雷帕霉素对甲氨蝶呤诱导急性淋巴细胞白血病EU-4细胞凋亡的影响

Influence of Rapamycin on apoptosis of acute lymphoblastic leukemia EU -4 cells induced by Methotrexate

目的探讨雷帕霉素（RAPA）对甲氨蝶呤（MTX）诱导急性淋巴细胞白血病（ALL）EU-4细胞凋亡的影响。方法分别使用5μg／L、10μg／L、25μg／L、50μg／L和100μg／L的RAPA预处理EU-4细胞0．5h，RAPA未处理组作为对照组，收集细胞，Westernblot检测LC-3蛋白，流式细胞技术检测凋亡率。观察不同质量浓度的RAPA预处理对Eu-4细胞自噬和凋亡的影响，确定RAPA预处理浓度。将Eu-4细胞分为RAPA预处理组和未处理组，分别使用0．05μmol／L、0．10／μmol／L和0．20μmol／L的MTX作用24h，流式细胞技术检测凋亡率，Westernblot检测LC-3蛋白，酶标仪测定吸光度（A_562），根据标准曲线计算样品的蛋白浓度。结果检测对照组和不同浓度RAPA预处理组的EU-4细胞凋亡率显示，对照组，5μg/L、10μg／L、25μg／L、50μg／L和100μg／LRAPA预处理组Eu-4细胞的凋亡率分别为（7．51±0．32）％、（7．33±0．41）％、（7．71±0．51）％、（7．63±0．38）％、（7．80±0．43）％和（16．66±0．87）％，5μg／L、10μg／L、25μg/L和50μg／LRAPA预处理组EU-4细胞的凋亡率与对照组比较差异均无统计学意义（t=0．427、-0．417、-0．297、-3．561，均P〉0．05），而100μ/gL RAPA预处理组Eu-4细胞的凋亡率显著高于对照组，差异有统计学意义（t=-28．815，P〈0．01）。根据Westernblot和流式细胞技术的检测结果综合考虑，选取50μg／L作为RAPA预处理的浓度。流式细胞技术检测RAPA预处理组和未处理组MTX作用24h后EU-4细胞的凋亡率显示，RAPA预处理组用0．05μmol／L、0．10μmol／L和0．20μmol／LMTX作用24h后，EU-4细胞的凋亡率分别为（50．23±2．11）％、（66．88±2．89）％和（73．11±2．67）％，未处理组0．05μmol／L、0．10μmol／L和0．20μmol／L MTX作用24h后，EU-4细胞的凋亡率分别为（22．53±1．67）％、（42．82±2．26）％和（53．22±1．93）％。相同浓度的MTX作用24h后，RAPA预处理组Eu-4细胞的凋亡率均显著高于未处理组，差异均有统计学意义（t=12．693、66．148、14．429，均P〈0．01）。结论RAPA预处理后，较低剂量的MTX即可诱导急性淋巴细胞白血病Eu-4细胞大量凋亡。

Objective To explore the influence of Rapamycin （RAPA） on apoptosis of acute lymphoblastic leukemia EU - 4 cells induced by Methotrexate （MTX）. Methods EU - 4 cells were pretreated O. 5 h with 5 μg/L, 10 μg/L,25μg/L,50 μg/L and 100 μg/L RAPA. RAPA untreated group was set up as control group. The cells were col- lected and the expression of LC - 3 was assayed by using Western blot. The apoptosis of EU - 4 cells was detected by using flow cytometry（FCM）. The effects of different concentrations of RAPA pretreatment on autophagy and apoptosis of EU - 4 cells were observed, and the pretreatment concentration of RAPA was determined. EU - 4 ceils were divided into RAPA pretreatment group and untreated group. The ceils were treated with 0.05 μmol/L,0. 10 μmol/L and 0.20 μmol/L MTX for 24 h, respectively. The apoptotic rate was detected by using FCM. Western blot was performed to test the expression of LC -3 protein,while the absorbance（A_562 ） was measured by using microplate reader,and the protein concentration in the sample was calculated according to the standard curve. Results The apoptotic rates of EU -4 cells in the control group and the different concentrations of RAPA pretreatment group showed that the control group, 5 μg/L, 10 μg/L,25 μg/L, 50 μg/L and 100 μg/L RAPA were （ 7.51 ± 0.32 ） %, （7.33 ± 0.41 ） %, （ 7.71 ± 0. 51 ） % , （7. 63 ± 0.38 ） %, （ 7.80 ± 0.43 ） % and （ 16.66 ± 0.87 ） % , respectively. The apoptotic rates of EU - 4 cells in the 5 μg,/L, 10 μg/L,25 μg/L and 50 μg/L RAPA pretreatment group had no significant difference from those in the control group（ t = 0.427, - 0. 417, - 0. 297, - 3. 561, all P 〉 0.05 ）. The apoptotie rate of EU - 4 cells in 100 μg/L RAPA pretreatment group was significantly higher than that in control group, and the difference was significant （t = - 28. 815 ,P 〈0. 01 ）. Combined with the results of Western blot and FCM ,50 μg/L was used as the pretreatment of RAPA. The apoptosis rates of EU - 4 cells in RAPA pretreatment group were （50.23 ± 2.11 ） %, （66.88 ± 2.89） % and （73.11 ± 2.67） % after treated with 0.05 μmol/L,0.10 μmol/L and 0.20 μmol/L MTX for 24 h, respectively. The apoptotic rates of EU - 4 cells in RAPA unpretreatment group were （22.53 ± 1.67） %, （42.82 ± 2.26） % and （53.22 ± 1.93）% after treated with 0.05 μmol/L,0. 10 μmol/L and 0.20 μmol/L MTX for 24 h, respectively. The apoptotic rates of EU -4 cells in RAPA pretreatment group were significantly higher than those in untreated group in the same concentration of MTX treatment after 24 h, and the differences were significant （ t = 12. 693,66. 148,14. 429 ; all P 〈0. 01 ）. Conclusion With RAPA pretreatment,relative low dose MTX can induce a great deal of acute lympho- blastic leukemia EU -4 cells apoptosis.