本文關(guān)鍵詞： 重組人熱休克蛋白70 樹突狀細(xì)胞 凍融抗原 肝癌　出處：《中國(guó)醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的 樹突狀細(xì)胞(Dendritic cells,DCs)是目前發(fā)現(xiàn)的功能最強(qiáng)大的抗原遞呈細(xì)胞(Antigen presenting cells,APC),能高效能遞呈抗原,且是唯一能激活初始型T淋巴細(xì)胞的抗原遞呈細(xì)胞,具有獨(dú)特的誘導(dǎo)初始免疫反應(yīng)對(duì)抗腫瘤的能力。目前在DCs疫苗抗腫瘤實(shí)驗(yàn)研究和臨床研究方面已取得了令人振奮的進(jìn)展,顯示了廣泛的應(yīng)用前景。但腫瘤疫苗研制中仍然存在著一個(gè)主要問(wèn)題:大多數(shù)腫瘤抗原的免疫原性很弱,不能正常的被呈遞以激活細(xì)胞毒性T淋巴細(xì)胞(cytotoxictlymphocytes,CTLs),為了提高其免疫原性,必須加入佐劑。研究已表明HSP70具有佐劑樣作用,HSP70可以促進(jìn)DCs成熟,刺激多種細(xì)胞因子的分泌而增強(qiáng)免疫功能。本研究對(duì)DCs被重組人熱休克蛋白70(recombinant human heat shock protein70,rhHSP70)和肝癌凍融抗原共同修飾前后的表型變化、形態(tài)變化及誘導(dǎo)CTLs產(chǎn)生的體外殺傷肝癌細(xì)胞作用進(jìn)行了觀察,以其明確rhHSP70聯(lián)合肝癌凍融抗原共同修飾DCs后,體外可誘導(dǎo)產(chǎn)生針對(duì)肝癌細(xì)胞的高效殺傷作用,并初步探討了rhHSP70增強(qiáng)DCs疫苗抗腫瘤能力的機(jī)制,為設(shè)計(jì)以DCs為基礎(chǔ)的針對(duì)肝癌的生物治療的高效瘤苗提供實(shí)驗(yàn)依據(jù)和理論基礎(chǔ)。 方法 分離健康人外周血獲得單個(gè)核細(xì)胞,經(jīng)粒-巨噬細(xì)胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF),白細(xì)胞介素4(interleukin-4,IL-4)誘導(dǎo)生成DCs,負(fù)載凍融抗原的同時(shí)加入rhHSP70,分為四組:①A組:DCs+凍融抗原(Ag);②B組:DCs+rhHSP70;③C組:DCs+Ag+rhHSP70;④D組:單獨(dú)DCs。流式細(xì)胞術(shù)(flow cytometry,FCM)檢測(cè)各組DCs表型(CD80,CD83,CD86)變化,倒置顯微鏡和掃描電鏡觀察各組DCs形態(tài)特點(diǎn)。不同分組修飾的DCs激活淋巴細(xì)胞生成CTLs,四甲基偶氮唑藍(lán)(MTT)法檢測(cè)DCs刺激淋巴細(xì)胞增殖能力以及CTLs對(duì)肝癌細(xì)胞的體外殺傷活性,熒光染色觀察各組肝癌細(xì)胞凋亡差異。 結(jié)果 rhHSP70,凍融抗原及二者聯(lián)合修飾的DCs表型較對(duì)照組相比差異顯著(P＜0.05)。CD80:A組為80.2±2.5%,B組為79.6±2.6%,C組為81.6±2.3%,D組為21.5±1.4%。CD83:A組為71.3±2.9%,B組為68.3±2.4%,C組為73.4±2.1%,D組為33.4±2.4%。CD86:A組為74.2±3.6%,B組為75.3±4.8%,C組為82.5±5.0%,D組為48.2±2.8%。 光鏡及掃描電鏡下可見rhHSP70,凍融抗原及二者聯(lián)合修飾的DCs較對(duì)照組樹枝狀突起明顯增加,表現(xiàn)出成熟DCs的形態(tài)特征,而對(duì)照組表現(xiàn)出相對(duì)不成熟DCs的形態(tài)特征。 不同分組修飾的DCs刺激淋巴細(xì)胞增殖能力不同,結(jié)果以增殖指數(shù)(proliferation index,PI,增殖指數(shù)=實(shí)驗(yàn)組OD值/對(duì)照組OD值)表示。其中A組為2.10±0.05,B組為2.06±0.05,C組為2.60±0.06,D組為1.20±0.05,PI以C組為最高,與其他兩試驗(yàn)組(A,B組)相比差異顯著(P＜0.05),其次為A組,但A組與B組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。 不同分組DCs呈遞腫瘤抗原誘導(dǎo)的CTL產(chǎn)生殺傷率不同,殺傷百分率=(1-實(shí)驗(yàn)組的OD值/對(duì)照組的OD值)×100%。在效靶比(效應(yīng)細(xì)胞:靶細(xì)胞,即CTLs:肝癌細(xì)胞)為10:1,20:1,50:1時(shí),A組殺傷率分別為40.27±4.89%,47.13±8.05%,51.64±6.73%,B組殺傷率分別為13.58±4.77%,12.13±4.26%,15.57±5.55%,C組殺傷率分別為61.09±7.89%,64.71±3.90%,74.59±3.34%,D組殺傷率分別為11.94±4.11%,13.90±4.11%,13.52±4.61%,在三種效靶比下殺傷率以C組為最高,與其他兩試驗(yàn)組(A組,B組)相比差異顯著(P＜0.05),更遠(yuǎn)強(qiáng)于對(duì)照組(P＜0.05);其次為A組;B組與D組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05)。 結(jié)論 rhHSP70聯(lián)合肝癌凍融抗原修飾DCs,能夠促進(jìn)DCs的成熟,增強(qiáng)DCs刺激淋巴細(xì)胞增殖的能力,誘導(dǎo)的CTLs在體外對(duì)肝癌細(xì)胞能產(chǎn)生高效殺傷。rhHSP70增強(qiáng)DCs抗腫瘤能力的機(jī)制可能與其促進(jìn)DCs成熟有關(guān)。
[Abstract]:objective
Dendritic cells (Dendritic cells, DCs) are the most potent antigen-presenting cells (Antigen, presenting, cells, APC) can efficiently present antigen, and only can activate T lymphocyte antigen presenting cells with distinct initial induction of immune response against the tumor at present. In the DCs vaccine against tumor experimental research and clinical research has made exciting progress, and has wide application prospect in development of tumor vaccine. But there are still a major problem: most of the immunogenicity of tumor antigen is very weak, can not be normal presentation to activate cytotoxic T lymphocytes (cytotoxictlymphocytes, CTLs), in order to improve its immunogenicity, must add adjuvant. Studies have shown that HSP70 can be used as an adjuvant, HSP70 can promote DCs maturation and stimulate a variety of cytokine secretion and immune enhancement The study of DCs function. The recombinant human heat shock protein 70 (recombinant human heat shock protein70, rhHSP70) and phenotypic changes before and after antigen modified freeze thawing liver morphological changes and in vitro cytotoxicity induced by CTLs producing hepatocellular carcinoma cells were observed in the rhHSP70 combined with liver frozen thawed antigen co modified after DCs in vitro, can be induced for efficient killing effect of hepatoma cells, and to explore the mechanism of rhHSP70 enhanced antitumor DCs vaccine ability, provide experimental and theoretical basis for the design of DCs based on biological treatment of hepatocellular carcinoma, tumor vaccine.
