论文部分内容阅读
目的探讨采用高强度聚焦超声(HIFU)辐照肿瘤细胞制备肿瘤抗原致敏树突状细胞(DC)和制备DC肿瘤疫苗的可行性。方法(1)采用1ng/mlIL-4和10ng/mlGM-CSF联合诱导小鼠骨髓细胞产生DC,并用流式细胞仪检测DC的CD80、CD86、H-2Kd和I-Ad表达情况;(2)固定辐照时间或超声声强,应用不同声强或辐照时间的HIFU辐照CT26细胞株后,用台盼蓝染色、MTT法检测活肿瘤细胞数,同时用台盼蓝染色观察肿瘤细胞的形态学变化,分析HIFU剂量与肿瘤细胞存活率的关系,了解剂量-效应关系;(3)固定辐照时间和超声强度,HIFU辐照CT26细胞悬液,制备肿瘤细胞抗原并致敏DC,致敏DC与T细胞共孵育48h后提取上清液,通过ELISA法检测上清中IL-12、γ干扰素(IFN-γ)的含量,并与冻融组、单纯CT26、对照组进行比较。结果(1)采用IL-4和GM-CSF联合诱导小鼠骨髓细胞可培养出典型的DC;(2)固定辐照时间,随着HIFU辐照剂量的增加肿瘤细胞的存活率迅速减少,肿瘤细胞的碎片逐渐增多,当声强为1000W/cm2,辐照30s时,无细胞存活,肿瘤细胞失去正常形态,全部被撕裂成碎片;(3)通过ELISA法检测HIFU、冻融及单纯CT26组上清液中IL-12、IFN-γ的含量显著高于对照组(P均<0.05),HIFU及冻融组上清液中IL-12、IFN-γ的含量高于单纯CT26组(P均<0.05),但HIFU、冻融组之间比较差异无统计学意义(P>0.05)。结论HIFU能灭活肿瘤细胞并能使肿瘤细胞破碎,HIFU制备的肿瘤抗原可体外致敏DC,并可使DC成熟分泌大量IL-12,同时诱导T细胞分泌大量IFN-γ。
Objective To investigate the feasibility of preparing tumor antigen-primed dendritic cells (DCs) and preparing DC tumor vaccines by irradiating tumor cells with high-intensity focused ultrasound (HIFU). Methods (1) DCs were induced by 1ng / ml IL-4 and 10ng / ml GM-CSF in mouse bone marrow cells and the expression of CD80, CD86, H-2Kd and I- Fixed irradiation time or ultrasonic intensity, the application of different intensity or irradiation time of HIFU CT26 irradiated cells, with trypan blue staining, MTT assay of live tumor cells, while observed by trypan blue staining of tumor cells Morphological changes, analysis of the relationship between HIFU dose and tumor cell survival rate, to understand the dose-effect relationship; (3) fixed irradiation time and intensity of ultrasound, HIFU radiation CT26 cell suspension to prepare tumor cell antigen and sensitized DC The DC and T cells were incubated 48 h after the supernatant was extracted, the supernatant by ELISA assay IL-12, IFN-γ (IFN-γ) content, and compared with the freeze-thawed group, pure CT26, control group. Results (1) The combination of IL-4 and GM-CSF induced the typical DCs induced by mouse bone marrow cells. (2) For fixed irradiation time, the survival rate of tumor cells decreased rapidly with the increase of HIFU irradiation dose. Cell debris gradually increased, when the sound intensity of 1000W / cm2, irradiated 30s, no cell survival, the tumor cells lose the normal morphology, all were torn into pieces; (3) by ELISA HIFU, freeze-thaw and simple CT26 The levels of IL-12 and IFN-γ in the supernatants were significantly higher than those in the control group (all P <0.05). The levels of IL-12 and IFN-γ in the supernatants of HIFU and freeze-thaw groups were higher than those in the CT26 group P <0.05). However, there was no significant difference between HIFU and freeze-thaw groups (P> 0.05). CONCLUSION HIFU can inactivate tumor cells and disrupt tumor cells. Tumor antigens produced by HIFU sensitize DCs in vitro and induce mature DCs to secrete large amounts of IL-12 and induce T cells to secrete large amounts of IFN-γ.