胶质瘤氩氦冻融产物负载树突状细胞对颅内胶质瘤大鼠的免疫治疗研究

Immunotherapy effect of dendritic cells pulsed by lysate of tumor cell Ar-He cryoablation on rat models of intracranial gliomas

目的观察C6胶质瘤细胞经氩氦冷冻后，冻融产物对Wistar大鼠骨髓源性树突状细胞（BM．DCs）成熟的影响，以及致敏的树突状细胞（DCs）对胶质瘤模型大鼠的抗肿瘤作用。方法体外常规培养C6胶质瘤细胞，双循环氩氦冷冻法冻融C6细胞悬液为实验组，只插入探针不作冷冻为阴性对照组，双循环氩氦冷冻法冻融等量PBS为空白对照组；应用重组大鼠白细胞介素-4（rmIL-4）、重组大鼠粒一巨噬细胞集落刺激因子（rmOM．CSF）和肿瘤坏死因子d（TNFd）诱导Wistar大鼠骨髓DCs成熟，培养第7天分别加入各组冷冻产物，48h后光镜下观察DCs细胞形态，流式细胞仪检测各组DCs细胞表面共刺激分子CD80和CD86的表达，EUSA法检测各组上清液中IL-12含量；将C6细胞接种于Wistar大鼠建立荷脑胶质瘤模型，第3、10天分别皮下注射空白对照组、阴性对照组、实验组DCs疫苗，比较各组大鼠的中位生存期。结果培养第7天未成熟DCs加入冷冻产物48h后实验组具有成熟DCs形态学特征，表面分子CD80、CD86阳性表达率、上清中IL-12的分泌量高于阴性对照组和空白对照组，差异有统计学意义（P〈0．05）；实验组DCs疫苗治疗后荷脑胶质瘤大鼠中位生存期高于阴性对照组和空白对照组，差异有统计学意义（P〈0．05）。结论胶质瘤氩氦冻融产物可以诱导DCs成熟，介导大鼠脑胶质瘤的免疫治疗，为冷冻免疫机制提供一定理论基础，同时该DCs疫苗为胶质瘤个体化治疗的研究提供一种新的方法。

Objective To investigate the role of C6 glioma cells mediated by rapid freezing and thawing ofAr-He cryoablation in the maturation of marrow-derived dendritic cells （BM-DCs） in Wistar rats, and the anti-tumor effect of these DCs on rat models ofintracranial gliomas. Methods C6 glioma cells were routinely cultured in vitro; rapid freezing and thawing of At-He cryoablation was employed in C6 glioma cells of the experimental group, and C6 glioma cells of the negative control group were only performed insertion of the probe; blank control group （using rapid freezing and thawing of At-He cryoablation on the same amount of PBS） was also employed. Bone marrow-derived mononuclear cells （MNCs） were first prepared from tibia and femur bones of Wistar rats. These cells were cultured with such cytokines as recombinant granulocyte-macrophage colony-stimulating factor （rmGM-CSF）, recombinant interleukin-4 （rmIL-4） and tumor necrosis factor-alpha （TNFc0 to induce their maturation; BM-DCs were pulsed with or without tumor cell lysate obtained by rapid freezing and thawing of At-He cryoablation at a ratio of （DC：tumor cells =1：3） 7 d after that. Morphological observation of BM-DCs was performed by light microscopy and the expression of DCs costimulatory molecules CD80 and CD86 were measured by flow cytometry 48 h after the addiction; the IL-12 level in the supernatant of DCs was detected by ELISA. In order to determine whether or not vaccination with C6 TP DCs can induce the therapeutic potential in the established glioma-bearing models, the C6 cells cultured in vitro were stereotaxically implanted into the left caudate nucleus of Wistar rat brain; glioma-bearing rats were injected with vaccination with DCs, cells from the blank control group and negative control group on the 3rd and 10th d. Survival time was observed and determined using the method of Kaplan-Meier and Log-Rank analysis. Results DCs from rats＇ bone marrow cells cultured with cytokines and pulsed with tumor lysates showed the characters of typical mature DCs. Morphologically, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, they expressed high levels of CD80 and CD86 antigens （71.81%±1.10% and 74.66%±1.48% in experimental group, 49.49%± 1.08% and 51.20%±2.06% in negative control group, and 48.47%±1.09% and 49.53%±1.89% in blank control group）; significant difference was noted between each 2 groups （P〈0.05）. Functionally, the IL-12 level in the supernatant of DCs showed obvious increment in the experimental group （245.99±3.20 pg/mL） as compared with that in the negative control group （138.68±3.20 pg/mL） and blank control group （135.16±2.88 pg/mL, P〈0.05）. These cells gained the capacity of mediating immunotherapy against intracranial gliomas in rats： the median survival in the experimental group （33 d） was significantly higher than that in the negative control and blank control groups （22 and 24 d, respectively, P〈0.05）. Conclusion C6 glioma cells mediated by rapid freezing and thawing ofAr-He cryoablation can induce maturation of BM-DCs in Wistar rats; these BM-DCs pulsed with tumor lysates, as new therapeutic vaccines, can mediate immunotherapy against intracranial gliomas in rats.