银杏叶提取物对放射性损伤耳蜗毛细胞株HEI-OC1增殖、凋亡及Survivin、Bax表达的影响
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摘要
目的探讨银杏叶提取物(EGB761)对放射性损伤耳蜗毛细胞株HEI-OC1增殖、凋亡及细胞存活素(Survivin)、Bcl-2家族前凋亡蛋白(Bax)表达的影响。方法体外培养耳蜗毛细胞株HEI-OC1,设置正常对照组、放射性损伤组及10 mg/L、20 mg/L、50mg/L、100 mg/L EGB761干预组,除正常对照组外其他组均选择对数生长期HEI-OC1细胞进行照射,建立放射性损伤模型,之后各干预组分别给予相应浓度EGB761进行干预。应用四甲基偶氮唑蓝(MTT)实验检测各组HEI-OC1细胞增殖能力,利用流式细胞仪(FCM)检测各组HEI-OC1细胞凋亡情况,采用实时荧光定量聚合酶链式反应(qRTPCR)检测各组细胞Survivin、Bax表达水平。结果 MTT结果显示,与正常对照组比较,放射性损伤组HEI-OC1细胞增殖能力明显下降(P <0. 05);与放射性损伤组比较,EGB761干预组HEI-OC1细胞增殖能力明显上升(P <0. 05),且呈一定剂量依赖性。FCM结果显示,与正常对照组比较,放射性损伤组HEIOC1细胞凋亡率明显增加(P <0. 05);与放射性损伤组比较,EGB761干预组HEI-OC1细胞凋亡率明显降低(P <0. 05),且呈一定剂量依赖性。qRT-PCR结果显示,与正常对照组比较,放射性损伤组HEI-OC1细胞中Survivin水平明显下降(P <0. 05),Bax水平明显升高(P <0. 05);与放射性损伤组比较,EGB761干预组HEI-OC1细胞中Survivin水平明显升高(P <0. 05),Bax水平明显下降(P <0. 05),且呈一定剂量依赖性。结论 EGB761可明显促进HEI-OC1细胞增殖,抑制HEI-OC1细胞凋亡。其作用机制可能为上调凋亡抑制基因Survivin表达及下调凋亡促进基因Bax表达。
Objective It is to investigate the effects of Ginkgo biloba extract(EGB761) on proliferation,apoptosis and expression of Survivin,Bcl-2 family pre-apoptotic protein(Bax) in radiation-injured cochlear hair cell line HEI-OC1.Methods Cochlear hair cell line HEI-OC1 was cultured in vitro,and to set up normal control group,radiation injury group,10 mg/L,20 mg/L,50 mg/L,100 mg/L EGB761 intervention group; Except for the normal control group,the logarithmic growth phase HEI-OC1 cells were selected to be irradiated and established a radiation damage model in the other groups.Then each intervention group was given corresponding concentration of EGB761 for intervention. The proliferation ability of HEI-OC1 cells was detected by four methazolam blue(MTT) assay,the apoptosis of HEI-OC1 cells was detected by flow cytometry(FCM),and the expression levels of Survivin and Bax was detected by Real time fluorescence quantitative polymerase chain reaction(qRT-PCR). Results The results of MTT experiment showed that the proliferation of HEI-OC1 cells in the radiation injury group was significantly decreased than the normal control group(P < 0. 05),the proliferation ability of HEI-OC1 cells in all EGB761 intervention groups were increased significantly than the radiation injury group(all P <0. 05),and showed a dose-dependent manner. FCM experimental results show that the apoptosis rate of HEI-OC1 cells in the radiation injury group was increased significantly than the normal control group(P < 0. 05),the apoptosis rate of HEIOC1 cells in all EGB761 intervention groups were significantly lower Compared with radiation injury group(all P < 0. 05),and showed a dose-dependent manner. QRT-PCR results showed that the level of Survivin in HEI-OC1 cells in radiation injury group was significantly lower and the level of Bax was significantly higher than the normal control group(all P < 0. 05),the levels of Survivin in HEI-OC1 cells in EGB761 intervention group was significantly higher and Bax was significantly lower than the radiation injury group(all P < 0. 05),and showed a dose-dependent manner. Conclusion EGB761 can obviously promote the proliferation of HEI-OC1 cells,inhibit HEI-OC1 cell apoptosis. the mechanism may be to up-regulate the expression of apoptosis inhibiting genes Survivin and down-regulate the expression of apoptotic genes Bax.
