黄芩苷通过激活JNK/p38 MAPK通路诱导ALL Reh细胞凋亡

目的 探究黄芩苷对急性淋巴细胞白血病(ALL)细胞株Reh增殖和凋亡的影响及c-Jun氨基蛋白激酶(JNK)/p38丝裂原活化蛋白激酶(p38 MAPK)通路在其机制中的作用。方法 黄芩苷处理或联合活性氧(ROS)清除剂NAC共处理Reh细胞;流式细胞术检测细胞凋亡、胞内ROS水平和线粒体膜功能(MMP);免疫荧光实验观察凋亡细胞的数目及其形态变化。Western blotting免疫印迹检测Reh细胞内凋亡蛋白和信号通路蛋白的表达水平。结果 与对照组比较,黄芩苷组Reh细胞增殖被抑制,细胞凋亡率、促凋亡蛋白、ROS、JNK和p38磷酸化水平显著上升,抗凋亡蛋白表达下调,MMP明显受损(P<0.01)。NAC清除ROS后明显恢复细胞增殖,JNK和p38磷酸化水平明显下降,Survivin表达恢复(P<0.01)。结论 黄芩苷经激活JNK/p38 MAPK通路活化Caspase3/7,诱导Reh细胞凋亡。

黏着斑激酶基因对REH白血病细胞生物学特性的影响

【目的】研究黏着斑激酶(FAK)基因对白血病细胞生物学特性的影响。【方法】构建FAK shRNA慢病毒载体,转染至急性淋巴白血病细胞株REH,同时建立空白载体对照细胞株。通过蛋白质印迹方法检测FAK蛋白表达情况,予荧光染料Annexin V标记细胞观察细胞凋亡情况。体外应用Transwell培养体系,观察白血病细胞在细胞因子SDF-1作用下的迁移能力。将REH细胞予绿色荧光CFSE进行标记,经尾静脉注射到SCID小鼠,观察白血病细胞在动物体内归巢及白血病生成情况。【结果】FAK shRNA慢病毒载体在REH白血病细胞蛋白水平的抑制率为75%。凋亡实验中,空白对照组和FAK基因沉默组Annexin V+细胞百分比分别为(2.19±0.36)%和(6.48±0.58))%。体外迁移实验发现空白对照组与FAK基因沉默组的细胞迁移百分比分别为(50.3±7.22)%和(7.6±3.15)%;动物体内归巢实验发现FAK基因沉默的细胞归巢于骨髓能力明显降低。白血病动物实验发现空白载体对照组小鼠生存时间是40～47 d,而FAK基因沉默组小鼠生存时间是72～80 d。白血病细胞输注后第45天,空白载体对照组与FAK基因沉默组小鼠骨髓中白血病细胞所占百分比分别为(65.5±8.20)%和(9.60±3.65)%。【结论】FAK基因沉默能诱导白血病细胞凋亡,并降低白血病细胞迁移及归巢能力,并抑制白血病生成,提示分子靶向FAK基因有望成为白血病治疗的新策略。

Dehydroabietic acid chemosensitizes drug-resistant acute lymphoblastic leukemia cells by downregulating survivin expression

Objective:To explore the mechanism of drug resistance in acute lymphoblastic leukemia and the anti-tumor effect of combination therapy of dehydroabietic acid and vincristine against acute lymphoblastic leukemia cells.Methods:Acute lymphoblastic leukemia cells REH and CCRFCEM were employed to detect the anti-tumor effect of vincristine and doxorubicin on proliferation and apoptosis using EdU assay,human active caspase-3 Quantikine ELISA kit,and flow cytometry.Vincristine-resistant REH cells(REH-R),survivin knockdown and overexpressing REH cells were established to verify the role of survivin in drug resistance.Additionally,in vitro and in vivo assays were performed to determine the effect of dehydroabietic acid on the cytotoxicity of vincristine.Results:Vincristine and doxorubicin markedly suppressed proliferation and induced apoptosis of REH and CCRF-CEM cells.Survivin expression was upregulated in REH-R cells compared with REH cells.Knockdown of survivin expression obviously restored the sensitivity of REH-R cells to vincristine.Akt phosphorylation was also increased in REH-R cells compared to REH cells.In addition,LY294002,a PI3k/Akt pathway blocker,inhibited survivin expression and enhanced cytotoxicity of vincristine to REH-R cells.Dehydroabietic acid effectively reduced survivin expression in REH-R cells,thereby enhancing the therapeutic effect of vincristine on drug-resistant cells.Survivin overexpression markedly reduced the effect of dehydroabietic acid on enhancing the anti-proliferation and inducing apoptosis effect of vincristine.Moreover,the combination of dehydroabietic acid with vincristine significantly extended the survival rate in a mouse xenograft model of acute lymphoblastic leukemia,compared with vincristine treatment alone.Conclusions:Dehydroabietic acid may be used as a potential candidate for the treatment of acute lymphoblastic leukemia in combination with vincristine.

胰岛素对Reh细胞胰岛素受体和胰岛素样生长因子Ⅰ型受体表达及细胞增殖的影响

本研究探讨胰岛素对Reh细胞胰岛素受体(IR)和胰岛素样生长因子Ⅰ型受体(IGF-ⅠR)表达的影响及对Reh细胞的促增殖作用。用CCK-8法检测Reh细胞的增殖;采用实时PCR法检测Reh细胞IR和IGF-ⅠRmRNA的表达。结果表明,胰岛素对Reh细胞具有浓度和时间依赖性的促增殖作用,当胰岛素浓度为10-9 mol/L时其促增殖作用最强,进一步提高浓度,胰岛素的促增殖作用不再增强。与对照组相比,胰岛素有明显的促增殖作用(P<0.05)。胰岛素10-9mol/L作用24、48及72 h,IR mRNA表达量与对照组相比的比值分别为2.2520±0.7431、1.9956±0.9692和3.9766±1.3189,IGF-ⅠR表达量与对照组相比的比值分别为1.0803±0.2238、1.6026±0.6158和3.1013±0.1008。胰岛素刺激后IR和IGF-ⅠR的mRNA相对表达量与对照组相比均明显增加,差别有统计学显著意义(P=0.022和P=0.00)。结论:胰岛素能促进Reh细胞的增殖,其机制可能与IR和IGF-ⅠR上调有关。IR和IGF-ⅠR的高表达与白血病细胞的生长有着密切关系。

FAK基因沉默诱导白血病细胞凋亡

目的:本研究靶向黏着斑激酶(FAK)基因,构建FAK-shRNA慢病毒载体,并研究FAK基因沉默对白血病细胞生长的影响。方法:化学合成人FAK基因的shRNA序列,应用分子生物学方法将该FAK shRNA序列插入含荧光素GFP慢病毒质粒。该FAK shRNA慢病毒载体在体外包装、浓缩,然后转染至人白血病细胞株。应用逆转录聚合酶链反应及蛋白质印迹方法检测FAK基因表达水平,予Annexin V标记检测细胞凋亡的情况。结果:经核苷酸序列分析提示FAK shRNA正确插入慢病毒质粒,该病毒载体在白血病细胞株的转染率为10%-25%。与对照组相比(无FAK shRNA的慢病毒载体),FAK shRNA慢病毒载体在细胞FAK mRNA及蛋白水平的抑制率分别为40%和70%。凋亡实验表明,对照组与FAK shRNA慢病毒载体组的Annexin V阳性率分别为(4.19±0.36)%和(8.48±0.58)%,两者差异明显(P<0.05)。结论:我们成功构建FAK shRNA慢病毒载体,该载体能抑制FAK基因表达并诱导白血病细胞凋亡。