丙戊酸钠增强骨髓瘤细胞对自然杀伤细胞敏感性及其机制的研究

Valproic acid enhances myeloma cell sensitivity to natural killer cell-mediated lysis and its mechanism

目的观察丙戊酸钠（VPA）对人类骨髓瘤细胞表面自然杀伤（NK）细胞活化性受体凝集素样同型二聚体（NKG2D）配体表达的影响，并研究VPA对NK细胞杀伤骨髓瘤细胞作用的影响及其机制，为骨髓瘤的免疫治疗提供实验依据。方法选用0．5、1、2、5mmol／LVPA处理人类骨髓瘤细胞ARK、OPM2，与无VPA处理的对照组进行比较，观察不同浓度VPA对细胞ARK、OPM2活力的影响。通过实时荧光定量PCR和流式细胞仪分别检测1mmol／LVPA处理的骨髓瘤ARK、OPM2细胞NKG2D配体在mRNA水平和蛋白质水平的变化。应用钙黄绿素释放法（CARE—LASS）检测VPA对NK细胞抗骨髓瘤活性的调节作用及机制。结果5mmol／LVPA处理ARK、OPM2细胞48h后，对骨髓瘤细胞活力有影响（P〈0．05）；与未处理细胞比较，1mmol／LVPA处理的两种骨髓瘤细胞MICA、MICB和ULBP2mRNA水平及蛋白质表达水平均增高（P〈0．05）。NK细胞对骨髓瘤细胞杀伤率增高，效靶比8：1时，VPA处理后ARK细胞杀伤率为60．2％，对照组为31．4％（P=0．018），VPA处理后OPM2细胞杀伤率为56．8％，对照组为22-3％（P=0．040）；且增强的杀伤率可以被NKG2D抗体特异性拮抗。NK细胞通过穿孔素和颗粒酶杀伤效应裂解肿瘤细胞。结论VPA明显上调骨髓瘤细胞的MICA／B、ULBP2表达，可增强NK细胞对骨髓瘤细胞的杀伤活性，NK细胞经穿孔素和颗粒酶杀伤途径杀伤骨髓瘤细胞，为骨髓瘤的临床免疫治疗提供了实验依据。

Objective To investigate the effect of valproic acid （VPA） on NKG2D-ligand expression in ARK, OPM2 human myeloma cell lines and their sensitization to natural killer （NK） cell-mediated Killing. Methods Different concentrations of VPA from 0-5.0 mmol/L were used to treat ARK, OPM2 cells respectively, then the cell viabilities were tested by flow cytometry （FCM）. Real-time quantitative-PCR and FCM were used to detect the changes in mRNA, protein levels of NKG2D-ligand respectively in the two cell lines treated with 1 mmol/L VPA for 48 hours. The caleein-release-assay （CARE-LASS） was carried out to detect cytotoxic changes of NK cells against myeioma cells after VPA treatment. Results VPA induced the expression of MICA/B, ULBP2 （P 〈 0.05） and in turn enhanced the NK cytotoxieity on myeloma cells. The enhancing effect of VPA was blocked by NK cells pretreated with anti-NKG2D mAb （P 〈 0.05）. The primary mechanism of NK cell killing of myeloma cells was perforin/granzyme-mediated. Conclusion VPA can induce the expression of MICA/B, ULBP2 in ARK, OPM2 cells, thereby enhancing the cytotoxicity against myeloma cells, which implies a new mechanism of anticancer approach and may be a new approach in myeloma immunotherapy.