维生素K_2对骨髓增生异常综合征细胞株MDS-JSN04生长抑制作用及其机理研究

Inhibition Effect of Vitamin K_2 on Human MDS-JSN04 Cell Line and Its Possible Mechanism

为了探讨维生素K2(VK2)对骨髓增生异常综合征细胞株MDSJSN04生长的抑制作用及其作用机制,采用细胞形态学方法观察VK2处理后细胞形态的改变;应用四甲基偶氮唑蓝(MTT)比色法检测VK2对细胞生长的抑制作用;流式细胞术分析细胞凋亡、细胞周期变化和髓系分化抗原CD11b、CD13的表达情况;RTPCR技术检测抗凋亡基因bcl2、survivin和促凋亡基因bax的表达;用发光比色法检测caspase3活性。结果表明:VK2作用细胞株72小时后,细胞呈现典型的凋亡细胞的形态特征;细胞的凋亡率随着VK2浓度的增加和作用时间的延长而逐渐增高,经统计学处理与对照组比较有显著性差异(P<0.01);S期和G2期细胞随着药物浓度的增加而逐渐减少,G0/G1期细胞逐渐增多,与对照组比较有显著性差异(P<0.01),细胞被阻滞在G0/G1期,而且在G0/G1期前出现明显的亚G1期细胞即凋亡峰;抗凋亡基因bcl2、survivin表达随着VK2浓度的增高而明显下调((P<0.05),而促凋亡基因bax表达无明显变化((P>0.05),caspase3的活性随着VK2浓度的增加和作用时间的延长而逐渐增强。结论:VK2主要是通过激活caspase3途径诱导MDSJSN04细胞发生凋亡,同时抗凋亡基因bcl2和survivin也参与了凋亡调控过程。

To study the effects and possible mechanism of Vitamin K2 （ VK2 ） in the treatment of MDS-JSN04 cells, the changes of morphologic features of MDS-JSN04 cells were investigated by cytomorphology, the apoptosis of MDS- JSN04 cells was observed by transmission electron microscope; cellular proliferation was determined by the MTT assay; cell apoptosis ,cell cycle shift and expression of myeloid-specific differentiation antigen （CD11 b, CD13 ） were analyzed by flow cytometry （FCM）. The expression of apoposis-related genes bcl-2 ,survivin and bax were detected by retrotranscriptase polymerase chain reaction （RT-PCR） ; the activity of caspase-3 was determined by chemiluminescence assay. The results showed that the typical apoptotic morphological features appeared in cells treated with VK2 for 72 hours ; VK2 induced apoptosis of MDS-JSN04 cells and in a dose-and-time- dependent manner, G0/G1 cell arrest and significantly down-regulated the expression of bcl-2 and survivin, but had no effect on the expression of bax; the activity of caspase-3 significantly increased. It is concluded that VK2 induces apoptosis of MDS-JSN04 cells through activating caspase-3 pathways and the apoptosis-related genes bcl-2, survivin may play an important role in this process.