Objective It is to investigate the effects of Ginkgo biloba extract(EGB761) on proliferation,apoptosis and expression of Survivin,Bcl-2 family pre-apoptotic protein(Bax) in radiation-injured cochlear hair cell line HEI-OC1.Methods Cochlear hair cell line HEI-OC1 was cultured in vitro,and to set up normal control group,radiation injury group,10 mg/L,20 mg/L,50 mg/L,100 mg/L EGB761 intervention group; Except for the normal control group,the logarithmic growth phase HEI-OC1 cells were selected to be irradiated and established a radiation damage model in the other groups.Then each intervention group was given corresponding concentration of EGB761 for intervention. The proliferation ability of HEI-OC1 cells was detected by four methazolam blue(MTT) assay,the apoptosis of HEI-OC1 cells was detected by flow cytometry(FCM),and the expression levels of Survivin and Bax was detected by Real time fluorescence quantitative polymerase chain reaction(qRT-PCR). Results The results of MTT experiment showed that the proliferation of HEI-OC1 cells in the radiation injury group was significantly decreased than the normal control group(P < 0. 05),the proliferation ability of HEI-OC1 cells in all EGB761 intervention groups were increased significantly than the radiation injury group(all P <0. 05),and showed a dose-dependent manner. FCM experimental results show that the apoptosis rate of HEI-OC1 cells in the radiation injury group was increased significantly than the normal control group(P < 0. 05),the apoptosis rate of HEIOC1 cells in all EGB761 intervention groups were significantly lower Compared with radiation injury group(all P < 0. 05),and showed a dose-dependent manner. QRT-PCR results showed that the level of Survivin in HEI-OC1 cells in radiation injury group was significantly lower and the level of Bax was significantly higher than the normal control group(all P < 0. 05),the levels of Survivin in HEI-OC1 cells in EGB761 intervention group was significantly higher and Bax was significantly lower than the radiation injury group(all P < 0. 05),and showed a dose-dependent manner. Conclusion EGB761 can obviously promote the proliferation of HEI-OC1 cells,inhibit HEI-OC1 cell apoptosis. the mechanism may be to up-regulate the expression of apoptosis inhibiting genes Survivin and down-regulate the expression of apoptotic genes Bax.
引文
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[11]韩倩,张淑香,原红艳,等.银杏叶提取物对突发性聋患者外周血清同型半胱氨酸和血脂浓度的影响[J].中国耳鼻咽喉头颈外科,2015,22(4):191-193
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[13]杨红,郎晓芳,王勇.银杏叶提取物联合高压)氧对局灶性脑缺血再灌注损伤大鼠外周血清氧化相关物质浓度的影响[J].解放军医药杂志,2015,27(10):34-38
[14]施雨露,刘照轩,陈强,等.银杏叶提取物辐射防护作用的研究[J].中国实验诊断学,2013,17(11):1973-1974
[15]姜梁,胡晓艳,黎万荣,等.银杏叶提取物对早期中耳放射性损伤防护作用的实验研究[J].中国中西医结合耳鼻咽喉科杂志,2013,21(3):161-164
[16]宋媛媛,吕欣,高春时,等.银杏叶提取物对电离辐射下大鼠骨髓间充质干细胞的保护作用机制研究[J].中国实验诊断学,2016,20(8):1235-1239
