CAJ | 학술논문

目的探讨骨形态发生蛋白质2 (BMP-2)在神经胶质瘤细胞株中的表达及BMP-2对细胞增殖的作用机制.方法传代培养人U8vIII、U87、T98G、LN229及U373神经胶质瘤细胞株.采用PCR法检测BMP-2及其受体BMPR1A、BMPR1B、Alk-2和Smad1在5种细胞株中的表达.MTS法检测0、25、50、100、250及500 ng/ml的BMP-2对U87细胞增殖的影响.Western blot方法分析用上述剂量的BMP-2处理U87细胞后,Smads和丝裂原活化蛋白激酶类(MAPKs)活性的变化;100 ng/ml的BMP-2处置U87细胞后0、5、15、30及60 min,Smads和MAPKs活性的变化;用100 ng/ml的BMP-2处置U87细胞后0、4、8、16及24 h,BMP通路下游基因cyclin B、cyclin D1、p27及p21的表达.结果 BMP-2及其受体BMPR1A、BMPR1B、Alk-2和Smad1在5种细胞株中均有表达,表达量差异有统计学意义(均P ＜0.01);BMP-2在U87中表达最高,U373中表达最低.MTS实验显示,BMP-2对U87细胞的增殖具有促进作用,在剂量为100 ～ 250 ng/ml时达高峰;与0 ng/mlBMP-2比较,随着BMP-2剂量的增加磷酸化的Smad1/5/8、p38激酶、c-Jun N-末端激酶(JNK)和细胞外信号调节蛋白激酶(ERK)的表达逐渐增高(均P＜0.01),100 ～ 250 ng/ml时达高峰.在BMP-2处理U87细胞5 min后磷酸化的Smad1/5/8、p38、JNK和ERK蛋白的表达开始升高,30 min达高峰,各磷酸化蛋白在不同时间点的表达差异均有统计学意义(均P ＜0.01).BMP-2(100 ng/ml)处置U87细胞后,cyclin B和cyclin D1的表达逐渐升高,p27和p21的表达逐渐下降,均于24 h达高峰,各个时间点的表达差异均有统计学意义(均P＜0.01).结论 BMP-2通过介导BMP/Smad和BMP/MAPK通路的活性而促进神经胶质瘤细胞的增殖.
목적 탐토골형태발생단백질2 (BMP-2)재신경효질류세포주중적표체급BMP-2대세포증식적작용궤제.방법 전대배양인U8vIII、U87、T98G、LN229급U373신경효질류세포주.채용PCR법검측BMP-2급기수체BMPR1A、BMPR1B、Alk-2화Smad1재5충세포주중적표체.MTS법검측0、25、50、100、250급500 ng/ml적BMP-2대U87세포증식적영향.Western blot방법분석용상술제량적BMP-2처리U87세포후,Smads화사렬원활화단백격매류(MAPKs)활성적변화;100 ng/ml적BMP-2처치U87세포후0、5、15、30급60 min,Smads화MAPKs활성적변화;용100 ng/ml적BMP-2처치U87세포후0、4、8、16급24 h,BMP통로하유기인cyclin B、cyclin D1、p27급p21적표체.결과 BMP-2급기수체BMPR1A、BMPR1B、Alk-2화Smad1재5충세포주중균유표체,표체량차이유통계학의의(균P ＜0.01);BMP-2재U87중표체최고,U373중표체최저.MTS실험현시,BMP-2대U87세포적증식구유촉진작용,재제량위100 ～ 250 ng/ml시체고봉;여0 ng/mlBMP-2비교,수착BMP-2제량적증가린산화적Smad1/5/8、p38격매、c-Jun N-말단격매(JNK)화세포외신호조절단백격매(ERK)적표체축점증고(균P＜0.01),100 ～ 250 ng/ml시체고봉.재BMP-2처리U87세포5 min후린산화적Smad1/5/8、p38、JNK화ERK단백적표체개시승고,30 min체고봉,각린산화단백재불동시간점적표체차이균유통계학의의(균P ＜0.01).BMP-2(100 ng/ml)처치U87세포후,cyclin B화cyclin D1적표체축점승고,p27화p21적표체축점하강,균우24 h체고봉,각개시간점적표체차이균유통계학의의(균P＜0.01).결론 BMP-2통과개도BMP/Smad화BMP/MAPK통로적활성이촉진신경효질류세포적증식.
Objective To investigate the BMP-2 expression in glioma cell lines and its promotive role and mechanism of proliferation in glioma cells.Methods Subculture of five glioma cell lines including U8vIII,U87,T98G,LN229 and U373,Semi-quantitative PCR was performed to analyze BMP-2,BMP-2 receptors (BMPR1A,BMPR1B and Alk-2) and Smadl expression in these glioma cell lines.MTS assay was used to investigate the glioma cell proliferation with BMP-2 (0,25,50,100,250 and 500 ng/ml) treatment.Western blot was performed to detect activation of canonical BMP pathway (BMP/Smad pathway) and non-canonical BMP pathway (BMP/MAPK pathway) under BMP-2 treatment with different doses and timepoints.At last,the expression of BMP pathway downstream genes cyclin B,cyclin D1,p27 and p21 were explored at 0,4,8,16 or 24 h posttreatment of BMP-2 (100 ng/ml).Results The expression of BMP-2,its receptors and Smad1 were significantly different among five glioma cell lines (P ＜ 0.01).The MTS assay showed that BMP-2 promoted U87 cell proliferation.It reached the maximum level when the BMP-2 concentration reached 100-250 ng/ml.The Western blot results revealed that BMP-2 activated phosphorylated Smad1/5/8,p38,JNK and ERK in dose and time dependent manners (P ＜ 0.01).The expression levels of BMP pathway downstream genes cyclin B and cyclin D1 were higher compared to those of control (P ＜ 0.01).However,the levels of p27 and p21 were lower (P ＜ 0.01).Conclusions BMP-2 could promote glioma cell proliferation via regulation of BMP/Smad and BMP/MAPK pathway.