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目的:探讨胃癌细胞抗原致敏的树突状细胞(DC)诱导T淋巴细胞(T lymphocyte)增殖和杀伤胃癌细胞的效率.方法:应用淋巴细胞分离液分离人外周血单个核细胞,经重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF)、重组人白细胞介素4(rhIL-4)和肿瘤坏死因子α(TNF-α),体外诱导培养获取成熟DC,流式细胞术检测DC表面CD83,CD80,CD86及HLA-DR表型;同时经重组人白细胞介素2(rhIL-2)诱导扩增T淋巴细胞,检测CD3,CD4,CD8及CD56表型.以人胃癌OCUM-2MD3细胞株冻融抗原致敏DC,然后刺激T淋巴细胞,得到肿瘤抗原特异的细胞毒性T淋巴细胞(CTL).MTT法检测致敏DC对T淋巴细胞增殖的影响和致敏DC活化的特异性CTL对胃癌细胞的体外杀伤效率.结果:体外诱导人外周血单个核细胞,可获得大量形态典型、具备强烈刺激增殖能力、且高表达CD80,CD86的DC及高表达CD3的T淋巴细胞;MTT法检测结果发现胃癌细胞抗原致敏DC组刺激T淋巴细胞增殖指数显著高于未致敏DC组和对照组(P<0.01);流式细胞术检测结果发现胃癌细胞抗原致敏DC主要刺激T淋巴细胞增殖为CD8+的CTL(60.58±8.43)%,经胃癌细胞抗原致敏DC刺激后,对胃癌细胞的杀伤效应显著增加,致敏DC组对胃癌细胞杀伤活性(71.57±4.83)%,较未致敏DC组(12.53±1.62)%和单纯T淋巴细胞组(10.45±1.40)%作用更为显著(P<0.01),而且随着效靶比增加,其杀伤效应亦显著增加.结论:胃癌细胞抗原致敏DC可显著刺激T淋巴细胞增殖为CD8+CTL,进而发挥其高效杀伤胃癌细胞的作用.
Objective: To investigate the efficiency of dendritic cells (DCs) sensitized by gastric cancer cells to the induction of T lymphocyte proliferation and to kill gastric cancer cells.Methods: Human peripheral blood mononuclear cells (PBMNCs) were isolated from lymphocytes by recombinant human RhGM-CSF, rhIL-4 and tumor necrosis factor-α (TNF-α) were cultured in vitro to induce mature DCs. Flow cytometry was used to detect DC surface CD83, CD80, CD86 and HLA-DR phenotype. At the same time, T lymphocytes were induced by recombinant human interleukin-2 (rhIL-2) to detect CD3, CD4, CD8 and CD56 phenotype. The strains were immunized with freeze-thaw antigen and then stimulated with T lymphocytes to obtain tumor antigen-specific cytotoxic T lymphocytes (CTLs) .MTT assay was used to detect the effect of sensitized DCs on T lymphocyte proliferation and sensitized DC-activated specific CTLs In vitro killing efficiency of gastric cancer cells.Results: In vitro induction of human peripheral blood mononuclear cells, can obtain a large number of typical morphological, with strong stimulation of proliferation, and high expression of CD80, CD86 of DC and high expression of CD3 T lymphocytes; MTT method The results showed that gastric cancer cell antigen - sensitized DC stimulation group The lymphocyte proliferation index was significantly higher than that of the non-sensitized DC group and the control group (P <0.01). Flow cytometry showed that the antigen-sensitized DCs of gastric cancer cells mainly stimulated the CTL proliferation of T lymphocytes to CD8 + (60.58 ± 8.43)% , The killing effect on gastric cancer cells was significantly increased after DC sensitized by gastric cancer cell antigen, the killing activity of gastric cancer cells in sensitized DC group was (71.57 ± 4.83)%, compared with that in non-sensitized DC group (12.53 ± 1.62)% and pure (10.45 ± 1.40)% (P <0.01), and the lethal effect was also significantly increased with the increase of effective target ratio.Conclusion: DC sensitized with gastric cancer cells can significantly stimulate the proliferation of T lymphocytes For CD8 + CTL, and then play its role in efficient killing of gastric cancer cells.