采用Calcein-AM评价白血病细胞膜P-gp泵功能及其逆转剂合理应用的研究

Evaluation of the P-gp pump function on leukemic cell membrane and proper application of its reversal agents with Calcein-AM and flow cytometry

目的通过荧光染料 Calcein-AM(C-AM)在 K562及其耐长春新碱(VCR)的细胞系K562/VCR 细胞中的变化以及异搏定(VER)、环孢霉素 A(CSA)对 C-AM 在 K562和 K562/VCR 细胞中摄取与排出的影响,研究 C-AM 在评价 P-gp 泵功能的作用,评价两种逆转剂的逆转作用,为临床合理应用逆转剂提供实验依据。方法以 K562及 K562/VCR 为实验对象,以流式细胞术测定两种细胞系内不同时间点(0、30、60、90和120 min)的 C-AM 荧光强度来反映 P-gp 泵功能以及 CSA、VER 对其的影响。结果 C-AM 在 K562和 K562/VCR 细胞中的变化以24h 内最为明显,24h 后 K562细胞和 K562/VCR 细胞内 C-AM 荧光强度分别下降至14.57%和15.64%。其在 K562细胞内各时间点的平均荧光强度(MFI)明显高于 K562/VCR 细胞(各时间点差异 P 值均<0.05);CSA、VER 和 CSA+VER 对 K562细胞内 C-AM 的摄取和外排无明显影响,但各组逆转剂、C-AM 与细胞共孵育120 min 后,对照组、CSA、VER 和 CSA+VER 组的 K562/VCR 细胞 MF1分别为3251±106.7、4014±219.0、3879±116.1和4158±302.6,与对照组相比,后三组细胞对 CAM 的摄取明显增加(P 均<0.05)。当外排120 min 后,对照组、CSA、VER 和 CSA+VER 组的 K562/VCR 细胞 MF1分别为1622±191.0、2237±155.2、1932±233.0和2231±146.7,与对照组相比,后三组的 CAM 残余量也明显增加(P均<0.05),表明 CSA、VER 和 CSA+VER 能增加 K562/VCR 细胞对 C-AM 的摄取和减少细胞内 C-AM 的外排。结论 P-gp 的表达能减少 K562/VCR 细胞对 C-AM 的摄取和增加其对 C-AM 外排,逆转剂CSA 与 VER 的联合应用对 K562/VCR 细胞摄取和外排 C-AM 的影响并不比单独使用 CSA 或 VER 明显,通过使用 C-AM 和流式细胞术,可以比较迅速、简便评价白血病细胞 P-gp 的泵功能以及逆转剂的逆转作用。

Objective Leukemia is the most common malignancy in children. Combined chemotherapy is currently the primary treatment modality. During the past decade, very high cure rates of childhood acute lymphoblastic leukemia （ALL） have been reported both at home and abroad. However, the cure rates of children with acute myeloid leukemia （AML） remain low due to the multiple-drug resistance （MDR）. P-glycoprotein （P-gp） is one of the most important mechanisms of MDR for leukemia cells. However, the function of the protein, the clinical application of its reversal agents and the efficacy of the combination of the reversal agents remain to be elucidated. The present study aimed to evaluate the P-gp pump function on leukemia cell membrane and the effects of the combined administration of the reversal agents cyclosporin A （CSA） and verapamil （VER） through the observation of Calcein-AM （C-AM） metabolism in the cell line K562 and its multi-drug resistant subline K562/VCR. Methods The mean fluorescence intensity （MFI） of C-AM inside the cytoplasm was analyzed with flow cytometry （FCM）. The events of K562 and K562/VCR ceils treated and untreated with CSA, VER and CSA ＋ VER were acquired at time points 0, 30, 60, 90 and 120 minutes, respectively, and the data obtained were analyzed with CellQuest software. Results The C-AM in the K562 and K562/VCR varied more apparently in the fist 24 hours. In addition, the MFI of the C-AM in K562 was significantly higher than that in K562/VCR cells indicating that the P-gp pump molecules were functioning. The MFIs of the CSA, VER and CSA ＋ VER groups Co-cuhrued with K562/VCR cells were 4014 ± 219, 3879 ± 116 and 4158 ± 302, respectively after 120 min of incubation, significantly higher as compared to that of control group （3251 ± 107, P 〈 0. 05 ）. On the other hand, significant inhibition of the efflux from the K562/VCR cell line was also noticed after the same time period of incubation with the MFIs of 2237 ± 155, 1932 ± 233 and 2231 ± 147, respectively in the three groups, which was significantly higher than that of control group （ 1622 ± 191, P 〈0. 05）. CSA, VER and CSA ＋ VER could increase the uptake and inhibit the efflux of C-AM by K562/VCR cells, while no evident influence on those functions inside the parental cell line K562 cells was noticed. Conclusions CSA, VER and CSA ＋ VER could increase the uptake and reduce the efflux of C-AM by K562/VCR cells while no significant difference between the CSA ＋ VER and CSA or VER was noticed. P-gp pump function and the effects of its reversal agents on leukemic cells can be rapidly and easily evaluated by using the C-AM and FCM.