摘要目的 将抗人P185erbB2 scFv-Fc-IL-2融合蛋白(HFI)分别作用于表达高、中、低3个水平erbB2受体的SKOV3、MCF-7、SGC-7901三株肿瘤细胞和健康人外周血单个核细胞(PBMC),探讨HFI调变肿瘤细胞表面分子,激活免疫效应细胞的机制;模拟体内肿瘤组织,将HFI-PBMC-肿瘤细胞混合培养,探讨HFI对分别表达高、中、低3个水平erbB2肿瘤细胞的淋巴因子激活的杀伤细胞(LAK)样和抗体依赖性细胞介导的细胞毒(ADCC)作用,为临床应用提供实验依据.方法 MTF法检测细胞增殖、杀伤活性;流式细胞术检测细胞表面分子的表达变化;应用非核素细胞毒试剂盒观察HFI介导ADCC杀伤作用.结果 HFI处理后SKOV3细胞表面杀伤相关分子细胞间黏附分子-1(ICAM-1)、Fas表达水平分别由24.85%、0.53%增高到85.36%、59.19%;SKOV3、MCF-7、SGC-7901三株肿瘤细胞erbB2表达水平分别由98.48%、42.60%、36.66%下降到94.01%、30.95%、12.36%.HFI刺激后人PBMC的增殖活性显著增强,呈时间依赖效应,CD3+ CD8+ T细胞和CD3- CD16+ CD56+NK细胞分别由24.37%、6.90%提高到38.80%、13.45%,CD25、淋巴细胞功能抗原-1(LFA-1)、FasL表达水平分别由3.99%、86.52%、5.02%提高到12.96%、99.06%、16.19%.HFI活化的人PBMC对不同erbB2表达水平肿瘤细胞的LAK样杀伤活性,在各效靶比均比对照组明显提高.HFI能够介导和增强人PBMC对表达erbB2肿瘤细胞的ADCC杀伤作用.结论 HFI上调SKOV3细胞表面杀伤相关分子ICAM-1、Fas表达,下调肿瘤细胞表面erbB2表达.HFI对人PBMC具有明显的激活增殖作用,活化的人PBMC对表达不同水平erbB2肿瘤细胞的LAK样杀伤作用均显著增强.HFI能够介导和增强人PBMC对肿瘤细胞的ADCC杀伤作用,且杀伤活性的高低与肿瘤细胞表面erbB2表达水平的高低呈平行关系.

abstractsObjective To explore the mechanism by which the anti-human P185erbB2 scFv-Fc-IL-2(HFI)modulates tumor surface molecules and activates immune effector cells in vitro. Methods MTT assay was used to test the proliferation and the LAK-like cytotoxicity. Flow cytometry assay was used to test the expression of ICAM-1, Fas and erbB2 receptors in tumor cells and the expression levels of CD molecules FasL and LFA-1 in human PBMC. Antibody-dependent cell-mediated cytotoxicity(ADCC)mediated by HFI against SKOV3, MCF-7 and SGC-7901 tumor cells was explored hv LDH release assay. Results The expression levels of ICAM-1 and Fas on SKOV3 cell treated with HFI were upregulated, from 24.85％ and 0.53％ to 85.36％ and 59.19％ respectively, while the expression levels of erbB2 on SKOV3, MCF-7 and SGC-7901 tumor cells treated with HFI were downregulated, from 98.48％, 42.60％ and 36.66％ to 94.01％,30.95％ and 12.36％ respectively. HFI could significantly enhance the proliferation activity of human PBMC, and CD3+ CD8+ T cells and CD3- CD16+ CD56+ NK cells were elevated, from 24.37％ and 6.90％ to 38.80％ and 13.45％ respectively. The expression levels of CD25, LFA-1 and FasL were significantly enhanced from 3.99％, 86.52％ and 5.02％ to 12.96％, 99.06％ and 16.19％. The LAK-like cytotoxicity of human PBMC treated with HFI against SKOV3, MCF-7,SGC-7901 tumor cells was significantly improved:HFI was effective in mediating ADCC against SKOV3,MCF-7 and SGC-7901 tumor cells which expressed high,medium and low levels of erbB2,respectively,and HFI-induced ADCC was correlated with the degrees of erbB2 expression on the tumor cells. Conclusion The expression levels of ICAM-1 and Fas on SKOV3 cell treated with HFI are significantly upregulated. The expression levels of erbB2 on SKOV3, MCF-7 and SGC-7901 tumor cells treated with HFI are downregulated. HFI can significantly enhance the proliferation activity of human PBMC. The LAK-like eytotoxicity of human PBMC treated with HFI against tumor cells is significantly enhanced. HFI iS effective in mediating ADCC and the activity of HFI-induced ADCC is correlated with the degrees of erbB2 expression on the tumor cells.