本文選題：胰腺癌 + HSP70多肽復(fù)合物　； 參考：《昆明醫(yī)學(xué)院》2009年碩士論文

【摘要】： 目的:探討從胰腺癌腫瘤塊中分離純化熱休克蛋白70-多肽復(fù)合物(heatshock protein 70-polypeptide complexes,HSP70-PCs)的方法,研究使用HSP70多肽復(fù)合物構(gòu)建樹(shù)突狀細(xì)胞(dendritic cell,DC)多肽疫苗,觀察其在體外對(duì)CTL的增殖和活化效果。并進(jìn)行免疫治療荷瘤小鼠,觀察小鼠的腫瘤大小及存活期。 方法:采用低滲裂解、ConA-Sepharose親和層析及ADP-Agarose親和層析法,從小鼠胰腺癌(MPC83)腫瘤組織中純化HSP70-PCs,所得蛋白經(jīng)聚丙烯酰胺凝膠電泳檢測(cè),Bradford法測(cè)定蛋白濃度,計(jì)算獲得率。用提純的HSP70-PCs激活小鼠骨髓來(lái)源的樹(shù)突狀細(xì)胞(DC),制備樹(shù)突狀細(xì)胞HSP70多肽腫瘤疫苗,用MTT法檢測(cè)混合淋巴細(xì)胞反應(yīng)(mixed lymphocyte response,MLR)中HSP70-PCs致敏DC對(duì)CTL的增殖及活化效果;并觀察DC誘導(dǎo)的腫瘤特異性細(xì)胞毒性T淋巴細(xì)胞(cytotoxic T lymphocyte,CTL)對(duì)腫瘤細(xì)胞的特異性殺傷活性,建立MPC83荷瘤小鼠模型,并將DC接種于荷瘤小鼠皮下,觀察樹(shù)突狀細(xì)胞HSP70多肽腫瘤疫苗對(duì)腫瘤生長(zhǎng)的抑制作用及對(duì)荷瘤小鼠生存期的影響。 結(jié)果:純化蛋白經(jīng)鑒定為分子量70KD的HSP70-PCs;平均1克濕重腫瘤組織中獲得79.95μg的HSP70-PCs;HSP70-PCs在10-12μg/ml范圍內(nèi)可達(dá)到刺激樹(shù)突狀細(xì)胞最強(qiáng)效果,用HSP70-PCs負(fù)載的DC能增強(qiáng)T細(xì)胞的增殖和活化能力,其誘導(dǎo)的CTL對(duì)小鼠胰腺癌MPC83腫瘤細(xì)胞具有特異性殺傷作用;應(yīng)用樹(shù)突狀細(xì)胞HSP70多肽腫瘤疫苗治療荷瘤小鼠能顯著抑制荷瘤小鼠腫瘤的生長(zhǎng),延長(zhǎng)荷瘤小鼠存活期。 結(jié)論:用上述層析法可分離出較高純度、保留腫瘤抗原信息的HSP70-PCs;體外將其負(fù)載經(jīng)聯(lián)合細(xì)胞因子誘導(dǎo)培養(yǎng)的DC可激活T淋巴細(xì)胞,產(chǎn)生對(duì)該腫瘤細(xì)胞的特異性殺傷效應(yīng)。體內(nèi)對(duì)胰腺癌荷瘤小鼠具有顯著的免疫治療效果。
[Abstract]:Objective: to investigate the method of isolation and purification of heat shock protein 70-polypeptide complex HSP70-PCsfrom pancreatic cancer tumor, to study the construction of dendritic cell dendritic cell dendritic cell DCS vaccine using HSP70 polypeptide complex, and to observe the effect of HSP70-PCS on the proliferation and activation of CTL in vitro.The tumor size and survival period were observed.Methods: HSP70-PCswere purified from mouse pancreatic carcinoma tissue by ConA-Sepharose affinity chromatography and ADP-Agarose affinity chromatography. The protein was detected by polyacrylamide gel electrophoresis to determine the protein concentration and the yield was calculated.Dendritic cells derived from mouse bone marrow were activated with purified HSP70-PCs to prepare HSP70 polypeptide tumor vaccine from dendritic cells. The proliferation and activation of HSP70-PCs sensitized DCs in mixed lymphocyte response to MLRs were detected by MTT method.The specific cytotoxic cytotoxic T lymphocytes (CTL) induced by DC on tumor cells were observed. The MPC83 tumor-bearing mice model was established, and the DC was inoculated subcutaneously into the tumor-bearing mice.To observe the inhibitory effect of dendritic cell HSP70 polypeptide tumor vaccine on tumor growth and the survival time of tumor-bearing mice.Results: the purified protein was identified as HSP70-PCs with molecular weight of 70KD, and 79.95 渭 g HSP70-PCS70-PCs were obtained from tumor tissues with an average of 1 g wet weight, which could stimulate dendritic cells in the range of 10-12 渭 g/ml. DC loaded with HSP70-PCs could enhance the proliferation and activation of T cells.The CTL induced by CTL has a specific killing effect on MPC83 tumor cells in mice, and dendritic cell HSP70 polypeptide tumor vaccine can significantly inhibit the tumor growth and prolong the survival period of tumor-bearing mice.Conclusion: HSP70-PCs with high purity and retained tumor antigen information can be isolated by the method mentioned above, and the DC induced by the combination of cytokines can activate T lymphocytes in vitro and produce specific killing effect on the tumor cells.In vivo, there is a significant immunotherapy effect on mice bearing pancreatic cancer.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【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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