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杀伤肿瘤细胞的机制。方法HBD4多肽通过基因工程方法表达并纯化获得,NB细胞来源于临床病理诊断为NB的女性患儿肿瘤标本,经细胞培养传代获得。将HBD4作用于NB细胞,根据HBD4终质量浓度将NB细胞分为A、B、C、D 4组,质量浓度分别为0μg.L-1、20μg.L-1、40μg.L-1、100μg.L-1;根据HBD4作用时间又分为Ⅰ、Ⅱ、Ⅲ3组,时间分别为6 h、12 h、24 h。倒置相差显微镜下观察NB细胞形态,噻唑蓝(MTT)法测定HBD4对NB细胞的杀伤作用,扫描电镜观察细胞表面变化。结果 HBD4处理6 h后细胞大小、形态和贴壁能力与A组有明显差异:B组细胞形态变化不大,贴壁尚可;C组细胞明显皱缩,突起少见,细胞内颗粒颜色加深,折光性增强,大量细胞脱落。D组作用12 h后,大部分细胞死亡。MTT结果显示:D组随HBD4作用时间的延长,细胞数显著减少(P<0.05);Ⅰ、Ⅱ、Ⅲ组:随HBD4浓度增加,细胞数均显著减少(P<0.01),提示HBD4对NB细胞的杀伤作用具有时间和剂量依赖性。扫描电镜结果提示:NB细胞经HBD4作用12 h,细胞表面微绒毛消失,出现不规则孔洞细胞膜骨架严重破坏,而A组细胞膜结构完整无损。结论 HBD4对NB细胞具有杀伤作用;HBD4可通过破坏细胞膜的结构杀伤肿瘤细胞。
Objective To examine the cytotoxieity of human βdefensin 4 （HBD4） on neuroblastoma （NB） cell and explore the underl- ying mechanism of eytotoxicity against tumor cells. Methods HBD4 was expressed and purified by genetic engineering. NB cells were derived from tumor specimens of a girl who was pathologically diagnosed with NB. Then NB ceils were treated with HBD4. According to final concen- tration of HBD4, NB cells were divided into 4 groups： A, B, C, and D groups with the corresponding concentrations of 0 μg·L^-1, 20 μg·L^-1 ,40 μg·L^-1 ,and 100 μg·L^-1 ,respectively. NB cells were also classified as I , Ⅱ and m groups with the corresponding acting time of HBD4 for 6 h, 12 h, and 24 h, respectively. NB cell morphology was carefully observed under the inverted phase contrast microscope. Methyhhiazolyldiphenyl - tetrazoliumbromide（MTF） assay was adopted to detect the eytotoxicity of HBD4 on NB cells. In addition ,cell surface changes were observed by scanning electron microscope. Results In the first place, through observation under inverted phase contrast micro- scope, the size, shape and adhesive capacity of NB cells treated with HBD4 were significantly different from NB cells in group A after NB cells treated with HBD4 for 6 h. NB cells in group B had no change in cell morphology and adhesive capacity. NB ceils in group C was characte- rized by wrinkled cells, fewer protrusions, deepened color saturation of the particles within the cells, increased refraction, a great quantity of ex- foliated cells. The majority of NB cells in group D died after having been treated with HBD4 for 12 h. In the second place, MTT assay showed that NB cells in group D decreased with the prolongation of acting time of HBD4 （ P 〈 0.05 ） and those in group I , Ⅱ and Ⅲ significantly de- creased with the increase of concentration of HBD4（ P 〈 0.01 ） ,which indicated that eytotoxicity of HBD4 on NB cells was in a time and dose dependent manner. In the third place, scanning electron microscopy showed disappearance of cell surface mierovillius, irregular cell membrane perforation, and severely damaged membrane skeleton, whereas membrane structure of the cells in group A was undamaged. Conclusions HBD4 can kill NB cells by means of destructing the cell membrane structure ,which may provide a new therapeutic strategy for children with NB.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2012年第22期1757-1759,共3页
Journal of Applied Clinical Pediatrics
基金
陕西省科技计划项目(2009K12-01)
