硼替佐米诱导ABCG_2^(High)耐药鼻咽癌细胞高表达NKG2D配体的实验研究

Induction of Expression of Ligands for NKG2D Receptor in ABCG_2^(High) Nasopharyngeal Carcinoma by Bortezomib

目的探讨硼替佐米诱导高表达ABCG2耐药鼻咽癌细胞对Allo-NK细胞杀伤敏感性的机制。方法利用免疫磁珠技术分离ABCG2HighCNE2/DDP细胞及Allo-NK细胞,流式细胞技术检测分离后细胞纯度及经硼替佐米处理前后靶细胞NKG2D配体表达率,LDH释放测定法检测经硼替佐米处理前后ABCG2HighCNE2/DDP细胞对Allo-NK细胞的杀伤敏感性。结果ABCG2HighCNE2/DDP细胞分离后ABCG2表达率为(91.40±2.32)%,分选后NK细胞CD3-CD16+CD56+细胞的纯度达90%以上。经硼替佐米处理之后靶细胞MICA、MICB、ULBP1、ULBP2、ULBP3表达率,由药物处理之前的(2.92±0.33)%、(4.27±0.33)%、(5.80±0.62)%、(11.10±3.15)%、(7.75±1.14)%分别上升到(17.52±2.04)%、(12.53±3.68)%、(15.24±2.91)%、(62.02±6.85)%、(35.69±3.23)%。在效靶比为10∶1、20∶1、Allo-NK细胞对硼替佐米处理前后ABCG2HighCNE2/DDP细胞的杀伤率分别为(15.32±13.86)%、(27.26±6.81)%及(35.06±5.10)%、(52.34±4.78)%。处理前后杀伤率差异有统计学意义(F=26.03,P=0.000)。结论硼替佐米通过诱导肿瘤细胞高表达NKG2D配体(MICA/B、ULBP1-3),使肿瘤细胞对Allo-NK细胞的杀伤敏感性增强。

Objective To investigate the mechanism on the effects of improving cytotoxic sensitivity of ABCG2^High CNE2/DDP cells to Allo-NK cells which exerted by bortezomib. Methods ABCG2^High CNE2/DDP cells and Allo-NK cells were isolated by magnetic activated cell sorting （MACS）. Flow cytometry was used to evaluate the purity of isolated cells and the expression of NKG2D-ligands on target cells before and after incubation with bortezomib. Subsequently, the eytotoxic sensitivity of treated and un-treated ABCG2^High CNE2/DDP cells to Allo-NK cells were measured by LDH releasing assay. Results The expressions of ABCG2 in ABCG2^High CNE2/DDP cells were （91.40±2. 32）%. More than 90% of isolated NK cells showed tobe CD3- CD16＋ CD56＋ cells which would definitely meet the needs of experiments. The expressions of MICA,MICB,ULBP1 ,ULBP2 ,ULBP3 on target cells incubated with bortezomib have respectively increased from （2. 92±0. 33）%, （4. 27±0. 33）%, （5.80±0. 62）%, （11.10±3.15）%, （7. 75±1. 14）% to （17. 52±2. 04）%, （12. 53±3.68）%, （15.24±2. 91）%,（62. 02±6. 85）%,（35.69±3.23）%. At the E：T ratio of 10 ： 1 and 20 ： 1, the cytotoxic sensitivity of ABCG2^High CNE2/DDP cells to Allo-NK cells increased from （15.32±13. 86）% and （27. 26±6. 81）% in un-treated groups to （35.06±5. 10）% and （52. 34±4. 78）% in bortezomib treated groups. Data above showed that cytotoxic sensitivity of target cells in each group before and after bortezomib treatment had significant differences （F = 26. 03 P = 0. 000）. Conclusion Bortezomib can up-regulate expressions of NKG2D-ligands （MICA/B, ULBP1-3 ） in ABCG2^High nasopharyngeal carcinoma cells, which resulted in higher cytotoxic sensitivity to Allo-NK cells.