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目的:使用全反式维甲酸(ATRA)诱导SH-SY5YNB(SY5Y)细胞的TrkB表达,加入BDNF激活TrkB/BDNF信号传导通路使TrkB磷酸化,然后用特异性酪氨酸激酶抑制剂K252a阻断该通路,观察NB细胞对化疗药物—顺铂(CDDP)敏感性的变化。方法:用ATRA诱导SY5Y细胞的TrkB高表达后,用BDNF、CDDP和TrkB信号传导通路的特异性阻断剂—K252a处理,MTT法检测细胞的存活率变化;流式细胞技术(FCM)检测细胞凋亡率;应用免疫组化法检测细胞磷酸化TrkB(p-TrkB)的表达水平。结果:ATRA能特异性诱导TrkB的表达,加入BDNF后可激活TrkB/BDNF信号传导通路并能引起TrkB的自动磷酸化;K252a能够阻断TrkB的磷酸化和TrkB/BDNF信号传导通路;经CDDP处理的SY5Y细胞的存活率下降、凋亡率上升。结论:阻断TrkB/BDNF信号传导通路可提高共表达TrkB/BDNF的NB细胞的化疗敏感性。因此,应用TrkB的抑制剂治疗NB有望提高该病的临床治愈率。
OBJECTIVE: To induce TrkB expression in SH-SY5YNB (SY5Y) cells by using all-trans retinoic acid (ATRA) and to phosphorylate TrkB by activating TrkB / BDNF signaling pathway by adding BDNF, and then blocked by specific tyrosine kinase inhibitor K252a This pathway was used to observe the changes of sensitivity of NB cells to chemotherapeutic drug cisplatin (CDDP). Methods: The high expression of TrkB in SY5Y cells was induced by ATRA. The cells were treated with K252a, a specific inhibitor of BDNF, CDDP and TrkB signaling pathways. The viability of cells was detected by MTT assay. Flow cytometry (FCM) The apoptotic rate was detected by immunohistochemistry. The expression of phosphorylated TrkB (p-TrkB) was detected by immunohistochemistry. Results: ATRA could specifically induce the expression of TrkB. BDNF could activate TrkB / BDNF signal transduction pathway and induce autophosphorylation of TrkB. K252a could block TrkB phosphorylation and TrkB / BDNF signal transduction pathway. After treatment with CDDP The survival rate of SY5Y cells decreased, and the apoptosis rate increased. Conclusion: Blocking TrkB / BDNF signal transduction pathway can enhance chemosensitivity of NB cells co-expressing TrkB / BDNF. Therefore, the use of TrkB inhibitors in the treatment of NB is expected to improve the clinical cure rate of the disease.