CAJ | 학술논문

目的研究白花蛇舌草多糖(PEHD)对多发性骨髓瘤(MM)细胞增殖的影响,为其应用于临床治疗MM提供实验依据.方法以MM细胞株RPMI 8226为研究对象.MTT法检测PEHD对RPMI 8226细胞增殖的影响.Annexin V-FITC/碘化丙锭(PI)双染法(Annexin V/PI)标记的流式细胞术检测PEHD诱导RPMI 8226细胞凋亡情况.Hoechst 33258染色法观察PEHD诱导RPMI 8226细胞凋亡的形态学改变.用Western blot检测PEHD处理后有关凋亡信号传导通路蛋白caspase-3、-8、-9和PARP蛋白的表达以及PEHD作用前后NF-κB蛋白以及其他通路蛋白的变化情况.结果 PEHD在一定浓度范围内对RPMI 8226细胞有明显的增殖抑制作用,最高抑制率达到了92.3％,在1～4 mg/ml浓度范围内呈时间-剂量依赖性.不同浓度PEHD分别作用24 h,RPMI 8226细胞均可见凋亡小体,且凋亡小体数量随PEHD浓度增加而增加.1、2、3 mg/ml PEHD诱导的细胞早期凋亡率分别为22.52％、62.31％、69.94％,高于空白对照组8.93％.1、2、3 mg/ml PEHD作用24 h,RPMI 8226细胞caspase 8、9、3和PARP蛋白表达上升,Mcl-1、Bcl-xl、Bid、Bim的表达随着PEHD浓度的增加而下降,而Bax、Bak、Bad表达上升,但p-AKT蛋白(相对分子质量60 000)的表达明显下降,NF-κB蛋白表达水平明显下调.结论在一定浓度范围内的PEHD(1 ～4 mg/ml)可明显抑制RPMI 8226细胞增殖,呈时间和浓度依赖性；其增殖抑制作用与诱导细胞凋亡有关；诱导细胞凋亡机制与其激活caspase-8、-9、-3及PARP蛋白表达,下调p-AKT及NF-κB蛋白表达有关.
목적 연구백화사설초다당(PEHD)대다발성골수류(MM)세포증식적영향,위기응용우림상치료MM제공실험의거.방법 이MM세포주RPMI 8226위연구대상.MTT법검측PEHD대RPMI 8226세포증식적영향.Annexin V-FITC/전화병정(PI)쌍염법(Annexin V/PI)표기적류식세포술검측PEHD유도RPMI 8226세포조망정황.Hoechst 33258염색법관찰PEHD유도RPMI 8226세포조망적형태학개변.용Western blot검측PEHD처리후유관조망신호전도통로단백caspase-3、-8、-9화PARP단백적표체이급PEHD작용전후NF-κB단백이급기타통로단백적변화정황.결과 PEHD재일정농도범위내대RPMI 8226세포유명현적증식억제작용,최고억제솔체도료92.3％,재1～4 mg/ml농도범위내정시간-제량의뢰성.불동농도PEHD분별작용24 h,RPMI 8226세포균가견조망소체,차조망소체수량수PEHD농도증가이증가.1、2、3 mg/ml PEHD유도적세포조기조망솔분별위22.52％、62.31％、69.94％,고우공백대조조8.93％.1、2、3 mg/ml PEHD작용24 h,RPMI 8226세포caspase 8、9、3화PARP단백표체상승,Mcl-1、Bcl-xl、Bid、Bim적표체수착PEHD농도적증가이하강,이Bax、Bak、Bad표체상승,단p-AKT단백(상대분자질량60 000)적표체명현하강,NF-κB단백표체수평명현하조.결론 재일정농도범위내적PEHD(1 ～4 mg/ml)가명현억제RPMI 8226세포증식,정시간화농도의뢰성；기증식억제작용여유도세포조망유관；유도세포조망궤제여기격활caspase-8、-9、-3급PARP단백표체,하조p-AKT급NF-κB단백표체유관.
Objective To explore the proliferation inhibition and apoptosis effects of polysaccharides extracts from Hedyotis diffusa (PEHD) on multiple myeloma (MM) cell line RPMI 8226 cells in vitro,so as to provide experimental theory for the clinical application in the treatment of MM.Methods MTT assay was used to examine the effects of PEHD on cell growth.The apoptotic cells were analyzed by flow cytometry with AnnexinV/PI staining.Hoechst staining was used to observe the morphological changes of RPMI 8226 cell apoptosis.The expression levels of caspase-3,-8,-9,PARP,nucleoprotein NF-κB protein and other channel protein were assayed by Western blotting method.Results The growth of RPMI 8226 cells were suppressed after treatment with PEHD,the highest inhibition rate reached to 92.3％,the results in the doses from 1 to 4 mg/ml showed a dose-and-time-dependent manner.The proportion of apoptotic cells in 1,2 and 3 mg/ml PE-HD treatment groups for 24 h were 22.52％,62.31％ and 69.94％,respectively,and significantly higher than that of control 8.93％.After treated with PEHD,apoptotic body appeared in RPMI 8226 cells nucleus and the number of apoptotic body increased in a dose-dependent manner.With the increasing of PEHD concentration,the expression of caspase-8,-9,-3 and PARP protein increased.The expression of Mcl-1,Bcl-xl,Bid and Bim protein decreased gradually,but the expression of Bax,Bak and Bad protein increased,and the expression of p-AKT protein (60 kDa) and NF-κB obviously decreased.Conclusion PEHD could inhibited the growth of RPMI 8226 cells and displayed a dose-and-time-dependent manner,its mechanism may involve cell apoptosis induction,which was associated with the activation of caspase-8,caspase-9,and caspase-3 protein and the down-regulation of p-AKT and NF-κB protein expression.