硼中子俘获疗法对胶质瘤干细胞增殖和细胞周期的影响

Effect of boron neutron capture therapy on cell proliferation and cell cycle arrest in glioma stem cells

目的 研究硼中子俘获疗法（BNCT）对体外培养人胶质瘤干细胞（GSCs）周期进程的影响及机制,比较GSCs和胶质瘤细胞株SHG-44对BNCT敏感性的差异.方法 首先检测人GSCsSU2和人胶质瘤细胞株SHG-44吸收含硼化合物二羟基苯丙氨酸硼（BPA）的情况,然后采用医院中子照射器（IHNI-1）对含硼（10B）细胞进行照射,克隆存活实验检测细胞的放射敏感性,MTT法检测细胞增殖率,流式细胞术检测细胞周期进程,Westem blot检测cyclin B1、CDK1和P21[WAF1]蛋白表达情况.结果 5 mmol/L BPA孵育SU2和SHG-44细胞24h,10B浓度分别可达（1.76±0.28）和（2.50±0.12）μg/107细胞,富含10B的细胞经IHNI-1照射后生存率和增殖率与不含10B的细胞比均明显下降（P＜ 0.05或P＜0.01）,SU2细胞经IHNI-1照射4 min时BNCT敏感性低于SHG-44细胞（P＜0.05）.与未加BPA而仅用中子照射组比较,经BNCT后G2/M期SU2和SHG-44细胞比例增高（P＜0.05）,cyclin B1和CDK1蛋白表达降低（P＜0.01）,P21[WAF1蛋白表达增加（P＜0.01）.结论 GSCs的BNCT敏感性低于胶质瘤细胞,BNCT可通过抑制细胞周期蛋白形成将细胞阻滞于G2/M期来抑制细胞更新和增殖.

Objective To study the effect and mechanism of boron neutron capture therapy （BNCT） on cell cycle procession in human glioma stem cells （GSCs） in vitro,and to analyze the difference of sensitivity for BNCT on GSCs and glioma cells.Methods Firstly,uptake of boronophenylalarine （BPA） on human GSCs SU2 and glioma cells SHG-44 was detected.Then the cells enriched boron-10 （10B） were irradiated by In-Hospital Neutron Irradiator （IHNI-1）.The radiation sensitivity was measured using clonogenic survival assay.The proliferation was examined by MTT assay.The cell cycle procession was determined using flow cytometry.The protein expression of cyclin B1,CDK1 and P21[WAF1] were detected by Western blot.Results 10B concentration of SU2 and SHG-44 cells arrived at （1.76 ±0.28） and （2.50 ±0.12） μg/107cells at 24 h in 5 mMBPA.After radiation by IHNI-1,the survival fraction and viability of cells enriched 10B were decreased significantly （P 〈 0.05 orP 〈 0.01）,compared with those of 10B-free.The BNCT sensitivity of SU2 cells was lower than that of SHG-44 cells （P 〈 0.05）.The proportion of G2/M phase of SU2 and SHG-44 cells was increased after BNCT compared with that of 10B-free（P 〈 0.05）.The protein levels of cyclin B1 and CDK1 were decreased（P 〈 0.01）,and that of P21[WAF1] was increased（P 〈 0.01）.Conclusions The BNCT sensitivity of GSCs was lower than that of glioma cells.BNCT could inhibite cell regeneration and proliferation and make G2/M to be blocked by inhibition of cell cycle protein formation.