不同大肠癌细胞裂解物负载健康人外周血DC瘤苗的体外试验研究

Research of dendritic cells from human peripheral blood in vitro loaded with different colorectal carcinoma cells lysate antigen

目的探讨3种大肠癌肿瘤细胞裂解物负载的健康人外周血树突状细胞(DC)刺激同体和异体T淋巴细胞增殖活化的作用。方法对血站来源的健康人外周血采用密度梯度离心法,分离获得DC前体细胞,用临床应用药物GM-CSF、TNF-α和试验用试剂rhIL-4联合诱导培养扩增DC,制备3种大肠癌细胞裂解物,体外致敏DC,显微镜观察DC形态演化过程,电镜拍摄微观形态特征,流式细胞仪鉴定DC表型,ELISA法检测肿瘤抗原致敏DC分泌IL-12水平,检测致敏DC刺激T淋巴细胞分泌的IL-10和IFN-γ水平,3H-TdR法检测成熟DC(mDC)诱导同体和异体T淋巴细胞增殖能力。结果 DC形态学呈现典型树突状分化特征,mDC表达特征性免疫表型CD1a、CD83,共刺激分子CD86、HLA-DR,分泌高水平IL-12,mDC与同体和异体T淋巴细胞共培养均能有效刺激T淋巴细胞增殖活化,T淋巴细胞不分泌IL-10,分泌较高水平IFN-γ,SW480细胞株在各项指标上都具优势,3H-TdR法检测提示mDC不仅可以刺激同体淋巴细胞增殖,同样有效激活异体T淋巴细胞。结论来源于血站的健康人外周血是一种方便的DC前体细胞原料。联合细胞因子的培养方法可以成功诱导出成熟的DC1型细胞,引起Th1型细胞免疫反应;3种大肠癌肿瘤细胞裂解物,尤其是SW480细胞株裂解物抗原性质稳定,能够有效促进DC分化,提高DC成熟度;负载裂解物的DC不仅刺激同体T淋巴细胞增殖,也可有效激活异体T淋巴细胞增殖,因此应用健康人异体来源的DC瘤苗刺激患者T淋巴细胞增殖,从而产生抗肿瘤免疫反应的设想是可行的。

Objective To investigate the effects on autogenous and allogeneic T lymphocyte proliferation induced by dendritic cells （DC） from human peripheral blood in vitro loaded with three kinds of colorectal carcinoma cells lysate antigen. Methods Peripheral blood mononuclear cells were isolated by density gradient centrifugation from healthy human peripheral blood were cultured with granulocyte macrophage colony stimulating factor （GM-CSF）, necrosis factor （TNF- a） and interleukin-4 （IL-4） to generate dendritic cells, which loaded with three kinds of eolorectal carcinoma cells lysate antigen. The morphological features and the cellular ultra structure characteristics were observed respectively by light mi- croscope and transmission electron microscopy. The surface antigen of the induced cells was analyzed by flow cytometry （FCM）. The level of IL-12 produced by DC was analyzed by ELISA~ T-cell proliferating activity stimulated by the in- duced cells was identified by 3H-TdR. The level of IL-10 and IFN-7 produced by T-cell were also detected by ELISA. Re- suits These cells bear typical morphologic and phenotypic characteristics of dendritic cells. DC expressed high level of CDla, CD83, CD86 and HLA-DR. high level IL-12 was detected by ELISA. 3H-TdR revealed that DC were potent to stimulate the proliferation of autogenous and allogeneic T cells , IL-10 cannot be detected by ELISA, but IFN-7 produced by T-cell were detected on high level, the result means these DCs was the type of DC1 can induce Thl-immunoreaetion. DC loaded with three kinds of colorectal carcinoma cells lysate antigen gained the powerful capacity to stimulate prolifera- tion of autogenous and allogeneic lymphocytes. Conclusion It is a reliable method to induce and proliferate DCs, DC loaded with three kinds of colorectal carcinoma cells lysate antigen especially SW480 carcinoma cells lysate antigen gained the powerful capacity to stimulate proliferation of autogenous and allogeneic lymphocytes, which may facilitate further studies of DCs and its clinical application.