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目的观察肝细胞生长因子(hepatocyte growth factor,HGF)对经凋亡诱导剂足叶乙苷(etoposide,VP-16)诱导后慢性髓细胞性白血病(CML)K562细胞凋亡的抑制作用,并分析其分子机制。方法采用苏木素-伊红(HE)染色、吖啶橙染色(AO)染色对凋亡细胞形态特征变化进行定性/半定量分析;采用Annexin V-FITC/PI双染、JC-1染色检测细胞膜表面的PS外翻和完整性及线粒体膜电位分析凋亡细胞生化特征变化;采用荧光定量聚合酶链反应(PCR)检测Bcl-2、Bax、Caspase-3、Caspase-9等凋亡相关基因mRNA表达的变化,综合评价HGF抗凋亡效应,并阐述其分子机制。结果 HE法、AO法发现HGF+VP-16组凋亡率明显低于VP-16组(P<0.05、P<0.05),提示HGF可显著抑制凋亡的发生;Annexin V-FITC/PI双染法、JC-1染色法发现HGF+VP-16组早期凋亡细胞明显低于VP-16组(P<0.05、P<0.001),提示HGF具有抗K562细胞早期凋亡效应;凋亡相关基因mRNA表达检测结果发现,HGF+VP-16组的Bcl-2 mRNA表达量明显高于VP-16组(P<0.001),而Bax mRNA、Caspase-3 mRNA、Caspase-9 mRNA表达量明显低于VP-16组(P<0.05、P<0.001、P<0.001),证实HGF抑制凋亡基因表达,同时促进抗凋亡基因的表达,提示HGF具抗凋亡效应。结论 HGF显著抑制经VP-16诱导的CML K562细胞的凋亡,该抗凋亡效应可能通过HGF/c-Met途径调控PI3K/AKT通路而实现。
Objective To investigate the inhibitory effect of hepatocyte growth factor (HGF) on apoptosis of K562 cells induced by apoptosis-inducing agent (VP-16) Its molecular mechanism. Methods The morphological changes of apoptotic cells were qualitatively and semi-quantitatively analyzed by hematoxylin-eosin (HE) staining and acridine orange staining (AO) staining. Annexin V-FITC / PI double staining and JC- PS eversion and integrity and mitochondrial membrane potential were used to analyze the biochemical characteristics of apoptotic cells. The mRNA expression of apoptosis-related genes such as Bcl-2, Bax, Caspase-3 and Caspase-9 were detected by fluorescence quantitative polymerase chain reaction The comprehensive evaluation of anti-apoptotic effect of HGF and its molecular mechanism. Results The apoptotic rate of HGF + VP-16 group was significantly lower than that of VP-16 group by HE method and AO method (P <0.05, P <0.05), suggesting that HGF could significantly inhibit the apoptosis. Annexin V-FITC / The results of JC-1 staining showed that the number of early apoptotic cells in HGF + VP-16 group was significantly lower than that in VP-16 group (P <0.05, P <0.001), suggesting that HGF could inhibit the early apoptotic effect of K562 cells and apoptosis The mRNA expression of Bcl-2 in HGF + VP-16 group was significantly higher than that in VP-16 group (P <0.001), while the expression of Bax mRNA, Caspase-3 mRNA and Caspase-9 mRNA was significantly lower In VP-16 group (P <0.05, P <0.001, P <0.001), it was confirmed that HGF can inhibit the expression of anti-apoptotic gene and promote the anti-apoptotic effect of HGF. Conclusions HGF significantly inhibits the apoptosis of CML K562 cells induced by VP-16, and this anti-apoptotic effect may be mediated by the HGF / c-Met pathway regulating the PI3K / AKT pathway.