本文選題：多發(fā)性骨髓瘤 + 樹(shù)突細(xì)胞�。� 參考：《昆明醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的：通過(guò)體外殺傷實(shí)驗(yàn),研究轉(zhuǎn)染粘蛋白核心肽MUC1-2VNTR(兩串聯(lián))基因的樹(shù)突細(xì)胞(DC)誘導(dǎo)的細(xì)胞毒T細(xì)胞(CTL)對(duì)人的多發(fā)性骨髓瘤細(xì)胞株(U266)的誘導(dǎo)殺傷效應(yīng),為研制多發(fā)生性骨髓瘤基因疫苗奠定基礎(chǔ)。方法：1、根據(jù)靶基因設(shè)計(jì),將MUC1-2VNTR的編碼基因作為目的基因,將人工合成的全基因克隆入載體pcDNA3.1/myc-hisA中。2、用去內(nèi)毒素質(zhì)粒小提試劑盒擴(kuò)增、提取質(zhì)粒、并保存。3、選取健康人的外周血單個(gè)核細(xì)胞,體外由重組人粒細(xì)胞-巨噬細(xì)胞集落刺激因子(rhGM-CSF)和重組人白細(xì)胞介素4(rhIL-4)誘導(dǎo)成熟的DC細(xì)胞。4、構(gòu)建pcDNA3.1-2VNTR重組質(zhì)粒后,采用Lipofectimine脂質(zhì)體介導(dǎo)法轉(zhuǎn)染DC細(xì)胞(DC-2VNTR),以轉(zhuǎn)染空載體質(zhì)粒pcDNA3.1 (DC-pcDNA3.1)及l(fā)ipofctamine處理的DC (DC-Lipo)作為對(duì)照組。5、Western印跡法檢測(cè)重組體在DC細(xì)胞內(nèi)的表達(dá)。6、在體外刺激同源T細(xì)胞,細(xì)胞毒實(shí)驗(yàn)檢測(cè)DC-2VNTR誘導(dǎo)的CTL對(duì)多發(fā)性骨髓瘤細(xì)胞株(U266)的殺傷作用。結(jié)果：1.成功構(gòu)建了重組質(zhì)粒載體pcDNA3.1-2VNTR/myc-hisA;2.人的外周血DC細(xì)胞的培養(yǎng)和鑒定；3. Western印跡法檢測(cè)到MUC1-2VNTR在DC細(xì)胞內(nèi)的表達(dá)；4.DC-2VNTR誘導(dǎo)的CTL對(duì)多發(fā)性骨髓瘤細(xì)胞(U266)有顯著殺傷效應(yīng),且顯著高于DC-pcDNA3.1組及DC-Lipo組(P0.05)。結(jié)論：以MUC1-2VNTR基因轉(zhuǎn)染的DC可以誘導(dǎo)CTL反應(yīng),并能特異地殺傷U266多發(fā)性骨髓瘤細(xì)胞株。
[Abstract]:Objective: to study the cytotoxic T cells (CTL) induced by dendritic cells (DC) transfected with MUC1-2VNTR gene in vitro on human multiple myeloma cell line (U266). To lay a foundation for the development of multiple myeloma gene vaccine. Methods according to the target gene design, the encoding gene of MUC1-2VNTR was used as the target gene, and the synthetic gene was cloned into the vector pcDNA3.1 / myc-hisA. In vitro, recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and recombinant human interleukin-4 (rhIL-4) were used to induce mature DC cells. The recombinant plasmid pcDNA3.1-2VNTR was constructed. DC cells were transfected with Lipofectimine liposome mediated transfection of DC cells (DC-2VNTR) and transfected with unloaded pcDNA3.1 (DC-pcDNA3.1) and lipofctamine treated DC (DC-Lipo) as control group. The expression of recombinant cells in DC cells was detected by Western blotting, and the homologous T cells were stimulated in vitro. Cytotoxicity assay was used to detect the cytotoxicity of CTL induced by DC-2 VNTR on multiple myeloma cell line (U266). The result is 1: 1. The recombinant plasmid pcDNA3.1-2VNTR / myc-hisA2 was successfully constructed. Culture and identification of human peripheral blood DC cells. The expression of MUC1-2VNTR in DC cells was detected by Western blot. CTL induced by DC-2VNTR had a significant killing effect on multiple myeloma cells (U266) and was significantly higher than that in DC-pcDNA3.1 group and DC-Lipo group (P0.05). Conclusion: DC transfected with MUC1-2VNTR gene can induce CTL response and specifically kill U266 multiple myeloma cell line.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R733.3

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1 秋玉珍;MUC1-2VNTR基因修飾的DC對(duì)骨髓瘤細(xì)胞的體外殺傷效應(yīng)的研究[D];昆明醫(yī)科大學(xué);2016年

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本文編號(hào)：2099140