肝细胞生长因子诱导慢性髓细胞性白血病K562细胞抗凋亡效应研究  

Anti-apoptosis of Hepatocyte Growth Factor on CML K562 Cells

作　　者：郑筱娇[1] 高洲[2] 沈蓉蓉[2] 赵行[2] 滑世轩[2] 吕建新[2] 岑东[2,3] ZHENG Xiao-jiao GAO Zhou SHEN Rong-rong ZHAO Hang HUA Shi-xuan Lü Jian-xin CEN Dong(Ningbo First Hospital, Ningbo 315010, China Zhefiang Provincial Key Laboratory of Medical Genetics, Wenzhou Medical College, Wenzhou 325035, China Ningbo Urinary and Kidney Hospital, Ningbo 315040, China)

机构地区：[1]宁波市第一医院,浙江宁波315010 [2]温州医科大学,浙江省医学遗传学重点实验室,浙江温州325035 [3]宁波市泌尿肾病医院,浙江宁波315040

出　　处：《中国药学杂志》2017年第3期201-205,共5页Chinese Pharmaceutical Journal

基　　金：浙江省医药卫生科技项目(2014KYB243) ;宁波市科技计划( 2014C50015;2013A610244;2013A610219;2010A610031)

摘　　要：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 protection of hepatocyte growth factor （HGF） on CML cell line K562 from apoptosis induced by etoposide （VP-16） and its molecular mechanism. METHODS Quantitative and qualitative analyses on cell morphological change of apoptosis were performed through acridine orange （AO） staining and HE staining, and fluorescent flow cytometry. The test analyzes membrane on the surface of the PS evagination and integrity of cell membrane surface and mitochondrial membrane potential changes were performed through Annexin V-FITC/PI double dyeing and JC-1 cell dyeing tests, and apoptotic factors such as Bcl-2, Box, Caspase-3 and Caspase-9 were measured by SYBR Green （Takara） qRT-PCR. RESULTS The HE and AO staining revealed that apoptotic rates in HGF ＋ VP-16 groups were significantly lower than those in VP-16 groups （P 〈 0.05 ,P 〈 0. 05 ）, HGF can inhib- it the apoptosis of cells induced by VP-16; FCM （ Annexin V-FITC/PI and JC-1 ） tests showed that cells apoptotic rates in HGF ＋ VP- 16 groups were significantly lower than those in VP-16 groups （P 〈 0.05, P 〈 0. 001 ）, indicating that HGF has the anti-apoptosis func- tion. Apoptosis related gene mRNA expression tests found that the Bcl-2 mRNA expression in HGF ＋ VP group was obviously higher than that in the VP-16 group （P 〈 0. 001 ）, while Box mRNA, Caspase-3 mRNA, and Caspase-9 mRNA expressions were significantly lower than those in the VP-16 group （ P 〈 0. 05, P 〈 0. 001, P 〈 0. 001 ）, suggesting that HGF possesses antiapoptotic effect through in- hibiting apoptosis gene expression and promoting the antiapoptotic gene expression simultaneously. CONCLUSION HGF can signifi- cantly protect K562 cells from apoptosis induced by VP-16 through the HGF/c-Met way to regulate PI3K/AKT pathway.

关 键 词：肝细胞生长因子 慢性髓细胞性白血病 K562细胞 凋亡 

分 类 号：R965[医药卫生—药理学]