本文選題：IL-21 + NKG2D��； 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：IL-21是2000年發(fā)現(xiàn)的新型Ⅰ型細(xì)胞因子,屬于IL-2家族一員,且共用一條γc鏈。IL-21主要由活化的CD4+T細(xì)胞(包括Th17細(xì)胞,濾泡性輔助性T細(xì)胞以及NKT細(xì)胞)產(chǎn)生,而同時(shí)IL-21也能誘導(dǎo)細(xì)胞活化,促使細(xì)胞增殖或者刺激細(xì)胞發(fā)揮細(xì)胞毒作用等等。IL-21的受體(IL-21R)是由IL-21Rα和普通γ鏈組成的異源二聚體,主要由淋巴細(xì)胞(比如NK細(xì)胞,T細(xì)胞,B細(xì)胞和NKT細(xì)胞)及骨髓細(xì)胞(比如巨噬細(xì)胞和樹(shù)突狀細(xì)胞)表達(dá),而非免疫細(xì)胞(比如腸成纖維細(xì)胞和角質(zhì)形成細(xì)胞)也能表達(dá)。NKG2D是一種C型凝集素樣活化受體,由NK,NKT,CD8 + T和γδT細(xì)胞廣泛表達(dá)。小鼠NKG2D配體由視黃酸早期轉(zhuǎn)錄因子(RAE-1),H60和鼠UL-16結(jié)合蛋白白樣轉(zhuǎn)錄物1(MULT1)組成,人類NKG2D配體是主要組織相容性復(fù)合物Ⅰ類相關(guān)蛋白A和B(MICA和MICB)以及ULBP6-1。在大多數(shù)正常組織中NKG2D配體通常不表達(dá),但病毒感染的細(xì)胞或腫瘤細(xì)胞能夠誘導(dǎo)NKG2D配體的表達(dá),因此,NKG2D的配體在免疫治療中能夠作為腫瘤靶向抗原�；贜KG2D配體能夠靶向腫瘤的特點(diǎn),本課題利用NKG2D的胞外區(qū)與IL-21基因片段重組形成dsNKG2D-IL-21重組基因,并插入到pcDNA3.1(一)質(zhì)粒中構(gòu)成重組質(zhì)粒,研究dsNKG2D-IL-21目的蛋白的抗腫瘤活性。要將重組基因遞送進(jìn)細(xì)胞內(nèi)必須借助生物載體,因此利用經(jīng)過(guò)化學(xué)方法修飾過(guò)的強(qiáng)離子型化合物——?dú)ぞ厶?HTCC)來(lái)包裹重組質(zhì)粒pcDNA3.1-dsNKG2D-IL21形成殼聚糖納米顆粒(NP),大小200-400nm,表面帶正電荷。MTS/PMS法發(fā)現(xiàn)納米顆粒對(duì)CT-26細(xì)胞、NIH-3T3細(xì)胞以及RAW264.7細(xì)胞的毒性很小,且經(jīng)納米顆粒轉(zhuǎn)染過(guò)的CT-26細(xì)胞能表達(dá)目的蛋白dsNKG2D-IL-21,而注射納米顆粒的正常Balb/c小鼠其血清中也檢測(cè)到了 IL-21的表達(dá),證明本課題組制備的納米顆粒能夠在細(xì)胞水平或者宿主體內(nèi)表達(dá)目的蛋白dsNKG2D-IL-21。注射納米顆粒的荷瘤小鼠,其腫瘤體積明顯比未注射的小鼠小,且腫瘤生長(zhǎng)更緩慢,其脾臟及腫瘤組織中CD4+、CD8+、DX5+亞群的頻率也明顯更高,而且CD4+CD44+、CD8+CD44+細(xì)胞頻率的升高表明T細(xì)胞處于活化的狀態(tài),另外腫瘤組織中DX5+CD69+、DX5+CD44+、DX5+CD107a+頻率比未注射組高,表明納米顆粒能夠激活NK和T細(xì)胞,抑制腫瘤的生長(zhǎng)。給小鼠二次荷瘤發(fā)現(xiàn),注射納米顆粒組的小鼠二次荷瘤后并未長(zhǎng)出明顯的腫瘤,暗示dsNKG2D-IL21 目的蛋白誘導(dǎo)小鼠發(fā)揮了記憶免疫應(yīng)答,并產(chǎn)生了腫瘤相關(guān)抗體。綜上所述,本研究中融合的NKG2D和IL-21基因片段通過(guò)殼聚糖遞送納米顆粒,納米顆粒轉(zhuǎn)染細(xì)胞后能夠分泌dsNKG2D-IL-21蛋白并激活CD4+T,CD8+T和NK細(xì)胞,在荷瘤部位能大量累積,并通過(guò)小鼠T細(xì)胞和NK細(xì)胞抑制腫瘤生長(zhǎng),因此本課題組制備的NKG2D-IL-21基因納米顆粒能夠發(fā)揮有效的抗腫瘤活性,有望用于治療癌癥。
[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.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R73-36

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