TrkB-BDNF信号传导通路对神经母细胞瘤细胞耐药和转移的影响
TrkB-BDNF신호전도통로대신경모세포류세포내약화전이적영향
Effects of TrKB-BDNF signaling pathway on chemoresistance and metastasis of neuroblastoma cells
  • 万方数据
    中华小儿外科杂志 중화소인외과잡지
    CHINESE JOURNAL OF PEDIATRIC SURGERY
    2013年 8期 607-612 ,共6页

    刘建英%李坤霞%李爱敏%高惠敏

    류건영%리곤하%리애민%고혜민

    神经母细胞瘤%信号传导%基质金属蛋白酶-9 神經母細胞瘤%信號傳導%基質金屬蛋白酶-9
    신경모세포류%신호전도%기질금속단백매-9
    Neuroblastoma%Signal transduction%Matrix metalloproteinase-9
    目的 TrKB-BDNF信号传导通路与神经母细胞瘤(neuroblastoma,NB)细胞的化疗耐药和转移密切相关.本研究探讨阻断TrkB-BDNF信号传导通路的三条下游信号传导通路后,NB细胞SH-SY5Y对化疗药物顺铂的敏感性及分泌基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)的影响.方法 常规培养SH-SY5Y NB细胞,用纳摩尔浓度全反式维甲酸(all-trans Retinoic Acid,ATRA)诱导酪氨酸激酶受体B(tyrosine kinase receptor B,TrkB)高表达,加入脑源性神经营养因子(brain-derived neurotrophic factor,BDNF),从而激活TrkB-BDNF信号传导通路.用磷脂酰肌醇-3激酶(PI3K)抑制剂LY294002、磷脂酶C(PLC)抑制剂U73122、丝裂原活化蛋白激酶(MAPK)抑制剂U0126阻断该信号通路的相应下游信号传导通路后,加入化疗药顺铂(CDDP).四甲基偶氮蓝比色(MTT)实验方法检测应用三种抑制剂前后细胞的存活率的变化；流式细胞仪(FCM)检测应用三种抑制剂前后细胞凋亡率的变化.酶联免疫吸附测定(ELISA)技术检测SH-SY5Y细胞培养上清中MMP-9含量.结果 ATRA+ BDNF+ CDDP组NB细胞的存活率及凋亡率与对照组相比,差异无统计学意义(P＞0.05).ATRA+ BDNF+LY294002+CDDP组细胞对顺铂的敏感性增加,细胞的存活率明显降低,凋亡率明显升高,与ATRA+ BDNF+ CDDP组相比,差异有统计学意义(P＜0.05)；而ATRA+ BDNF+ U73122+ CDDP组、ATRA+BDNF+ U0126+ CDDP组细胞对顺铂的敏感性降低,细胞的存活率明显升高,凋亡率明显降低,与ATRA+ BDNF+ CDDP组相比,差异无统计学意义(P＞0.05).ELISA结果显示,ATRA+ BDNF组MMP-9含量明显高于对照组及ATRA组(P＜0.05)；ATRA+BDNF+ LY294002组MMP-9含量明显低于ATRA+ BDNF组(P＜0.05),与对照组及ATRA组比较差异无统计学意义(P＞0.05)；ATRA+ BDNF+ U73122组MMP-9含量明显高于对照组及ATRA组(P＜0.05),与ATRA+ BDNF组比较差异无统计学意义(P＞0.05).ATRA+ BDNF+ U0126组MMP-9含量明显高于对照组及ATRA组(P＜0.05),与ATRA+ BDNF组比较差异无统计学意义(P＞0.05).结论 激活TrkB-BDNF信号传导通路可增强NB细胞耐药及促进其合成、分泌MMP9.TrkB-BDNF信号传导通路可能通过进一步激活其下游PI3K/Akt通路增强耐药及促进NB细胞合成、分泌MMP-9,用LY294002阻断PI3K/Akt通路后可逆转耐药及抑制NB细胞合成、分泌MMP-9,从而抑制NB的侵袭、转移.
    목적 TrKB-BDNF신호전도통로여신경모세포류(neuroblastoma,NB)세포적화료내약화전이밀절상관.본연구탐토조단TrkB-BDNF신호전도통로적삼조하유신호전도통로후,NB세포SH-SY5Y대화료약물순박적민감성급분비기질금속단백매-9(matrix metalloproteinase-9,MMP-9)적영향.방법 상규배양SH-SY5Y NB세포,용납마이농도전반식유갑산(all-trans Retinoic Acid,ATRA)유도락안산격매수체B(tyrosine kinase receptor B,TrkB)고표체,가입뇌원성신경영양인자(brain-derived neurotrophic factor,BDNF),종이격활TrkB-BDNF신호전도통로.용린지선기순-3격매(PI3K)억제제LY294002、린지매C(PLC)억제제U73122、사렬원활화단백격매(MAPK)억제제U0126조단해신호통로적상응하유신호전도통로후,가입화료약순박(CDDP).사갑기우담람비색(MTT)실험방법검측응용삼충억제제전후세포적존활솔적변화；류식세포의(FCM)검측응용삼충억제제전후세포조망솔적변화.매련면역흡부측정(ELISA)기술검측SH-SY5Y세포배양상청중MMP-9함량.결과 ATRA+ BDNF+ CDDP조NB세포적존활솔급조망솔여대조조상비,차이무통계학의의(P＞0.05).ATRA+ BDNF+LY294002+CDDP조세포대순박적민감성증가,세포적존활솔명현강저,조망솔명현승고,여ATRA+ BDNF+ CDDP조상비,차이유통계학의의(P＜0.05)；이ATRA+ BDNF+ U73122+ CDDP조、ATRA+BDNF+ U0126+ CDDP조세포대순박적민감성강저,세포적존활솔명현승고,조망솔명현강저,여ATRA+ BDNF+ CDDP조상비,차이무통계학의의(P＞0.05).ELISA결과현시,ATRA+ BDNF조MMP-9함량명현고우대조조급ATRA조(P＜0.05)；ATRA+BDNF+ LY294002조MMP-9함량명현저우ATRA+ BDNF조(P＜0.05),여대조조급ATRA조비교차이무통계학의의(P＞0.05)；ATRA+ BDNF+ U73122조MMP-9함량명현고우대조조급ATRA조(P＜0.05),여ATRA+ BDNF조비교차이무통계학의의(P＞0.05).ATRA+ BDNF+ U0126조MMP-9함량명현고우대조조급ATRA조(P＜0.05),여ATRA+ BDNF조비교차이무통계학의의(P＞0.05).결론 격활TrkB-BDNF신호전도통로가증강NB세포내약급촉진기합성、분비MMP9.TrkB-BDNF신호전도통로가능통과진일보격활기하유PI3K/Akt통로증강내약급촉진NB세포합성、분비MMP-9,용LY294002조단PI3K/Akt통로후가역전내약급억제NB세포합성、분비MMP-9,종이억제NB적침습、전이.
