活性,有望用于治疗癌症。
[Abstract]:IL-21 is a new type of cytokines found in 2000, is a member of the IL-2 family, and share a gamma C chain.IL-21 mainly by activated CD4+T cells (including Th17 cells, follicular helper T cells and NKT cells), while IL-21 can also induce cell activation, promote cell proliferation or stimulate cell play the cytotoxic effect of.IL-21 receptor (IL-21R) and so on is composed of heterologous IL-21R alpha and gamma chain two common polymers, mainly composed of lymphocytes (NK cells, T cells, B cells and NKT cells) and bone marrow cells (such as macrophages and dendritic cells) expression, and non immune cells (such as the gut into fibroblasts and keratinocytes) can also express.NKG2D is a C type lectin like receptor activation, by NK, NKT, CD8 + T and gamma delta T cells are widely expressed. Mouse NKG2D ligand by retinoic acid early transcription factor (RAE-1), H60 and UL-16 binding protein like white rat Transcript 1 (MULT1), human NKG2D ligand is a major histocompatibility complex class related protein A and B (MICA and MICB) and ULBP6-1. NKG2D in most normal tissues did not express ligand expression, but usually, the virus infected cells or tumor cells can induce NKG2D ligands so NKG2D ligands in immunotherapy can be used as tumor targeting antigen. NKG2D ligand can target the tumor based on the characteristics of the subject of the use of the extracellular domain of IL-21 gene and recombinant NKG2D to form recombinant of dsNKG2D-IL-21 gene, and inserted into pcDNA3.1 (a) a recombinant plasmid plasmid, dsNKG2D-IL-21 protein of the antitumor activity of recombinant gene delivery. The inside of the cell must have the aid of biological carrier, therefore the use of strong ionic compounds - chitosan chemically modified (HTCC) to package recombinant plasmid pcDNA3.1-dsNKG2D-IL21 Into chitosan nanoparticles (NP), the size of 200-400nm, with positive charge on the surface of.MTS/PMS showed that the nanoparticles on CT-26 cells, NIH-3T3 cells and RAW264.7 cells toxicity is very small, and the nanoparticles transfection of CT-26 cells can express dsNKG2D-IL-21 protein, and the injection of nanoparticles in normal Balb/c mice serum was also detected in the the expression of IL-21, the research group that nanoparticles prepared at the cellular level or the host can express the target protein dsNKG2D-IL-21. nanoparticles into tumor bearing mice, the tumor volume significantly injection in mice, and the tumors grew more slowly, CD4+, spleen and tumor tissue CD8+, DX5+ subgroup the frequency is significantly higher, and CD4+CD44+, CD8+CD44+ cells showed that T cells in the higher frequency activation state, the addition of DX5+CD69+, DX5+CD44+ in tumor tissues, the frequency of DX5+CD107a+ than before The injection group, showed that the nanoparticles can activate NK and T cells, inhibit the growth of tumor. The tumor bearing mice two times to find nanoparticles into a group of tumor bearing mice two times did not grow significantly tumor, played an implied response memory immunity in mice induced by dsNKG2D-IL21 protein, and tumor associated antibodies in conclusion, NKG2D and IL-21 fusion gene fragments in this study through the delivery of chitosan nanoparticles, nanoparticles transfection cells can secrete dsNKG2D-IL-21 protein and activation of CD4+T, CD8+T and NK cells, a large number of accumulated in the tumor site, and through the mouse T cells and NK cells inhibit the growth of tumor, so NKG2D-IL-21 gene nanoparticles group of preparation of this issue can play an effective anti-tumor activity, is expected to be used in the treatment of cancer.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-36

【参考文献】