索拉非尼通过下调细胞周期蛋白D1诱导急性髓细胞白血病细胞凋亡的实验研究

目的观察索拉非尼对U937、HL60细胞株和1例急性髓细胞自血病（AML）患者（AML—M2）白血病细胞的细胞周期蛋白D1（CCND1）表达情况的影响，探讨索拉非尼对白血病细胞的作用机制。方法常规培养U937、HL60细胞株，密度梯度离心法分离白血病患者的白血病细胞。将加有索拉非尼的细胞培养组设为实验组，将加有空白培养液的培养组设为对照组，常规MTT法检测细胞生长抑制率。使用AnnexinV-FITC试剂盒，流式细胞术检测实验组及对照组0h、12h、24h、48h的细胞凋亡率。运用实时定量PCR（SYBR Green1荧光染料法）测定实验组和对照组在0h和24h两个时间点CCND1基因的表达量。结果索拉非尼各浓度均可抑制细胞生长，抑制率由1．27％升至89．88％。药物共同培养，实验组凋亡率高于对照组。实验组24h凋亡率在（19．89±5．73）％。定量PCR测定实验组CCND1表达下调达5．88倍，对照组CCND!表达上调达1．18倍。结论索拉非尼促进AML细胞凋亡和CCND1基因表达下调，CCNDI基因参与了索拉非尼促进AML细胞凋亡的调控过程。

Sorafenib induces apoptosis of AML cells via CCND1-mediated activation of the intrinsic apoptotic pathway

Objective To explore the apoptosis of U937 cells, HL60 cells and leukemic blasts from a patient with AML-M2 via CCNDI-mediated activation of the intrinsic apoptotic pathway induced by Sorafenib. Methods The human AML cell line U937, HL60 and leukemic blasts from a patient with AML- M2 were cultured routinely, the leukemia blasts were isolated with density gradient centrifugation, the group with Sorafenib as test group, and without Sorafenib as control group. Cell viability analysis was assessed using MTI＇, and apoptosis rate was measured by flow cytometry using Annexin V staining. The expression level of CCND1 was analyzed by real-time PCR. Results AML cells were suppressed significantly by Sorafenib in different concentrations,the rate of suppression ranged from 1.27% to 89.88%. Apoptosis rate in the test group was higher than that in control group. The apoptosis rate was （ 19.89 ±5.73 ） % in test group in 24 hour（ P 〈 0. 05 ）. The expression level of CCND1 was downregulated by Sorafenib. Conclusions Sorafenib may induce apoptosis of AML cells by suppression of CCNDI exprssion via Ras/MAP pathway.