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目的观察人慢性B淋巴细胞性白血病(B-CLL)细胞的独特型抗原Id-ScFv与热休克蛋白70(HSP70)形成复合物修饰的树突状细胞(DC)体外诱导特异性抗肿瘤作用,并初步探讨其机制。方法将HSP70与Id-ScFv体外结合形成复合物HSP70-Id,修饰自人外周血单核细胞获取的DC。倒置相差显微镜观察DC的形态特征,流式细胞仪检测修饰前后DC的表型变化,酶联免疫吸咐试验(ELISA)检测DC分泌的白细胞介素12(IL-12)和肿瘤坏死因子-α(TNF-α),四甲基偶氮唑蓝(MTT)法检测修饰的DC对自身淋巴细胞的激活和增殖作用,流式细胞仪检测激活的自身淋巴细胞T细胞亚群的变化,台盼蓝染色法检测其对Daudi、K562和HepG2等细胞的杀伤作用。结果DC体外诱导培养成功,HSP70-Id复合物可使DC成熟,镜下可见典型的DC形态,其CD1a表达率为20％～30％,CD83表达率>72％,CD86和HLA-DR表达显著增加(P<0．05),上清中IL-12、TNF-α亦显著高于DC对照组(P<0．01)。HSP70-Id复合物修饰的DC激活自身淋巴细胞,对Daudi细胞的杀伤率为71．24％,而对K562细胞杀伤作用较弱,对HepG2细胞无明显作用。其淋巴细胞亚群中,CD4+T细胞、CD8+T细胞的比例均显著增加,分别为56．51％和70．21％,CD4+T细胞／CD8+T细胞比值由空白对照组的1．49倒置为0．81。结论HSP70-Id复合物修饰的DC生物学活性增强,经其刺激后,传代培养的淋巴细胞可产生高效而特异性的抗肿瘤免疫效应,可能是CD4+T细胞、CD8+T细胞及DC协同作用的结果。
Objective To observe the specific antitumor activity of dendritic cells (DCs) modified by Id-ScFv and heat shock protein 70 (HSP70) in human chronic lymphocytic leukemia (B-CLL) And preliminary discussion of the mechanism. Methods HSP70 and Id-ScFv were combined in vitro to form the complex HSP70-Id, which modified DC from human peripheral blood mononuclear cells. The morphological characteristics of DCs were observed under an inverted phase contrast microscope. The phenotypic changes of DCs were detected by flow cytometry before and after modification. The levels of IL-12 and TNF-α secreted by DC were detected by enzyme-linked immunosorbent assay (ELISA) (TNF-α) and MTT assay were used to detect the activation and proliferation of autologous lymphocytes. Flow cytometry was used to detect the changes of activated T cell subsets in activated lymphocytes. Cytotoxicity of Daudi, K562 and HepG2 were detected by blue staining. Results DCs were successfully induced in vitro. HSP70-Id complex could mature DCs. Typical DCs were seen under microscope. The expression of CD1a was 20% -30%, the expression of CD83 was> 72%, and the expression of CD86 and HLA-DR was significant (P <0.05). The levels of IL-12 and TNF-α in the supernatant were also significantly higher than those in the DC control group (P <0.01). HSP70-Id complex-modified DCs activated their own lymphocytes, killing Daudi cells was 71.24%, while killing K562 cells weaker, no significant effect on HepG2 cells. The proportion of CD4 + T cells and CD8 + T cells in lymphocyte subsets were significantly increased (56.51% and 70.21% respectively). The ratio of CD4 + T cells / CD8 + T cells was significantly increased from 1 .49 inverted to 0.81. Conclusions The biological activity of DCs modified by HSP70-Id complex is enhanced. After stimulated by HSP70-Id complex, lymphocytes from subcultures can produce high-efficiency and specific antitumor immunity effects, which may be that CD4 + T cells, CD8 + T cells and DCs cooperate The result of the action.