半乳糖凝集素-9在白血病微血管内皮细胞上的表达及其对CIK细胞影响的初步研究

Preliminary study of galactin-9 expression on leukemic microvascular endothelial cells and its effect on cytokine induced killer cells

目的:探讨内皮细胞在高表达半乳糖凝集素-9(Gal-9)对CIK细胞杀伤活性、凋亡率和跨内皮迁移的影响。方法:收集确诊的9例急性髓系白血病(AML)患者(不包括AML-M3)、8例急性淋巴细胞白血病(ALL)患者、5例慢性淋巴细胞白血病(CLL)患者和6例健康对照者(HDs)的骨髓标本,利用流式细胞术检测Gal-9的表达。用慢病毒载体构建高表达Gal-9的病毒颗粒转染脐静脉内皮细胞系(EAhy 926细胞),应用PCR、Western Blot进行验证转染效率,将不同表达水平Gal-9的内皮细胞与CIK细胞共培养,应用CCK-8、流式细胞术、免疫荧光强度检测法检测共培养不同时间后CIK细胞的杀伤活性、凋亡率和跨内皮迁移率的变化。结果:入组患者的年龄、性别比较差异无统计学意义,AML和ALL患者骨髓中Gal-9表达明显高于CLL患者和HDs[AML(83.41±24.38)%,ALL(89.06±16.27)%vs CLL(59.33±34.43)%,HDs(4.48±3.62)%,P=0.0008]。与未转染高表达Gal-9的内皮细胞比较,高表达Gal-9的内皮细胞与CIK细胞共培养后,CIK细胞杀伤活性相对降低(135.10±37.51 vs 187.70±57.59),凋亡率增加[(28.45±6.15)%vs(23.93±3.47)%],并且CIK细胞跨内皮细胞迁移率降低。结论:白血病细胞中Gal-9高表达,CIK细胞表面Tim-3的表达增加,提示Gal-9/Tim-3途径可能影响CIK细胞治疗白血病的生物学效应;而内皮细胞高表达Gal-9则可能促进CIK细胞凋亡,影响CIK细胞的迁移和细胞杀伤效应,Tim-3单抗干预后可在一定程度上缓解相应的抑制效应。因此,CIK细胞治疗疗效的差异可能与Gal-9/Tim-3途径相关。

Objective:To investigate the effect of over-expression of Galectin-9(Gal-9)in microvascular endothelial cells(MEC)on the cytotoxicity,apoptosis and trans-endothelial migration(TEM)of cytokine induced killer cells(CIKs).Methods:Nine acute myeloid leukemia(AML)patients,8 acute lymphoblastic leukemia(ALL)patient,5 chronic lymphoblastic leukemia(CLL)patients and 6 healthy donors(HDs)were enrolled in the present study.Flow cytometry was applied to detect the expression of Gal-9 in the corresponding bone marrow samples.The viral particles with over-expression of Gal-9 were constructed by lenti-virus vector and transfected into umbilical vein endothelial cell line(EAhy 926 cells).The transfection efficiency was verified by PCR and Western Blot.The endothelial cells with different expression levels of Gal-9 were co cultured with CIK cells,and the cytotoxicity,apoptosis rate and TEM rate of CIK cells after co culture for different time were detected by Cell Counting Kit-8(CCK-8),flow cytometry,and immunofluorescence intensity detection.Results:There was no statistcially significant difference of age or gender distribution among the enrolled patients.The Gal-9 expression in AML and ALL patients were significantly higher than those in CLL patients and HDs(AML[83.41±24.38]%,ALL[89.06%±16.27]%vs CLL[59.33±34.43]%,HDs[4.48±3.62]%,P=0.0008).Compared to the blank control group,the co-culture with high Gal-9 expressing endothelial cells significantly decreased the cytotoxicity of CIK cells(135.10±37.51 vs 187.70±57.59),increased the apoptosis rate([28.45±6.15]%vs[23.93±3.47]%)and decreased the TEM rate.Moreover,addition of Tim-3 monoclonal antibody to some extent counteracted the inhibitory effect of Gal-9 over-expression in MECs on the cytotoxicity and TEM rate of CIK cells.Conclusion:High Gal-9 expression in leukemia cells and elevated expression of Tim-3 on the surface of CIK cells indicated the important regulatory role of Gal-9/Tim-3 axis in the CIK cell therapy for leukemia.Moreover,high Gal-9 expression in MECs co-culture might promote the apoptosis of CIK cells,inhibit the migration and cytotoxicity of CIK cells,while Tim-3 monoclonal antibody treatment somehow impaired the inhibitory effects.In summary,Gal-9/Tim-3 axis plays a crucial role in affecting the CIKs immunotherapy.