摘要
目的探讨酪氨酸激酶受体B（tyrosine kinase receptor B，TrkB）对鼻咽癌细胞系C666-1失巢凋亡特性的影响和调控。方法用反转录聚合酶链反应（PT-PCR）以及蛋白质印迹法检测培养的C666-1细胞中TrkB以及其配体脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）的表达。采用TrkB的抑制剂K252a以及其配体BDNF分别作用于C666-1细胞，采用软琼脂集落形成实验观察细胞的集落形成能力以及悬浮培养后用活细胞计数kit-8（cell counting kit-8，CCK-8）法和膜联蛋白V碘化丙啶（annexin V-propidium iodinate，Annexin V-PI）双染色法检测对细胞的增殖和凋亡的影响。通过蛋白质印迹法观察TrkB、BDNF、磷酸化的丝氨酸苏氨酸激酶（serine threonine kinase，Akt）等蛋白在实验前后的表达变化的情况。结果培养的C666-1细胞表达TrkB及BDNF。C666-1细胞受K252a作用后在软琼脂中集落细胞数减少，而在BDNF作用下集落细胞数增加。在悬浮培养状态，K252a对C666-1细胞的增殖具有抑制作用，经K252a作用后C666-1细胞的TrkB表达下调，并同时下调磷酸化的Akt蛋白（p-Akt）；而BDNF作用后，内源性BDNF表达增强，且TrkB和p-Akt的表达上调。结论TrkB具有抑制鼻咽癌细胞C666-1失巢凋亡的作用，K252a能够抑制TrkB的表达从而诱导凋亡，是具有潜力的酪氨酸激酶抑制剂。
Objective To study the regulation of anokis by tyrosine kinase receptor B（TrkB） in human nasopharyngeal carcinoma lines. Methods Expression levels of TrkB and brain-derived neurotrophic factor （BDNF） were evaluated by RT-PCR and Western blot. Colony formation ability of C666-1 was observed in soft agar. Proliferation rate and apoptosis, that change in cells by treating the TrkB inhibitor K252a and specificity ligand BDNF respectively under suspension culture, were measured by cell counting kit-8 （CCK-8） assay and the flow cytometry assay. The expression change of TrkB, BDNF and phosphorylation of serine threonine kinase （p-Akt） were investigated by Western blot analysis. Results TrkB and BDNF were identified in C666-1 cells. C666-1 cells could be decreased the proliferation of colony in soft agar by effect of K252a, but BDNF could make the colony prolific. K252a can inhibit the expression of TrkB in C666-1, and prevent p-Akt activation. And exogenous BDNF stimulated up-regulation TrkB and p-Akt, induced anokis resistance. Conclusion TrkB inhibits anoikis in nasopharyngeal carcinoma cells. Inhibiton of TrkB by K252a can induce anoikis, and may prove particularly effective in treatment of nasopharyngeal carcinoma.
出处
《中华耳鼻咽喉头颈外科杂志》
CAS
CSCD
北大核心
2008年第8期606-611,共6页
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
基金
广西自然科学基金（桂科自0728120）
