论文标题:Hsp70-肽复合物致敏的树突状细胞抗肿瘤效果的实验研究
Antitumor Effect of Dendritic Cells Modified by Stomach Neophasma Hsp70-petide Complexes
论文作者 刘学政
论文导师 袁庆文,论文学位 硕士,论文专业 外科学
论文单位 江西医学院,点击次数 13,论文页数 41页File Size270k
2005-05-01论文免费下载 http://paper.dic123.com/lunwen_106383472/ 胃癌; Hsp70-肽复合物; 树突状细胞; 抗肿瘤免疫
stomach neophasms ; Heat-shock proteins70-tumor peptide complexes ; Dendritic cells ; antitumor immunity
目的:探讨从胃癌组织中分离Hsp70-肽复合物,并致敏树突状细胞(DC),观察其在体外的抗肿瘤作用,为DC 介导的肿瘤免疫治疗提供理论实验依据。方法:采用GM-CSF、IL-4 刺激,培养胃癌患者外周血单个核细胞(PBMC),增殖产生大量DC,用SephadexG-100 凝胶层析法从临床胃癌组织中提取Hsp70-肽复合物,SDS-聚丙烯酰胺凝胶电泳测定其分子量并行western-bolt鉴定。用此复合物修饰DC,通过ELISA 法对修饰DC 和未被Hsp70-肽复合物修饰DC 上清液的IL-12、TNF-α、IL-1β含量进行检测,用DC 激活自体混合T 淋巴细胞,观察其在体外对胃癌细胞的杀伤作用,用未被Hsp70-肽复合物修饰DC 作对照。结果:用SephadexG-100 凝胶层析法从临床胃癌组织中提取的蛋白经SDS-聚丙烯酰胺凝胶电泳和western-bolt 鉴定为Hsp70,从胃癌患者外周血获得的PBMC 经GM-CSF、IL-4 刺激培养7d 后电镜观察细胞不规则形,细胞表面粗糙突起明显,细胞核呈肾形和马蹄形,可确定为DC。Hsp70 修饰DC 后分泌IL-12、TNF-α、IL-1β等细胞因子的含量为(305.02±20.70)pg/ml 、(480.61±32.52)pg/ml、(519.60±39.12)pg/ml,而未被Hsp70 修饰DC 分泌的细胞因子IL-12 、TNF-α、IL-1β的含量为(3.81±0.84)pg/ml、(3.50±0.21)pg/ml、(50.11±2.86)pg/ml.两者比较具有明显的差异(P﹤0.01)。Hsp70 修饰DC 激活的T 淋巴细胞后对胃癌细胞的杀伤力为(93.5±8.2)%,与对照组的杀伤力(6.2±0.3%)比较具有显著差异(P﹤0.01)。结论:从胃癌组织中提取蛋白为Hsp70,该蛋白保留了Hsp70 蛋白的原有生物活性,具有肿瘤的抗原信息。Hsp70 修饰DC 后,产生IL-12 、TNF-α、IL-1β等细胞因子的水平明显增加,修饰的DC 激活T 淋巴细胞后对肿瘤细胞具有很强的杀伤作用。
Objective: To study the antitumor effect of DC modified byHsp70-tumor peptide complexes derived from stomach neophasms fissuein vivo.Methods: Peripheral blood PBMC of stomach cancer were cultured withgranulocytc/macrophage colonystimulating factor(GM-CSF) andinterlenkin 4(IL-4) stimulating .thus proliferated to produce plentyof DC. Extract Hsp70-tumor peptide complexes from stomach nephasms withSephadexG-100 chromatography of protein and SDS-PAGE thus comfirmedby western-blot ,Afer Dc were modified by HSP70 tumor peptide complexes,Dc supematant (IL-12 、TNF-α、IL-1β)were detected using cytokinedetected kit,T lymphocytes was stimulated and their ability of killedtomor cells was investigated.Results: The obtained protein shown to be Hsp70 by western-blot.Peripheral blood PBMC of stomach cancer were cultured with GM-CSF andIL-4 ,After a week ,The cells is irregulate ,surface rough and tuberobvious ,The cells nucleus is kidney shape and horse unguis shape ,The cells is proved Dc. Dc modified by stomach neophasmsHsp70-peptidecomplexes could plentifully secret IL-12、TNF-α、IL-1β,Their contentis (305.02±20.70)pg/ml 、(480.61±32.52)pg/ml 、(519.60±39.12)pg/ml。The contents of IL-12、TNF-α、IL-1βsecreted by unmodifiedDc is(3.81 ±0.84)pg/ml 、(3.50 ±0.21)pg/ml 、(50.11 ±2.86)pg/ml ,Their difference is obvious (P﹤0.01). The T lymphocytesinduced with modified DC had strong killing role to tumor cells, killingrate is (93.5±8.2)%,Their difference is obvious compared with thecontrast group (6.2±0.3)% (P﹤0.01)。 Conclusion : The obtained protein shown to be Hsp70 derived fromstomach naplasm tissue ,and retain protein bioactivity and has strongimmunogeneicity.Dc modified by stomach neophasmsHsp70-peptidecomplexes could plentifully secret IL-12 、TNF-α、IL-1β.The Tlymphocytes induced by modified DC had strong killing role to tumorcells .
|