[17]张灏龙,张成瑶,朱方玉,等. Survivin基因过表达对血管内皮细胞增殖及抗凋亡能力的影响[J].重庆医科大学学报,2015,40(4):588-593
[18]李鸿飞,唐卫中,黄庆录,等.奥沙利铂对二甲肼诱导的结肠癌大鼠CD44V6、VEGF、survivin、Caspase-3和Caspase-7的影响[J].肿瘤防治研究,2015,42(6):560-563
[19]Gro Be L,Wurm CA,Brüser C,et al. Bax assembles into large ringlike structures remodeling the mitochondrial outer membrane inapoptosis[J]. Embo J,2016,35(4):402-404
[20]Karch J,Molkentin JD. Regulated necrotic cell death:The passive aggressive side of Bax and Bak[J]. Circul Res,2015,116(11):1800-1801
[21]沈晓咏,吕帆真,武韫,等. bcl-2,bax和p53在小细胞肺癌中的表达及对细胞凋亡的影响[J].四川医学,2013,34(7):915-917
[22]袁风菊,卢璐,刘根林,等.银杏叶提取物对1型糖尿病心肌病大鼠心肌Bcl-2、Bax和caspase-3表达的影响[J].医学研究杂志,2014,43(7):43-48
[2] Cho KS,Lee IM,Sim S,et al. Ginkgo biloba Extract(EGb 761@)Inhibits Glutamate-induced Up-regulation of Tissue Plasminogen Activator Through Inhibition of c-Fos Translocation in Rat Primary Cortical Neurons[J]. Phytoth Res,2016,30(1):58-65
[3]汪素娟,康安,狄留庆,等.银杏叶提取物主要活性成分药动学研究进展[J].中草药,2013,44(5):626-631
[4]陈昊鹰,张龙城.银杏叶提取物片对鼻咽癌放疗患者听力受损的影响[J].中国当代医药,2017,24(18):90-92
[5] Singh N,Krishnakumar S,Kanwar RK,et al. Clinical aspects for survivin:a crucial molecule for targeting drug-resistant cancers[J]. Drug Discov Today,2015,20(5):578-587
[6]李丹,骆志国.人工耳蜗植入配合颞骨次全切除治疗鼻咽癌放疗后颞骨骨坏死效果分析[J].肿瘤研究与临床,2017,29(3):151-154
[7]唐隽,施思斯,于青青,等.鼻咽癌放疗后继发慢性鼻窦炎的临床特征[J].山东大学耳鼻喉眼学报,2015,29(2):52-54
[8]张翔.咽鼓管置管在鼻咽癌放疗后并发分泌性中耳炎治疗中的临床应用[J].陕西医学杂志,2014,43(3):306-307
[9]李杰,林洪生.《中药新药治疗恶性肿瘤临床研究技术指导原则》修订过程及解析[J].中国新药杂志,2016,25(16):1833-1837
[10]Rapin JR,Zaibi M,Drieu K. In vitro and in vivo effects of an extract of Ginkgo biloba(EGb 761),ginkgolide B,and bilobalide on apoptosis in primary cultures of rat hippocampal neurons[J].Drug Developm Res,2015,45(1):23-29
[11]韩倩,张淑香,原红艳,等.银杏叶提取物对突发性聋患者外周血清同型半胱氨酸和血脂浓度的影响[J].中国耳鼻咽喉头颈外科,2015,22(4):191-193
[12]Zhang C,Ren C,Chen H,et al. The Analog of Ginkgo biloba,Extract 761 is a Protective Factor of Cognitive Impairment Induced by Chronic Fluorosis[J]. Bio Tra Elem Res,2013,153(1/3):229-236
[13]杨红,郎晓芳,王勇.银杏叶提取物联合高压)氧对局灶性脑缺血再灌注损伤大鼠外周血清氧化相关物质浓度的影响[J].解放军医药杂志,2015,27(10):34-38
[14]施雨露,刘照轩,陈强,等.银杏叶提取物辐射防护作用的研究[J].中国实验诊断学,2013,17(11):1973-1974
[15]姜梁,胡晓艳,黎万荣,等.银杏叶提取物对早期中耳放射性损伤防护作用的实验研究[J].中国中西医结合耳鼻咽喉科杂志,2013,21(3):161-164
[16]宋媛媛,吕欣,高春时,等.银杏叶提取物对电离辐射下大鼠骨髓间充质干细胞的保护作用机制研究[J].中国实验诊断学,2016,20(8):1235-1239
[17]张灏龙,张成瑶,朱方玉,等. Survivin基因过表达对血管内皮细胞增殖及抗凋亡能力的影响[J].重庆医科大学学报,2015,40(4):588-593
[18]李鸿飞,唐卫中,黄庆录,等.奥沙利铂对二甲肼诱导的结肠癌大鼠CD44V6、VEGF、survivin、Caspase-3和Caspase-7的影响[J].肿瘤防治研究,2015,42(6):560-563
[19]Gro Be L,Wurm CA,Brüser C,et al. Bax assembles into large ringlike structures remodeling the mitochondrial outer membrane inapoptosis[J]. Embo J,2016,35(4):402-404
[20]Karch J,Molkentin JD. Regulated necrotic cell death:The passive aggressive side of Bax and Bak[J]. Circul Res,2015,116(11):1800-1801
[21]沈晓咏,吕帆真,武韫,等. bcl-2,bax和p53在小细胞肺癌中的表达及对细胞凋亡的影响[J].四川医学,2013,34(7):915-917
[22]袁风菊,卢璐,刘根林,等.银杏叶提取物对1型糖尿病心肌病大鼠心肌Bcl-2、Bax和caspase-3表达的影响[J].医学研究杂志,2014,43(7):43-48