本文選題：熱休克蛋白70 + NY-ESO-1肽�。� 參考：《福建醫(yī)科大學》2011年碩士論文

【摘要】：【目的】 將原核表達純化獲得的人熱休克蛋白70(heat shock protein70, HSP70)與腫瘤睪丸抗原NY-ESO-1多肽體外連接制備HSP70-NY-ESO-1多肽復合物,檢測HSP70-NY-ESO-1多肽復合物負載的樹突狀細胞(dendritic cells, DC)對NY-ESO-1陽性腫瘤細胞的體外殺傷效率,探討增強腫瘤免疫治療的有效策略。 【方法】 1.人HSP70的原核表達、鑒定及純化:RT-PCR技術(shù)獲得人HSP70全長cDNA,將PCR產(chǎn)物克隆到原核表達載體PET-30a質(zhì)粒上,酶切與DNA測序鑒定后,將PET-30a-HSP70重組質(zhì)粒轉(zhuǎn)化至大腸桿菌Rosetta(DE3),IPTG誘導蛋白表達,表達產(chǎn)物行SDS-PAGE和Western-blot鑒定,His-tag層析柱純化HSP70蛋白。 2.人臍帶血樹突狀細胞的培養(yǎng)及鑒定:聯(lián)合應用重組人粒細胞/巨噬細胞集落刺激因子(recombination human granulocyte/macrophage colony-stimulating factor, rhGM-CSF)和重組人白細胞介素4(recombination human interleukin-4, rhIL-4)培養(yǎng)人臍帶血單個核細胞來源的DC,腫瘤壞死因子α(tumor necrosis factor-α, TNF-α)誘導其成熟。通過倒置顯微鏡、光學顯微鏡及透射電鏡觀察DC形態(tài),流式細胞術(shù)鑒定DC表型,混合淋巴細胞反應鑒定DC功能。 3.HSP70-NY-ESO-1多肽復合物負載DC的體外殺傷實驗:HSP70與NY-ESO-1多肽體外連接,獲得HSP70-NY-ESO-1多肽復合物。分別用HSP70-NY-ESO-1多肽復合物負載的DC、NY-ESO-1多肽負載的DC、HSP70負載的DC以及未經(jīng)負載的DC刺激淋巴細胞,并以未經(jīng)DC刺激的淋巴細胞作為對照組,共5組淋巴細胞,乳酸脫氫酶(lactic dehydroge-nase, LDH)釋放法檢測淋巴細胞對NY-ESO-1陽性腦膠質(zhì)瘤細胞的體外殺傷效率。 【結(jié)果】 1.人HSP70的原核表達、鑒定及純化:RT-PCR擴增出大小約2000bp的目的片段,經(jīng)酶切和DNA測序證實,HSP70基因成功地克隆到原核表達載體PET-30a質(zhì)粒上;轉(zhuǎn)入重組質(zhì)粒的大腸桿菌經(jīng)IPTG誘導后,SDS-PAGE顯示,在分子量為約72KD處有表達量明顯增多的蛋白條帶,Western-blot證實其為目的條帶,純化后蛋白純度達95%。 2.人臍帶血樹突狀細胞的培養(yǎng)及鑒定:培養(yǎng)第8d的成熟DC(mature DC, mDC)形態(tài)學觀察:細胞體積大,形態(tài)不規(guī)則,有典型的樹枝狀突起;流式細胞術(shù)鑒定:mDC表達HLA-DR(80.65%)、CD86(76.59%)、CD83(74.81%),較不成熟DC(immature DC, imDC)表達明顯上升(HLA-DR、CD86與CD83分別為25.20%、17.58%、1.50%);混合淋巴細胞反應證實:mDC體外激發(fā)混合淋巴細胞反應能力較強,imDC則較弱。 3.HSP70-NY-ESO-1多肽復合物負載DC的體外殺傷實驗:HSP70-NY-ESO-1多肽復合物負載DC組的淋巴細胞對NY-ESO-1陽性腦膠質(zhì)瘤細胞的殺傷活性明顯高于其他四組,差別有顯著性意義(P0.001);NY-ESO-1多肽負載的DC組淋巴細胞殺傷活性高于HSP70負載的DC組、未經(jīng)負載的DC組以及對照組,差別有顯著性意義(P0.001);HSP70負載的DC組、未經(jīng)負載的DC組與對照組相比殺傷率無顯著差別(P0.05)。 【結(jié)論】 1.成功構(gòu)建HSP70的原核表達載體,并高效表達HSP70蛋白,純化可獲得大量高純度的HSP70蛋白。 2.聯(lián)合應用細胞因子rhGM-CSF、rhIL-4和TNF-α可以從人臍帶血單個核細胞中培養(yǎng)出足夠數(shù)量的成熟DC。 3.HSP70-NY-ESO-1抗原肽復合物負載DC能在體外有效激活和擴增腫瘤特異性的細胞毒性T淋巴細胞(cytotoxic T lymphocyte, CTL),HSP70能夠協(xié)同NY-ESO-1抗原肽對NY-ESO-1陽性腦膠質(zhì)瘤細胞的殺傷,增強NY-ESO-1抗原肽的抗腫瘤免疫作用。
[Abstract]:[Objective]
HSP70-NY-ESO-1 polypeptide complex was prepared by human heat shock protein 70 (heat shock protein70, HSP70) derived from the prokaryotic expression and purification of human heat shock protein protein70 (HSP70), and the killing efficiency of HSP70-NY-ESO-1 polypeptide complex loaded with dendritic cells (dendritic cells, DC) on the in vitro killing efficiency of NY-ESO-1 positive tumor cells was investigated. An effective strategy for enhancing tumor immunotherapy.
