【作者】 王军民；

【导师】 袁先厚；

【作者基本信息】 武汉大学 ， 外科学， 2010， 博士

【摘要】 目的：目前通过树突状细胞诱导机体抗肿瘤免疫已经成为肿瘤免疫治疗的一种重要途径,通过热休克凋亡脑胶质瘤细胞抗原制备树突状细胞疫苗,可增强树突状细胞抗原提呈功能,诱导细胞毒性T淋巴细胞产生肿瘤杀伤作用；同时探讨胶质瘤细胞系U251细胞中肿瘤干细胞的培养和鉴定方法；研究应用脑胶质瘤干细胞抗原制备的树突状细胞疫苗在体外对胶质瘤细胞的杀伤作用并探讨其机制。方法：采用改进Inaba法在rmGM-CSF和rhIL-4作用下体外诱导扩增小鼠骨髓来源的树突状细胞,电镜、流式细胞仪鉴定其生物学特性；经恒温43℃、44℃、45℃分别处理U251细胞1h、2 h、3 h、4 h继续常规培养12小时,进一步在体外制备凋亡U251细胞致敏的DC瘤苗,后观察疫苗的混合淋巴细胞反应和细胞杀伤作用；将U251细胞置于DMEM/F12+10%FBS的培养基中培养,对数生长期时0.25%胰蛋白酶消化,洗涤,加入无血清DMEM/F12培养基(含LIF 10ng/ml, EGF 20ng/ml,FGF 20ng/ml,B27 20ul/ml)培养。每日观察细胞球生长情况,每周传一代。收集培养4周的细胞球,行免疫细胞化学染色,检测神经干细胞标志Nestin和CD133的表达。使用流式细胞仪分别检测含血清培养条件下的肿瘤细胞,无血清培养1周、2周、4周和8周的肿瘤细胞中CD133＋和nestin+细胞含量。收集培养4周的细胞球在DMEM/F12+10%FBS培养基中培养5-10天,应用免疫细胞化学染色,检测NSE、MAP2和GFAP的表达；从脑胶质瘤临床标本中培养获得肿瘤干细胞,应用反复冻融法获得胶质瘤干细胞冻溶物。诱导小鼠间充质干细胞分化为树突状细胞,将冻溶物和树突状细胞共培养,获得胶质瘤干细胞疫苗,流式细胞仪检测树突状细胞表面标志的变化。应用CCK-8法检测疫苗的促T细胞增殖能力以及对胶质瘤细胞的杀伤作用,应用ELISA法检测培养基中干扰素-Y的含量。结果：在细胞因子作用下,培养6-7天即可诱导出DCs,经倒置显微镜和电镜证实膜表面具有典型树突状结构；流式细胞仪证实其表达特征性表面标记CDllc,同时高表达功能相关抗原CD80、CD86、MHC-Ⅱ；应用倒置显微镜和流式细胞仪确定热诱导凋亡胶质瘤细胞的最佳条件是44℃处理3小时并常规培养12小时；凋亡肿瘤细胞能更好的负载DCs,混合淋巴细胞反应表明负载后DC疫苗有较强的刺激同种淋巴细胞反应的能力,DC疫苗活化抗原特异性CTL具有很强的杀伤肿瘤细胞能力；U251细胞在无血清培养基中1周可形成细胞团。传代后,能形成新的细胞球,并随传代次数的增多,细胞球逐渐变得规则。U251细胞系中部分细胞具有单克隆性质。培养4周的细胞球,免疫细胞化学染色提示Nestin和CD133均呈阳性表达。流式分析,CD133阳性率在肿瘤细胞0.5%,悬浮培养1周2.4%,悬浮培养2周3.0%,悬浮培养4周10.0%,悬浮培养8周可达17.7%。细胞球在含血清培养基中培养5-10天,免疫细胞化学染色提示神经元特异性标志MAP2与NSE和胶质细胞标志GFAP阳性表达,部分细胞MAP2和GFAP均表达，细胞异质性明显；经脑胶质瘤干细胞胞溶物的强烈刺激,树突状细胞表面标志表达显著提高,包括CD80、CD86、CD11C和MHCⅡ；胶质瘤干细胞疫苗能强烈刺激T细胞增殖；该疫苗诱导的免疫反应,能有效杀灭胶质瘤细胞,并能促进干扰素-γ的分泌。结论：在rmGM-CSF和rmIL-4的作用下体外成功诱导、扩增小鼠骨髓源性功能性的DC,热诱导凋亡胶质瘤细胞负载的DC疫苗在体外能有效刺激自体T淋巴细胞增殖,并诱导抗原特异性CTL产生较强的杀伤胶质瘤细胞功能；U251细胞系中存在少量肿瘤干细胞,具有神经干细胞样特性,可在无血清培养基中培养扩增,对胶质瘤的发生和进展有重要作用；应用胶质瘤干细胞制备的树突状细胞疫苗能有效诱导特异性细胞毒性T细胞,表现出较强的抗肿瘤特性。更多还原