Method
The separation of human peripheral blood mononuclear cells by granulocyte macrophage colony stimulating factor (granulocyte-macrophage colony-stimulating, factor, GM-CSF), interleukin 4 (interleukin-4, IL-4) induced the formation of DCs load, frozen thawed antigen and joined the rhHSP70, divided into four groups: group A: DCs+ (frozen thawed antigen Ag); B group: DCs+rhHSP70; group C: DCs+Ag+rhHSP70; D group: single DCs. flow cytometry (flow cytometry, FCM) were detected in DCs (CD80, CD83, CD86 phenotype changes), inverted microscope and scanning electron microscope DCs. Morphological characteristics of different groups of modified DCs activated lymphocytes to generate CTLs four, methyl thiazolyl tetrazolium (MTT) method was used to detect the killing activity of DCs to stimulate lymphocyte proliferation and CTLs on hepatocellular carcinoma cells in vitro, observe the apoptosis of hepatocellular carcinoma cell differences in fluorescence staining.
Result
RhHSP70, frozen thawed antigen and combination of the two modified DCs phenotype was significantly higher than the control group (P < 0.05) of.CD80:A group is 80.2 + 2.5%, 79.6 + 2.6% B group, C group is 81.6 + 2.3%, D group is 21.5 + 71.3 + 2.9% 1.4%.CD83:A group, B group is 68.3 + 2.4%. The C group is 73.4 + 2.1%, D group is 33.4 + 2.4%.CD86:A group is 74.2 + 3.6%, 75.3 + 4.8% B group, C group is 82.5 + 5%, D + 2.8%. group was 48.2
Under light microscope and scanning electron microscope, rhHSP70, frozen thawed antigen and two co modified DCs increased significantly compared with the control group, showing the morphological characteristics of mature DCs, while the control group showed relatively immature DCs morphological characteristics.
Different groups of modified DCs stimulated lymphocyte proliferation results in different proliferation index (proliferation index, PI, proliferation index = experimental group control group od / OD). The A group is 2.10 + 0.05, 2.06 + 0.05 B group, C group is 2.60 + 0.06, D group is 1.20 + 0.05, PI in the C group was the highest, and the other two experimental groups (A, B) compared with significant difference (P < 0.05), followed by the A group, but no significant difference between A group and B group (P > 0.05).
Different groups of DCs induced tumor antigens CTL killing rate, killing percentage (OD / OD = 1- experimental group control group) * 100%. (in effect target ratio of effector cells and target cells, i.e. CTLs: cells) of 10:1,20:1,50:1, A group killing rate was 40.27 + 4.89%, 47.13 + 8.05% 51.64, + 6.73%, B group killing rate were 13.58 + 4.77%, 12.13 + 4.26%, 15.57 + 5.55%, C group killing rate were 61.09 + 7.89%, 64.71 + 3.90%, 74.59 + 3.34%, D group killing rate were 11.94 + 4.11%, 13.90 + 4.11%, 13.52 + 4.61%, in the three target than the killing rate of C group is highest, and the other two experimental groups (A group, B group) compared with significant difference (P < 0.05), far higher than in the control group (P < 0.05); followed by A group; B group and D group had no significant difference (P > 0.05).
conclusion
RhHSP70 combined with liver cancer freeze-thaw antigen modified DCs can promote the maturation of DCs and enhance the ability of DCs to stimulate lymphocyte proliferation. The mechanism that induced CTLs can produce high effective killing.RhHSP70 and enhance DCs antitumor ability in vitro can be related to the promotion of DCs maturation.

【學(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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