摘要
目的制备热诱导凋亡胶质瘤细胞致敏树突状细胞疫苗，探讨其增强树突状细胞抗原递呈、诱导细胞毒性T淋巴细胞产生更强肿瘤细胞杀伤作用的机制。方法采用改良Inaba法，在重组小鼠粒细胞巨噬细胞集落刺激因子（rmGM—CSF）和重组小鼠白细胞介素-4（rmIL-4）作用下，体外诱导扩增小鼠骨髓来源的树突状细胞，电子显微镜和流式细胞术检测其生物学特征；分别以不同加热温度处理U251细胞，制备凋亡U251细胞致敏树突状细胞疫苗，^3H-TdR掺入法和MTT法检测T淋巴细胞增殖能力和活化细胞毒性T淋巴细胞杀伤率。结果在rmGM-CSF和rmIL-4作用下，小鼠骨髓来源细胞体外培养6～7d即可诱导出树突状细胞，经倒置相差显微镜和电子显微镜证实细胞表面呈典型树突状结构；流式细胞术检测证实其表达特异性表面标记物CD11c，同时表达功能相关性抗原CD80、CD86和MHCⅡ。倒置相差显微镜和流式细胞术确定热诱导U251细胞凋亡的最佳条件为：44℃处理3h再常规培养12h，凋亡U251细胞可更好地负载树突状细胞，负载的树突状细胞可以激发T淋巴细胞增殖，诱导活化细胞毒性T淋巴细胞具有更强的杀伤肿瘤细胞的能力。结论在rmGM—CSF和rmIL-4作用下，经体外成功制备的小鼠骨髓来源的树突状细胞，可热诱导凋亡胶质瘤细胞致敏树突状细胞疫苗于体外有效激发T淋巴细胞增殖，诱导细胞毒性T淋巴细胞产生较强的杀伤肿瘤细胞能力。
Objective To prepare dendritic cells （DCs） vaccine sensitized by hyperthermia-induced apoptotic glioma cells, and to explore the mechanism of enhancement of DCs antigen in transmitting and inducing stronger killer action of cytotoxic T lymphocyte （CTL） on tumor cells. Methods The murine bone marrow-derived DCs were induced and amplificated by recombinant murine granuloeyte-macrophage colony stimulating factor （rmGM-CSF） and recombinant murine interleukin-4 （rmIL-4） with modified Inaba method in vitro, and the biological characteristics of DCs were indentified by electron microscope and flow cytometry. The apoptotic U251 cell-sensitized DCs vaccine was prepared by heating treatment of U251 cells. Tritium-labelled thymine deoxyribonueleoside （3H-TdR） endosmosis and methyl-thiazolyl-tetrazolium （MTT） method were used to detect the reproduction potentiality of T lymphocyte and the killing rate of activated CTL. Results DCs were generated after 6-7 d induced with rmGM-CSF and rmIL-4. The typical dendritic structure on membrane surface was indentified by inverted phase contrast microscope and electron microscope. The flow cytometry confirmed that DCs expressed the typical surface marker CDllc and functional related antigen CD80, CD86 and MHC Ⅱ in high level. The examination of inverted phase contrast microseope and flow cytometry comfirmed that the best thermal condition for inducing apoptotic U251 cells was 44 ℃ for 3 h and then routinely cultured for 12 h. Thus, the apoptotic tumor cells could preferably load DCs, and the loaded DCs could stimulate higher reproduction potentiality and induce the activated CTL to produce stronger ability for killing tumor cells. Conclusion The functional DCs from murine bone marrow were successfully induced and amplificated by rmGM-CSF and rmIL-4 in vitro. The DCs Vaccine loaded by hyperthermia= inducd apopto!iC glioma cells can stimulate the proliferation of autologous T lymphocyte, and induce CTL to produce strong killer action.
出处
《中国现代神经疾病杂志》
CAS
2009年第6期548-556,共9页
Chinese Journal of Contemporary Neurology and Neurosurgery
基金
国家自然科学基金资助项目(项目编号:30772223)
湖北省自然科学基金资助项目(项目编号:2006ABA211)
关键词
高温
诱发
细胞凋亡
脑肿瘤
神经胶质瘤
树突细胞
癌症疫苗
免疫疗法
Hyperthermia, induced
Apopt0sis
Brain neoplasms
Glioma
Dendritic ceils
Cancer vaccines
Immunotherapy
