摘要
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 possibility that dendritic cells （DCs） loaded with high intensity focused ultrasound （HIFU） prepared tumor antigen can serve as a tumor vaccine. Methods （ 1 ） DCs were derived from mouse bone marrow cultured with 1 ng/ml IL-4 and 10 ng/ml GM-CSF. The explessions of CD80, CD86, H-2K^d and I-A^d were tested by flow-eytometric analysis. （2） CT26 cells were irradiated with different sound intensity or irradiation time of HIFU, the number of survival cells was detected with typan blue staining and MTT colorimetry, and the dosage-effect of HIFU therapy was analyzed. （3） Spleen T cells were cultured with DC pulsed with tumor antigen prepared by HIFU with fixed irradiation time and sound intensity for 48 h,and the levels of IL-12 and IFN-γ in supernatants were detected with ELISA. Results （ 1 ） The morphology of DCs appeared on the 3rd day after culture and DCs had the expressions of CD80 and CD86, H-2K^d and I-A^d on their surface. （2） With increasing dose of sound intensity, the cell survival rate decreased sharply and more cell debris was observed. At sound intensity of 1000 W/cm^2 for 30 s, the cells were induced to complete destruction, and no tumor cell survived, （3） Higher levels of IL-12 and IFN-γ were detected in HIFU, freezing melting and CT26 groups compared with control group, the differences were significant （P 〈0.05 for all）. The levels of IL-12 and IFN-γ in HIFU and freezing melting groups were significantly higher than those in CT26 group （ P 〈 0.05 for both）. However, there was no significant difference in IL-12 and IFN-γ levels between HIFU and freezing melting groups （ P 〉 0.05 ）. Conclusions The resuits suggest that high intensity focused ultrasound can destroy and fracture living tumor cells. HIFU-prepared tumor antigen can mediate the maturation of DCs, which can secrete IL-12 and stimulate T cells to secrete a large amount of IFN-γ.
出处
《中华医学超声杂志（电子版）》
2006年第1期5-10,共6页
Chinese Journal of Medical Ultrasound(Electronic Edition)
