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目的采用血清饥饿法诱导K562细胞凋亡,比较K562细胞与人白血病骨髓间充质干细胞(LMSC)共培养前后,K562细胞凋亡的变化,并进一步探讨其可能涉及到的信号转导通路(PI3K/Akt/Bad),为临床靶向治疗白血病提供依据。方法采用膜联蛋白V/碘化丙啶(Annexin V/PI)荧光标记法检测血清饥饿后和与LMSC共培养后K562细胞凋亡的变化。蛋白印迹法检测与LMSC共培养后及加入磷脂酰肌醇-3-激酶(PI3K)特异性阻断剂LY294002后,K562细胞中Akt、p-Akt、Bad、p-Bad蛋白的变化。结果 10%胎牛血清(FBS)培养时,K562细胞凋亡率为(4.87±0.09)%,FBS饥饿培养24h后,K562细胞凋亡率为(10.14±0.50)%,较10%FBS培养明显升高(P<0.05)。与LMSC共培养后,K562细胞凋亡率为(7.15±0.06)%,细胞凋亡率较FBS饥饿培养下降(P<0.05)。进一步蛋白印迹法检测发现,K562细胞在单独悬浮培养条件下,即有Akt、p-Akt、Bad、p-Bad蛋白的表达;与LMSC共培养后,p-Akt、p-Bad的表达量显著升高(P<0.05),在共培养体系中加入PI3K特异性抑制剂LY294002后,p-Akt、p-Bad的表达量明显下降(P<0.05);而3组间Akt、Bad蛋白的表达量未见明显变化(P>0.05)。结论①LMSC能够抑制血清饥饿诱导的K562细胞凋亡。②其涉及到的信号转导通路可能为PI3K/Akt/Bad。③PI3K/Akt/Bad可作为白血病靶向治疗的新的作用靶点。
OBJECTIVE: To induce the apoptosis of K562 cells by serum starvation and to compare the changes of apoptosis of K562 cells before and after the co-culture of K562 cells and human leukemia bone marrow mesenchymal stem cells (LMSC), and to further explore its possible signal transduction pathway (PI3K / Akt / Bad), provide the basis for clinical targeted therapy of leukemia. Methods The apoptosis of K562 cells after serum starvation and co-culture with LMSC was detected by Annexin V / PI fluorescent labeling method. The changes of Akt, p-Akt, Bad and p-Bad protein in K562 cells after co-culture with LMSC and LY294002 specific inhibitor of phosphatidylinositol 3-kinase (PI3K) were detected by Western blotting. Results The apoptosis rate of K562 cells was (4.87 ± 0.09)% when incubated with 10% fetal bovine serum (FBS). The apoptosis rate of K562 cells after (10.14 ± 0.50)% of FBS starvation culture was significantly higher than that of 10% FBS Increased (P <0.05). After co-cultured with LMSC, the apoptosis rate of K562 cells was (7.15 ± 0.06)%, and the apoptosis rate of K562 cells was lower than that of FBS starvation culture (P <0.05). Further Western blotting showed that the expression of Akt, p-Akt, Bad and p-Bad protein in K562 cells under suspension culture alone was significantly higher than that in LMSC alone (P <0.05). The expression of p-Akt and p-Bad in PI3K-specific inhibitor LY294002 decreased significantly after co-culture (P <0.05). However, the expression of Akt and Bad proteins No significant changes in the amount of (P> 0.05). Conclusion ① LMSC can inhibit serum-induced K562 cell apoptosis. ② The signaling pathway involved may be PI3K / Akt / Bad. ③ PI3K / Akt / Bad can be used as a new target of leukemia targeted therapy.