    Objective To present the changes in the sensitivity of SH-SY5Y cells to cisplatin (CDDP) and the synthesis and secretion of matrix metalloproteinases-9(MMP-9) before and after blockage of TrkB-BDNF downstream signaling pathways.Methods Human neuroblastoma (NB) cell line SH-SY5Y (SY5Y) was cultured.High expression of tyrosine kinase receptor B (TrkB) was induced by nM all-trans retinoid acid (ATRA).Ectogenid brain-derived neurotrophic factor (BDNF)was then added to activate the TrkB-BDNF signaling pathway.The three downstream signaling pathways were respectively inhibited by LY294002 (PI3K pathway inhibitor),U73122(PLC pathway inhibitor) and U0126(MAPK pathway inhibitor).Cell viability was assessed by methyl thiazolyl tetrazolium (MTT) assay.Cell apoptosis rate was detected by flow cytomctry (FCM).MMP-9 concentrations in the SY5Y cell culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA).Results The viability and apoptosis rate of SY5Y cells treated with ATRA,BDNF and CDDP were not different from the control group (P＞0.05).However,the sensitivity of SY5Y cells to CDDP increased in the ATRA + BDNF + LY294002 + CDDP group,the viability rate decreased and the apoptosis rate increased in SY5Y cells when compared to the ATRA + BDNF + CDDP group (P＜0.05).However,sensitivity of SY5Y cells to CDDP decreased in the ATRA + BDNF + U73122 + CDDP group and in the ATRA + BDNF + U0126 + CDDP group,the viability and the apoptosis rate of the two groups had no significant difference when compared to the ATRA + BDNF + CDDP group (P＞0.05).The results of ELISA implied that MMP-9 protein levels in neuroblastoma cells cultured in serum-free media in the ATRA + BDNF group were significantly higher than those of the control group and ATRA alone group (P＜0.05).MMP-9 protein levels in the ATRA + BDNF + LY294002 group were significantly lower than those of the ATRA + BDNF group and showed no significant difference when compared to the control group and the ATRA alone group.MMP-9 protein levels in neuroblastoma cells cultured in serum-free media in the ATRA + BDNF + U73122 group were significantly higher than those of the control group and ATRA alone group and had no significant difference when compared with the ATRA + BDNF group.MMP-9 protein levels in neuroblastoma cells cultured in serumfree media in the ATRA + BDNF + U0126 group were significantly higher than those of the control group and ATRA alone group and had no significant difference when compared to the ATRA + BDNF group.Conclusions Activation of TrkB-BDNF signaling pathway can increase the chemoresistence and the synthesis and secretion of MMP-9 in human neuroblastoma cells.TrkB-BDNF signaling pathway may act through activating its downstream PI3K/Akt pathway to increase the sensitivity of NB cells to CDDP and promote the synthesis and secretion of MMP-9.Blocking the TrKP-BDNF downstream signaling pathway PI3K/Akt with LY294002 can reverse chemoresistance and inhibit the synthesis and secretion of MMP-9,thus limiting neuroblastoma tumor invasion and metastasis.
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