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目的将抗人P185erbB2scFv-Fc-IL-2融合蛋白(HFI)作用于人卵巢癌细胞株SKOV3细胞和人外周血单个核细胞(PBMC),通过体外实验阐明HFI调变肿瘤细胞表面分子和激活免疫效应细胞的抗肿瘤机制,为HFI临床应用提供实验依据。方法MTT法检测细胞增殖、杀伤活性;流式细胞术观察细胞表面分子的表达水平;生物活性法检测细胞膜相关TNF杀伤活性;RT-PCR检测细胞穿孔素表达水平。结果HFI处理后,未观察到对SKOV3细胞增殖活性的直接抑制作用;SKOV3细胞表面杀伤相关分子ICAM-1、Fas表达率分别由24.85%、0.53%增高到85.36%、59.19%(P<0.01);人PBMC的增殖活性增强,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%(P<0.01);CD25、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%(P<0.01);穿孔素基因、膜相关TNF均表达增强,LAK样、NK样杀伤活性在各效靶比时均明显增高(P<0.01)。结论HFI提高SKOV3细胞杀伤相关分子ICAM-1、Fas表达水平,并且对人PBMC有明显的增殖活化作用,通过激活LFA-1/ICAM-1、Fas/FasL途径提高杀伤介质穿孔素和膜相关TNF的释放,增强LAK样、NK样杀伤活性。
OBJECTIVE: To investigate the effects of HFI on the proliferation of human ovarian cancer cell line SKOV3 and human peripheral blood mononuclear cells (PBMCs) by in vitro anti-human P185erbB2scFv-Fc-IL-2 fusion protein (HFI) The anti-tumor mechanism of effector cells provides experimental evidence for the clinical application of HFI. Methods MTT assay was used to detect cell proliferation and cytotoxicity. Flow cytometry was used to observe the expression of cell surface molecules. Cell viability was assayed by bioactivity assay. Perforin expression was detected by RT-PCR. Results No direct inhibition of SKOV3 cell proliferation was observed after treatment with HFI. The expression of ICAM-1 and Fas in SKOV3 cells increased from 24.85% and 0.53% to 85.36% and 59.19%, respectively (P <0.01) ; The proliferation activity of human PBMC was enhanced, the levels of CD25 + T cells and CD3 + CD16 + CD56 + NK cells increased from 24.37% and 6.90% to 38.80% and 13.45% respectively (P <0.01) The expression levels of perforin gene and membrane-associated TNF were increased from 3.99%, 86.52% and 5.02% to 12.96% and 99.06% to 16.19% (P <0.01), respectively. LAK-like and NK- The target was significantly higher than the average (P <0.01). Conclusion HFI can increase the expression of ICAM-1 and Fas in SKOV3 cells and have a significant effect on human PBMC proliferation. Activation of LFA-1 / ICAM-1 and Fas / FasL can increase the expression of perforin and membrane-associated TNF Release, enhance LAK-like, NK-like killing activity.