人β防御素4对神经母细胞瘤细胞增殖凋亡及Caspase-3蛋白表达的影响*

曹玉红1张静怡2张光运3曹艳华4

(1. 第四军医大学西京医院儿科，陕西 西安 710032; 2. 西安交通大学医学院第一医院，陕西 西安 710061；

3.第四军医大学西京医院神经内科，陕西 西安 710032;4.济南军区总医院儿科，山东 济南250031)

【摘要】目的观察人β防御素4(HBD4)对神经母细胞瘤(NB)细胞的杀伤作用,探讨HBD4对NB细胞增殖、凋亡及Caspase-3蛋白表达的影响。方法将HBD4作用于NB细胞，根据HBD4终浓度将NB细胞分为A、B、C 3组，浓度分别为5、10、20 mg·L-1；HBD4作用时间分别为12、24、48h。噻唑蓝(MTT)法测定HBD4对NB细胞增殖的抑制作用，流式细胞仪检测HBD4作用于NB细胞后细胞凋亡情况；分光光度法检测HBD4对NB细胞Caspase-3蛋白表达的影响。结果MTT检测结果:NB细胞抑制率均随HBD4浓度的升高而升高(P<0.01),随HBD4作用时间的延长而升高(P<0.01)，提示HBD4对NB细胞的抑制作用具有时间和剂量依赖性；流式细胞仪检测结果显示：A、B、C三组NB细胞凋亡率均高于对照组(P<0.01)。三组NB细胞凋亡率比较亦有显著差异(P<0.01)，说明随药物浓度的增加，细胞凋亡率呈增高趋势； 分光光度法检测结果显示：A、B、C三组NB细胞Caspase-3含量均高于对照组(P<0.01)。三组NB细胞Caspase-3含量比较亦有显著差异(P<0.01)，说明随药物浓度的增加，细胞Caspase-3含量呈增高趋势。结论HBD4对NB细胞增殖具有抑制作用；HBD4可诱导NB细胞凋亡;HBD4可能是通过激活Caspase-3诱导NB细胞凋亡。

【关键词】人β防御素4； 神经母细胞瘤； Caspase-3； 细胞凋亡

【中图分类号】R 329.2+8【文献标志码】A

基金项目:陕西省自然科学基金(2012JM4054); 陕西省科技攻关计划(2009K12-01)

通讯作者：张光运,E-mail: zhgyun@fmmu.edu.cn

收稿日期：( 2014-07-01； 编辑： 张文秀)

Influence of human β-defensin 4 on the proliferation, apoptosis and Caspase-3 protein expression of neuroblastoma cellsCAO Yuhong1, ZHANG Jinyi2, ZHANG Guangyun3,etal

(1.DepartmentofPediatrics,XijingHospital,TheFourthMilitaryMedicalUniversity,Xi'an710032,China;

2.TheFirstAffiliatedHospitalMedicineCollegeofXi'anJiaoTongUniversity,Xi'an710061,China;

3.DepartmentofNeurology,XijingHospital,TheFourthMilitaryMedicalUniversity,Xi'an710032,China;

4.DepartmentofPediatrics，GeneralHospitalofJinanMilitaryDistrict，Jinan250031，China)

Abstract【】ObjectiveTo examine cytotoxicity of human β defensin 4(HBD4)on neuroblastoma (NB) cell and explore the influence of human β-defensin 4 on proliferation, apoptosis and Caspase-3 protein expression of NB cells. MethodsIn this research, NB cells were treated with HBD4. According to final concentration of HBD4, NB cells were divided into 3 groups: A, B and C with the corresponding concentrations of 5, 10, and 20 mg.L-1, respectively. The reaction times were 12, 24 and 48h respectively. Methyhhiazolyldiphenyl- tetrazoliumbromide(MTT)assay was adopted to detect the inhibitory effect of HBD4 on NB cell proliferation. In addition, NB Cell apoptosis was assessed by flow cytometry and Caspase-3 activity was detected by spectrophotometry. ResultsMTT assay showed the inhibitory rate of NB cells treated with HBD4 increased with the increase of HBD4 concentration and the prolongation of acting time of HBD4(P<0.01), which indicated that HBD4 inhibited the proliferation of NB cells in a time and dose dependent manner. Flow cytometry assay showed that NB cell Apoptosis rates of group A, B and C were all higher than that of the control group(P<0.01), and there were statistically significant differences among the three groups(P<0.01), which suggested that the apoptosis rate increased with the increase of HBD4 concentration. In the third place, Spectrophotometry assay showed that Caspase-3 activity of group A, B and C were all higher than that of the control group(P<0.01), and the differences among the three groups were statistically significant(P<0.01), which suggested that the Caspase-3 activity increased with the increase of HBD4 concentration. ConclusionHBD4 inhibits the proliferation of NB cells by means of inducing cellular apoptosis via increasing Caspase-3 activity.

【Key words】Human β defensin 4; Neuroblastoma; Caspase-3; Apoptosis

神经母细胞瘤(NB)是儿童一种常见的恶性实体肿瘤，大部分患儿在就诊时已广泛转移，病死率极高。传统的放疗或化疗毒副作用大，对肿瘤细胞和正常细胞均有不同程度的杀伤作用，不可避免地造成多脏器损害。另外肿瘤细胞对多种抗癌药物产生抗药性，是肿瘤化疗失败的主要原因之一[1-4]。因此研究开发新一代毒副作用小、又能克服耐药性的抗肿瘤药物成为目前肿瘤防治研究的焦点。近几年研究发现，一些具有广谱杀菌活性的防御素，可以特异性杀伤某些肿瘤细胞，抑制肿瘤生长和增殖，但对正常人体细胞不造成破坏[5-12]。……