【Abstract】 OBJECTIVE:At present,DC-inducing anti-tumors immune has become one of important cancer immunotherapy.The DC vaccine loaded by hyperthermia-induing apoptotic glioma cells antigens can enhance differentiation and maturation of the dendritic cells, strengthen its function of antigen presentation and induce stronger tumor killing effect of cytoxicity T lymphocytes. To explore the method of culturing human glioma stem cell in U251 cell line in vitro with reformed neural stem cell culturing medium, meanwhile to study biological characters on proliferation and differentiation, and so on. To explore anti-glioma cells ability and mechanism of specific T cells induced by dendritic cells loaded with antigen of glioma stem cells,in vitro.METHODS:The murine bone marrow-derived DCs were induced and amplified by rmGM-CSF and rhIL-4 using improved Inaba way in vitro, the biological characteristics of which were indentificated by electron microscope and flow cytometer. Human U251 glioma cells were cutured in vitro and induced by hyperthermia 43℃、44℃、45℃for 1、2、3、4 hours respectively and then routine culture for 12 hours.The DCs were sensitized by apoptotic U251 cells to prepare DC vaccine in advance which was charactirizated through the mixed lymphocyte responses and cell killing experiment. Human glioma cells U251 were cultured in serum free mediam DMEM/F12, plus LIF(10ng/ml), EGF(20ng/ml), FGF(20ng/ml) and B27(20ul/ml), examined daily and subcultured weekly. Cell spheres were collected after 4 weeks.The expressions of neural stem cell marker Nestin and CD133 were detected by immunocytochemistry. CD133+ cells and nestin+ cells were detected with flow cytometry, in adherent U251 and non-adherent cell spheres, which were cultured for 1 to 8 weeks. After 4 weeks, Cell spheres were collected at 4 weeks and incubated for 5 to 10 days for differentiation in medium containing serum. Then, the expressions of NSE, MAP2 and GFAP on differentiation cells were examined by immunocytochemistry. Cancer stem cells were cultured from clinical specimen of glioma,lysate of glioma stem cells was obtained by the repeated freezing and thawing method,and DC stemed from mesenchymal stem cells(MSCs). Glioma stem cell vaccine was got by mixing tumor lysate with DC,then,surface molecules of DC were checked by flowcytometry. CCK-8 method was used to delect the DC vaccine’s ability of promoting T cells proliferation and killing glioma cells. Level of IFN-yin the supernatant was checked by ELISA.RESULTS:The DCs were generated after 6-7 days induce with rmGM-CSF and rhIL-4, the typical dendritic structure on membrane surface was indentificated by inverted microscope and electron microscope.The flow cytometry confirmed that DCs expressed the typical surface marker CD11c and functional correlation antigen CD80,CD86 and MHC-II in high level meanwhile. The best condition of glioma cell inducing apoptosis was 44℃for 3 hours and then routine culture for 12 hours,which was defined by inverted microscope and flow cytometry. Apoptotic tumor cells could preferably load DCs.The DCs vaccine had strong ablity to stimulate the homogeneity mixed lymphocyte responses.Apoptotic tumor cells loaded DCs also could stongly active antigen specific CTL to kill glioma cells. A small amount of U251 cells had capacity of continuous proliferating and renewing for long time, in vitro, with positive immunohistochemistry-staining on Nestin and CD 133. The percentage of CD 133+ cells were 0.1% in adherent U251,1.5% after 1 week,3.0% after 2 weeks and 10.0% after 8 weeks in non-adherent cell spheres. Glioma stem cells could differentiate into neurons and astrocytes. Differentiation cells were positive for neural marker MAP2/NSE or glial marker GFAP, some expressed both markers. After stimulation of lysate of glioma stem cell, expression of surface molecules of DC was up-regulated,including CD80、CD86、CD11c and MHC II. Glioma stem cell vaccine could stimulate T cell proliferation strongly. The vaccine could induce effective immune response, kill tumor cells and boost the secretion of interferon-y.CONCLUSIONS:The functional DCs were successfully indced and amplificated by cytokines rmGM-CSF and rmIL-4 in vitro.The DCs vaccine loaded by hyperthermia-inducing apoptotic tumor cells could stimulate autologous T lymphocyte proliferation, and induce antigen specific CTL produce strong killer function. Human glioma stem cells could be cultured from U251,and could be differentiated into neurons and astrocytes, as neural stem cells,which played an important pole in the pathogenesis of glioma. Dendritic cell vaccine can effectively induce specific cytotoxic T cells, and show strong anti-tumor properties, loaded with antigen of glioma stem cells,in vitro.更多还原