黏着斑激酶基因对白血病细胞生物学特性的影响

 【目的】本实验研究黏着斑激酶（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REH白血病细胞株，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72～80d。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。

Effect of FAK Gene on Biochemical Characteristics of REH Leukemic Cells

【Objective】 The study was aimed to investigate the effect of focal adhesion kinase(FAK) gene on the biochemical characteristics of leukemic cells.【Methods】 Lentiviral-FAK-shRNA was transduced into REH leukemic cells,while empty vector was transduced into the same cells used as control.FAK protein was detected by western blot.Cell apoptosis was tested by labeling with Annexin V.The leukemic cells were incubated with SDF-1 in a transwell system,and the migration rate was detected in vitro.Moreover,leukemic cells labeled with CFSE were injected into SCID mice,and the homing rate to different tissues and leukemogenesis were monitored.【Results】 FAK protein expression was knocked down by up to 75% relative to empty vector in REH cells by FAK shRNA.The apoptosis experiment showed that the percentage of Annexin V+ cells in empty vector group and FAK gene silencing group were(2.19 ± 0.36)% and(6.48 ± 0.58)%,respectively.Moreover,the migration percent in empty vector group and FAK gene silencing group were(50.3 ± 7.22)% and(7.6 ± 3.15)%,respectively.The results of homing experiment in vivo showed that FAK gene silencing significantly inhibited the homing rate of leukemia in bone marrow.The mice in vector control group died between day 40 and day 47,while the mice in FAK shRNA group died between day 72 and day 80.Moreover,45 days post injection of leukemic cells,the percentage of leukemic cells in bone marrow of leukemic mice in vector control and FAK gene silencing group were(65.5 ± 8.20)% and(9.60 ± 3.65)%,respectively.【Conclusion】 FAK gene silencing was cable of inducing cell apoptosis,reducing migration and homing ability,and inhibiting leukemogenesis.Our results indicated that targeting FAK gene might be considered as a new therapeutic strategy for leukemia.