摘要
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RPMI8226为研究对象。MTT法检测PEHD对RPMI8226细胞增殖的影响。AnnexinV—F｜TC／碘化丙锭（PI）双染法（AnnexinV／PI）标记的流式细胞术检测PEHD诱导RPMI8226细胞凋亡情况。Hoechst33258染色法观察PEHD诱导RPMI8226细胞凋亡的形态学改变。用Westernblot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—KB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RPMI8226细胞有明显的增殖抑制作用，最高抑制率达到了92．3％，在1—4mg／ml浓度范围内呈时间一剂量依赖性。不同浓度PEHD分别作用24h，RPMI8226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、3mg／mlPEHD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、3mg／mlPEHD作用24h，RPMI8226细胞caspase8、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60000）的表达明显下降，NF—KB蛋白表达水平明显下调。结论在一定浓度范围内的PEHD（1—4mg／m1）可明显抑制RPMI8226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、-3KPARP蛋白表达，下调p-AKT及NF—KB蛋白表达有关。
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, nueleoprotein NF-KB 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 ceils nucleus and the number of apoptotic body increased in a dose-dependent manner. With the increasing of PEHD con- centration, 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-KB obviously decreased. Conclusion PEHD could inhibited the growth of RPMI 8226 cells and displayed a dose-and-time-dependent manner, its mechanism may involvecell 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-kB protein expression.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2013年第4期337-340,共4页
Chinese Journal of Hematology
