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

郑筱娇, 高洲, 沈蓉蓉, 赵行, 滑世轩, 吕建新, 岑东

中国药学杂志 ›› 2017, Vol. 52 ›› Issue (3) : 201-205.

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中国药学杂志 ›› 2017, Vol. 52 ›› Issue (3) : 201-205. DOI: 10.11669/cpj.2017.03.008
论著

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

  • 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*
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Anti-apoptosis of Hepatocyte Growth Factor on CML K562 Cells

  • ZHENG Xiao-jiao1, GAO Zhou2, SHEN Rong-rong2, ZHAO Hang2, HUA Shi-xuan2, Lü Jian-xin2, CEN Dong2,3*
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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。

Abstract

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, Bax, 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 inhibit 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 function. 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 Bax 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 inhibiting apoptosis gene expression and promoting the antiapoptotic gene expression simultaneously. CONCLUSION HGF can significantly 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

Key words

hepatocyte growth factor / chronic myelocytic leukemia / K562 cell / apoptosis

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郑筱娇, 高洲, 沈蓉蓉, 赵行, 滑世轩, 吕建新, 岑东. 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[J]. 中国药学杂志, 2017, 52(3): 201-205 https://doi.org/10.11669/cpj.2017.03.008
ZHENG Xiao-jiao, GAO Zhou, SHEN Rong-rong, ZHAO Hang, HUA Shi-xuan, Lü Jian-xin, CEN Dong. Anti-apoptosis of Hepatocyte Growth Factor on CML K562 Cells[J]. Chinese Pharmaceutical Journal, 2017, 52(3): 201-205 https://doi.org/10.11669/cpj.2017.03.008
中图分类号: R965   

参考文献


[1] NAKAMURA K, NISHIZAWA T, HIGIYA M, et al. Molecular cloning and expression of human hepatocyte growth factor[J]. Nature, 1989, 342(6248):440-443.
[2] JIANG W G, DAVIES G, MARTIN T A, et al. The potential lymphangiogenic effects of hepatocyte growth factor/scatter factor in vitro and in vivo [J]. Int J Mol Med, 2005, 16(4):723-728.
[3] VANRIET I. Adhesion of myeloma cells to the bone marrow stroma matrix protein fibronectin is stimulated by hepatocyte growth factor[J]. Haematologica, 2005, 90(4):436-472.
[4] TJIN E P, GROEN R W, VOGELZANG I, et al. Functional analysis of HGF/MET-signaling and aberrant HGF activator expression in diffuse large B cell lymphoma[J]. Blood, 2005, 107(2):760-768.
[5] SUN B C, ZHANG S W, NI C S, et al. The clinical significance study of vasculogenetic mimicry in 337 cases of bi-directional differential maiignant tumors[J]. Chin J Clin Oncol(中国肿瘤临床), 2005, 32(2):64-67.
[6] ZHENG X Q, GAO Z, SHEN R R, et al. Expression, purification and preliminary activity study of recombinant hepatocyte growth factor protein in E. coli [J]. Chin J Microbiol Immunol(中华微生物学与免疫学杂志), 2012, 32(11):967-971.
[7] HEIDEMAN D A, OVERMEER R M, VAN BEUSECHEM V W, et al. Inhibition of angiogenesis and HGF-c-MET-elicited malignant processes in human hepatocellular carcinoma cells using adenoviral vector-mediated NK4 gene therapy[J]. Cancer Gene Ther, 2005, 12(12):954-962.
[8] TJIN E P, GROEN R, VOGELZANG I, et al. Functional analysis of HGF/MET-signaling and aberrant HGF activator expression in diffuse large B cell lymphoma[J]. Blood, 2006, 107(2):760-768.
[9] YE Y X, ZHOU J, ZHOU Y H, et al. Clinical significance of BCR-ABL fusion gene subtypes in chronic myelogenous and acute lymphoblastic leukemias[J]. Asian Pac J Cancer Prev, 2014,15(22):9961-9966.
[10] LI B, SUN B, ZHU J, et al. Expression of RKIP in chronic myelogenous leukemia K562 cell and inhibits cell proliferation by regulating the ERK/MAPK pathway[J]. Tumour Biol, 2014, 35(10):10057-10066.
[11] MORGAN C W, JULIEN O, UNGER E K, et al. Turning on caspases with genetics and small molecules[J]. Methods Enzymol, 2014, 54(4):179-213.
[12] SUEN D F, NORRIS K L, YOULE R J. Mitochondrial dynamics and apoptosis[J]. Genes Dev, 2008, 22(12):1577-1590.
[13] SHEN R R, ZHENG X J,ZHAO H, et al. Protection effect of hepatocyte growth factor on tumor cells from apoptosis and its mechanism [J]. Chin Pharm J(中国药学杂志), 2012, 47(18):1478-1482.

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)
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