论文部分内容阅读
目的:探讨舒尼替尼促进肝癌细胞自然杀伤细胞2族成员D配体(natural killer group 2 member D ligands,NKG2DLs)表达及提高NK细胞杀伤肿瘤细胞的作用。方法:常规体外培养HepG2细胞,免疫磁珠法从健康志愿者外周静脉血中分选NK细胞,流式细胞技术检测NK细胞纯度及1μmol/L舒尼替尼孵育24 h前后肝癌细胞NKG2DLs表达率;LDH释放测定法检测NK细胞对药物处理前后靶细胞的杀伤活性,实时荧光定量PCR检测药物处理前后HepG2细胞NKG2DLs mRNA的表达情况。结果:分选后NK细胞(CD3~-CD16~+CD56~+细胞)的纯度达78%以上;经舒尼替尼处理后靶细胞MHC-Ⅰ类链相关分子A或B(MHC classⅠ-related chain molecules A/B,MICA/MICB)、人巨细胞病毒糖蛋白UL16结合蛋白(UL16-binding proteins,ULBPs)的表达率均有升高,以MICA、MICB和ULBP2升高最明显(F=17.73,P=0.000)。当效靶比为10∶1、20∶1时,NK细胞对舒尼替尼处理前后HepG2细胞的杀伤活性分别从(9.47±1.11)%、(20.45±1.94)%上升到(28.88±1.23)%、(44.93±1.57)%,舒尼替尼处理前后NK细胞对靶细胞杀伤活性的差异有明显统计学意义(P<0.05)。舒尼替尼处理HepG2细胞后,MICA、MICB和ULBP2 mRNA表达水平明显升高(F=62.628,P=0.000)。结论:舒尼替尼能选择性诱导肿瘤细胞高表达NKG2DLs(MICA/B和ULBP2),并增强NK细胞杀伤活性。
OBJECTIVE: To investigate the effect of sunitinib on the expression of natural killer group 2 member D ligands (NKG2DLs) and to enhance the cytotoxicity of NK cells to tumor cells. Methods: HepG2 cells were cultured in vitro routinely. NK cells were sorted from peripheral blood of healthy volunteers by immunomagnetic beads method. The purity of NK cells was detected by flow cytometry and the expression of NKG2DLs in 24 hours after incubation with 1 μmol / L Sunitinib The killing activity of NK cells on target cells before and after drug treatment was detected by LDH release assay. The expression of NKG2DLs mRNA in HepG2 cells was detected by real-time fluorescence quantitative PCR. Results: The purity of NK cells (CD3 ~ -CD16 ~ + CD56 ~ +) after sorting was above 78%. After MHC class I-related molecules A or B were treated with sunitinib, (MICA / MICB), and UL16-binding proteins (ULBPs) were all increased, the most obvious increase was MICA, MICB and ULBP2 (F = 17.73 , P = 0.000). The killing activity of NK cells on HepG2 cells before and after sunitinib treatment increased from (9.47 ± 1.11)% and (20.45 ± 1.94)% to (28.88 ± 1.23)% respectively when the target ratio was 10:1, %, (44.93 ± 1.57)% respectively. There were significant differences in the killing activity of NK cells before and after sunitinib treatment (P <0.05). Sunitinib treatment of HepG2 cells, MICA, MICB and ULBP2 mRNA expression was significantly increased (F = 62.628, P = 0.000). Conclusion: Sunitinib can selectively induce NKG2DLs (MICA / B and ULBP2) highly expressed in tumor cells and enhance NK cell killing activity.