甲氨蝶呤与长春新碱双载红细胞的体外抗肿瘤活性研究

In vitro antineoplastic effect of erythrocytes co-encapsulated with methotrexate and vincristine

目的研究甲氨蝶呤(methotrexate,MTX)与长春新碱(vincristine,VCR)双载红细胞体外抗肿瘤效果。方法改良低渗预膨胀技术制备MTX+VCR双载红细胞、MTX单载红细胞和VCR单载红细胞。分别采用制备的载体红细胞、未载药红细胞、MTX+VCR工作液及RPMI1640培养液与K562细胞共培养。MTT法检测K562细胞增殖,PI染色流式细胞仪分析K562细胞周期,Annexin Ⅴ-FITC/PI染色流式细胞仪分析K562细胞凋亡情况。结果双载红细胞明显抑制K562细胞的增殖并促进其凋亡作用,且随共培养时间的延长而作用增强,效果优于MTX单载红细胞(P<0.05),与未载药红细胞相比差异显著(P<0.01);细胞周期结果显示,双载红细胞组G0/G1期K562细胞明显减少,G2/M期细胞逐渐增多,S期K562细胞在作用12h后明显增多,24h后略有减少,与未载药红细胞的作用相比差异显著,与MTX+VCR原药组的作用基本相同。结论与未载药红细胞和单载红细胞相比,MTX+VCR双载红细胞杀伤肿瘤细胞作用更强。

Objeetive： To study the in vitro anti-tumor effect of methotrexate （MTX） and vincristine （VCR）-loaded erythrocytes. Methods： MTX-4-VCR-loaded, MTX-loaded, and VCR-loaded erythrocytes were prepared by modified hypoosmotic swelling technique. K562 cells were treated with the prepared erythrocytes, pure erythrocytes, MTX＋VCR solution, and RPMI 1640 medium. MTT assay was used to examine the proliferation of K562 cells. The cell cycle of K562 was observed by flow cytometer using PI dyeing. The apoptosis of co-cultured K562 cells was analyzed by flow cytometer with Annexin VFITC/PI kit. Results： MTX ＋ VCR-loaded erythrocytes efficiently inhibited the proliferation and induced apoptosis of co-cultured K562 cells in a time-dependent manner, and its effects were better than those mono-loaded ones （P〈0. 05） and unloaded ones（P〈0.01）. Cellular cycle analysis demonstrated that, compared with control groups, proportion of K562 cells at G0/G1 phase decreased whereas that at S phase increased 12 hours after co-cultured with MTX＋VCR-loaded erythrocytes. 24 hours after co-cultured with MTX ＋ VCR-loaded erythrocytes the cells at G2/M phase increased and the cells in S phase decreased compared with those of 12 hours; there were significant difference between MTX＋VCR group and unloaded group; and the MTX＋VCR-loaded erythrocytes had a similar effect to that of MTX＋VCR solution. Conclusion： Compared with pure erythrocytes and mono-loaded erythrocytes, MTXA-VCR-loaded erythrocytes have more potent anti-tumor effect.