Li Ai-min, Zhang Ji-hong, Zhang Jin-hua, Zhang Ke-ren, Rong Dao-jian
Department of Hematology, The Second Affiliated Hospital of China Medical University, Shenyang 110004, China.
Zhonghua Er Ke Za Zhi. 2006 Jul;44(7):535-9.
Neuroblastoma (NB) is a pediatric solid tumor derived from neural crest precursor cells. It is resistant to current therapeutic protocols, including high dose chemotherapy. The mechanisms of chemoresistance are very complex. The recent studies have shown that the levels of tyrosine kinase receptor B (TrkB) and brain-derived neurotrophic factor (BDNF) are high in NB tumors with poor prognosis. The aim of this research was to explore the effects of TrkB and BDNF levels on the chemotherapeutic sensitivity in neuroblastoma by using the NB cell line SH-SY5Y in vitro.
The expression of TrkB protein was detected with Western-blot after the treatment with different concentrations of all trans-retinoic acid (ATRA). Cell survival rate was analyzed using MTT. Apoptosis was detected using flow cytometry (FCM) and a transmission electron microscope (TEM).
(1) The expression of TrkB protein was undetectable in SY5Y. It was positive, however, after the treatment with ATRA (1, 10, 100 nmol/L) for five days. The level of TrkB protein was increased with adding of ATRA at different concentrations. (2) The difference of the survival and apoptotic rate between the BDNF (10 ng/ml) + ATRA (10 nmol/L) + cisplatin (CP, 5 microg/ml) group (survival rate 46.51% +/- 13.44%, apoptosis rate 11.79% +/- 1.53%) and the CP alone group (survival rate 38.51% +/- 9.66%, apoptosis rate 14.95% +/- 2.06%) was not statistically significant (P > 0.05). The survival rate of the BDNF (50 ng/ml and 100 ng/ml) + ATRA (10 nmol/L) + CP (5 microg/ml) group (66.85% +/- 18.39%, 94.30% +/- 10.71%) was greatly higher than CP alone group (P < 0.05, P < 0.01), whereas the apoptotic rate (9.36% +/- 1.03%, 5.20% +/- 1.99%) was significantly lower than that of the CP alone group (P < 0.01, P < 0.01). The survival rates of BDNF (100 ng/ml) + ATRA (10 nmol/L) + CP (5 microg/ml) group were higher than those of BDNF (50 ng/ml) + ATRA (10 nmol/L) + CP (5 microg/ml) group (P < 0.01), whereas the apoptotic rates were lower than those of BDNF (50 ng/ml) + ATRA (10 nmol/L) + CP (5 microg/ml) group (P < 0.05). There were no significant difference between the ATRA (1 nmol/L) + BDNF (50 ng/ml) + CP group (survival rate 45.33% +/- 11.83%, apoptosis rate 12.48% +/- 2.48%) and the CP alone group in the survival and apoptotic rates (P > 0.05). The survival rates of the ATRA (10 nmol/L, 100 nmol/L) + BDNF (50 ng/ml) + CP (61.62% +/- 18.53%, 105.02% +/- 5.55%) group were greatly higher than those of the CP alone group (P < 0.05, P < 0.01), whereas the apoptotic rate (9.36% +/- 1.03%, 5.05% +/- 1.88%) was significantly lower than that of the CDDP alone group (P < 0.05, P < 0.01). The survival rates of the ATRA (100 nmol/L) + BDNF (50 ng/ml) + CP group were higher than those of the ATRA (10 nmol/L) + BDNF (50 ng/ml) + CP group (P < 0.01), whereas the apoptotic rates were lower than the ATRA (10 nmol/L) + BDNF (50 ng/ml) + CP group (P < 0.01). (3) Some of the cells showed apoptotic changes in the CP alone group, whereas the intranuclear chromoplasma was well-distributed, the nuclear membrane was clear, and mitochondria, ribosome and solvent were present in the ATRA (10 nmol/L) + BDNF (50 ng/ml) + CP group.
The sensitivity of SY5Y to CP was affected by TrkB and BDNF. The higher the level of TrkB and BDNF was, the lower the sensitivity of SY5Y to CP.
神经母细胞瘤（NB）是一种源自神经嵴前体细胞的儿科实体瘤。它对包括大剂量化疗在内的当前治疗方案具有抗性。化疗耐药机制非常复杂。最近的研究表明，在预后较差的NB肿瘤中，酪氨酸激酶受体B（TrkB）和脑源性神经营养因子（BDNF）的水平较高。本研究的目的是通过体外使用NB细胞系SH-SY5Y来探讨TrkB和BDNF水平对神经母细胞瘤化疗敏感性的影响。
用不同浓度的全反式维甲酸（ATRA）处理后，通过蛋白质免疫印迹法检测TrkB蛋白的表达。使用MTT分析细胞存活率。使用流式细胞术（FCM）和透射电子显微镜（TEM）检测细胞凋亡。
（1）在SY5Y中未检测到TrkB蛋白的表达。然而，用ATRA（1、10、100 nmol/L）处理五天后呈阳性。随着不同浓度ATRA的添加，TrkB蛋白水平升高。（2）BDNF（10 ng/ml）+ ATRA（10 nmol/L）+顺铂（CP，5 μg/ml）组（存活率46.51%±13.44%，凋亡率11.79%±1.53%）与单独CP组（存活率38.51%±9.66%，凋亡率14.95%±2.06%）之间的存活率和凋亡率差异无统计学意义（P>0.05）。BDNF（50 ng/ml和100 ng/ml）+ ATRA（10 nmol/L）+ CP（5 μg/ml）组（66.85%±18.39%，94.30%±10.71%）的存活率显著高于单独CP组（P<0.05，P<0.01），而凋亡率（9.36%±1.03%，5.20%±1.99%）显著低于单独CP组（P<0.01，P<0.01）。BDNF（100 ng/ml）+ ATRA（10 nmol/L）+ CP（5 μg/ml）组的存活率高于BDNF（50 ng/ml）+ ATRA（10 nmol/L）+ CP（5 μg/ml）组（P<0.01），而凋亡率低于BDNF（50 ng/ml）+ ATRA（10 nmol/L）+ CP（5 μg/ml）组（P<0.05）。ATRA（1 nmol/L）+ BDNF（50 ng/ml）+ CP组（存活率45.33%±11.83%，凋亡率12.48%±2.48%）与单独CP组在存活率和凋亡率方面无显著差异（P>0.05）。ATRA（10 nmol/L，100 nmol/L）+ BDNF（50 ng/ml）+ CP（61.62%±18.53%，105.02%±5.55%）组的存活率显著高于单独CP组（P<0.05，P<0.01），而凋亡率（9.36%±1.03%，5.05%±1.88%）显著低于单独顺铂组（P<0.05，P<0.01）。ATRA（100 nmol/L）+ BDNF（50 ng/ml）+ CP组的存活率高于ATRA（10 nmol/L）+ BDNF（50 ng/ml）+ CP组（P<0.01），而凋亡率低于ATRA（10 nmol/L）+ BDNF（50 ng/ml）+ CP组（P<0.01）。（3）单独CP组中一些细胞显示出凋亡变化，而在ATRA（10 nmol/L）+ BDNF（50 ng/ml）+ CP组中，核内染色质分布均匀，核膜清晰，并存在线粒体、核糖体和溶酶体。
TrkB和BDNF影响SY5Y对CP的敏感性。TrkB和BDNF水平越高，SY5Y对CP的敏感性越低。
