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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 observe the killing effect of human β-defensin 4 (HBD4) on neuroblastoma (NB) cells and to explore the mechanism of HBD4 killing tumor cells. Methods The HBD4 polypeptide was expressed and purified by genetic engineering method. The NB cells were derived from the tumor samples of female patients with clinically and pathologically diagnosed NB, and were passaged by cell culture. NB cells were treated with HBD4, and NB cells were divided into groups A, B, C and D according to the final concentration of HBD4, the concentrations were 0μg.L-1, 20μg.L-1, 40μg.L-1, 100μg .L-1; According to the role of HBD4 time is divided into Ⅰ, Ⅱ, Ⅲ 3 group, the time was 6 h, 12 h, 24 h. The morphology of NB cells was observed under inverted phase contrast microscope. The killing effect of HBD4 on NB cells was determined by MTT assay. The changes of cell surface were observed by scanning electron microscopy. Results After 6 h HBD4 treatment, the cell size, morphology and adherent ability of HBD4 were significantly different from those of group A. The cell morphology of group B was not changed much and adherent was acceptable. The cells of group C were obviously contracted, the processes were rare, the color of intracellular granules was deepened, Refractive enhancement, a large number of cells off. In group D, after 12 h, most of the cells died. The MTT results showed that the number of cells in group D was significantly decreased (P <0.05) with the prolongation of HBD4 treatment time in group D. The number of cells in group Ⅰ, Ⅱ and Ⅲ was significantly decreased with the increase of HBD4 concentration (P <0.01) The cytotoxicity of cells is time-and dose-dependent. Scanning electron microscopy results showed that: NB cells treated with HBD4 for 12 h, cell surface microvilli disappeared, irregular pore cell skeleton serious destruction, and A group of intact cell membrane structure. Conclusion HBD4 has a killing effect on NB cells. HBD4 can kill tumor cells by disrupting the structure of the cell membrane.