Objective: To study the molecular mechanism of PI3K-Ⅲ like functional domain inducing programmed cell death of leukemia cell line K562.
Methods: The purified PI3K-Ⅲ like functional domain protein was obtained by Pichia pastoris expression system. MTT assay and colony-forming assay were used to detect the effects of PI3K-Ⅲ like functional domain protein on K562 cell proliferation. The effects of PI3K-Ⅲ like functional domain protein on apoptosis and cell cycle of on K562 cells were detected by flow cytometry. The ultrastructural changes were detected by transmission electron microscopy. The expression of caspase-3 was detected by ELISA. The protein expressions of ATG4B, Beclin-1, Bcl-2 and LC3-II were evaluated by Western blot.
Results: PI3K-Ⅲ like functional domain protein could inhibit the proliferation and clony formation of K562 cells, which was significantly higher than the control group (P<0.05). In the experimental group, apoptosis and autophagosome were shown in K562 cells. The proportion of cells in G0/G1 phase increased significantly, while in S phase decreased significantly. Cell growth mostly stagnated in G0/G1 phase, which was significantly different from the control group (P<0.05). With the increase of concentration, the expression of caspase-3 protein increased significantly compared with the control group (r=0.966, P<0.05). The expression of ATG4B and beclin-1 appeared from increase to decrease, LC3-II increased while Bcl-2 decreased at different time points.
Conclusion: PI3K-Ⅲ like functional polypeptide could induce programmed cell death of leukemia cell K562. Beclin-1/Bcl-2 and caspase pathway may be involved in this way, which suggesting meant autophagy and apoptosis may work together at the same time.
题目: PI3K-Ⅲ样功能多肽对白血病细胞K562程序性死亡的作用机制研究.
目的: 研究PI3K-Ⅲ样功能多肽诱导白血病细胞K562程序性死亡的作用机制。.
方法: 采用毕赤酵母表达系统获得纯化PI3K-Ⅲ样功能多肽；采用MTT法与平板克隆形成试验检测PI3K-Ⅲ样功能多肽对K562细胞增殖能力的影响；流式细胞术检测K562细胞的凋亡与细胞周期；透射电子显微镜检测细胞超微结构的变化；ELISA方法检测Caspase-3表达；Western blot检测K562细胞ATG4B、Beclin-1、Bcl-2、LC3-II等蛋白表达。.
结果: PI3K-Ⅲ样功能多肽能够抑制K562细胞的增殖及克隆形成，与对照组细胞相比抑制率明显增高（P<0.05）；功能域蛋白作用后，K562细胞呈现凋亡状态，且伴随自噬体形成；细胞G0/G1 期所占比例明显升高，S期所占比例明显降低，细胞生长多停滞于G0/G1 期，与对照组细胞比例存在显著性差异（P<0.05）；随着多肽作用浓度增加，Caspase-3蛋白表达显著增加，与对照组相比有显著差异（r=0.966，P<0.05）；功能域蛋白作用K562细胞后不同时间点ATG4B、Beclin-1的表达呈先升后降、LC3-II的表达呈上升、Bcl-2的表达呈下降趋势。.
结论: PI3K-Ⅲ样功能多肽可诱导白血病细胞K562发生程序性死亡，Beclin-1/Bcl-2与Caspase通路可能参与其中，自噬与凋亡可能均有发挥作用。.
Keywords: PI3K-Ⅲ; apoptosis; autophagy; leukemia.