[method]
The prokaryotic expression, identification and purification of 1. people HSP70: RT-PCR technology obtained the full length cDNA of human HSP70, and cloned the PCR product on the prokaryotic expression vector PET-30a plasmid. After the enzyme digestion and DNA sequencing, the recombinant plasmid was transformed to the Escherichia coli Rosetta (DE3), and the egg white expression was induced, and the expression product was identified and identified. HSP70 protein was purified by G chromatography column.
Culture and identification of 2. human umbilical cord blood dendritic cells: combined use of recombinant human granulocyte / macrophage colony stimulating factor (recombination human granulocyte/macrophage colony-stimulating factor, rhGM-CSF) and recombinant human interleukin 4 (recombination human interleukin-4, rhIL-4) to cultivate human umbilical cord blood mononuclear cells DC, tumor necrosis factor alpha (tumor necrosis factor- a, TNF- alpha) induced its maturation. The morphology of DC was observed by inverted microscope, optical microscope and transmission electron microscope. The DC phenotype was identified by flow cytometry and the function of DC was identified by mixed lymphocyte reaction.
The in vitro killing experiment of the 3.HSP70-NY-ESO-1 polypeptide complex loaded with DC: HSP70 and NY-ESO-1 polypeptide were connected in vitro, and the HSP70-NY-ESO-1 polypeptide complex was obtained. The DC of the HSP70-NY-ESO-1 polypeptide complex, DC of the NY-ESO-1 polypeptide loaded, DC on HSP70 load, and the unloaded DC to stimulate the lymphocyte. As a control group, a total of 5 groups of lymphocytes and lactic dehydroge-nase (LDH) release assay were used to detect the killing efficiency of lymphocytes against NY-ESO-1 positive glioma cells in vitro.
[results]
1. human HSP70 prokaryotic expression, identification and purification: RT-PCR amplification of the size of 2000bp of the target fragment, confirmed by enzyme digestion and DNA sequencing, the HSP70 gene was successfully cloned to the prokaryotic expression vector PET-30a plasmid, and the recombinant plasmid was induced by IPTG and SDS-PAGE showed a significant increase in the molecular weight of about 72KD. The protein bands and Western-blot confirmed that they were the target bands, and the purity of the purified protein reached 95%.
Culture and identification of 2. human umbilical cord blood dendritic cells: mature DC (mature DC, mDC) morphological observation of culture 8D: large cells, irregular morphology, typical dendritic protuberances, flow cytometry identification: mDC expression HLA-DR (80.65%), CD86 (76.59%), CD83 (74.81%), and less mature DC (immature DC) increased significantly CD83 was 25.20%, 17.58%, 1.50%, respectively. Mixed lymphocyte reaction confirmed that mDC had strong ability to stimulate mixed lymphocyte reaction in vitro, while imDC was weak.
3.HSP70-NY-ESO-1 polypeptide complex loaded DC in vitro killing experiment: HSP70-NY-ESO-1 polypeptide complex loaded DC group lymphocytes to NY-ESO-1 positive brain glioma cells were significantly higher than the other four groups, the difference was significant (P0.001); NY-ESO-1 polypeptide loaded DC group lymphocyte killing activity was higher than the HSP70 load DC Groups, unloaded DC group and control group, the difference was significant (P0.001), HSP70 loaded DC group, unloaded DC group compared with the control group, there was no significant difference in killing rate (P0.05).
[Conclusion]
1. the prokaryotic expression vector of HSP70 was successfully constructed, and HSP70 protein was efficiently expressed, and a large quantity of high purity HSP70 protein could be obtained.
2. the combined use of cytokines rhGM-CSF, rhIL-4 and TNF- a can produce enough mature DC. from human umbilical cord blood mononuclear cells.
The 3.HSP70-NY-ESO-1 antigen peptide complex can effectively activate and amplify the tumor specific cytotoxic T lymphocyte (cytotoxic T lymphocyte, CTL) in vitro. HSP70 can cooperate with NY-ESO-1 antigen peptide to kill NY-ESO-1 positive glioma cells and enhance the anti-tumor immunity of NY-ESO-1 antiproto peptide.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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本文編號：2000980