三七皂苷对人骨髓造血细胞凋亡相关蛋白表达的影响

Expression of Apoptosis-related Proteins in the Human Bone Marrow Hematopoietic Cells Treated by Panax Notoginosides

本文研究观察三七皂苷(PNS)对造血细胞促进凋亡相关蛋白(Daxx、Fas)和抑制凋亡的核转录因子(NFkB、c-Rel)表达的影响,探讨PNS促进造血细胞增殖,支持生长存活的作用机制。采用半固体集落培养法观察PNS对人骨髓红系、粒系祖造血细胞(CFU-GM、CFU-E)的增殖作用,台盼蓝拒染法观察细胞的存活率,涂片染色法观察细胞形态学的变化,用流式细胞术分析细胞凋亡状况。同时,用PNS处理粒系HL-60、红系K562、巨核系CHRF-288和Meg-014种细胞株后,提取胞浆蛋白和核蛋白,Western免疫印迹观察Daxx、Fas、NFkB和c-Rel蛋白的表达水平。结果发现:①PNS能够刺激人骨髓红系、粒系祖细胞和HL-60等4种细胞株增殖。②HL-60等4种株细胞经PNS和饥饿处理后,活性良好,镜下未观察到凋亡的形态学特征;AnnexinⅤ分析也未见凋亡的阳性细胞。③Westernblot结果显示,经PNS处理的4株细胞Daxx蛋白表达水平与对照相比,下降幅度分别为33.3%-61·5%;HL-60细胞本身表达Fas蛋白低下,PNS处理后也无明显下降,而K562、CHRF-288和Meg-01细胞Fas蛋白表达与对照相比,下降幅度分别为33.3%-71.4%。④NFkB、c-Rel蛋白除HL-60细胞对PNS诱导无明显变化外,其余细胞均出现不同程度地增加,分别提高了2.0-2.7倍和1.5-2.3倍。结论:PNS在某种程度上能够抑制Daxx、Fas蛋白表达,而相应减少造血细胞的凋亡,同时也能通过上调NFkB、c-Rel转录因子,促进细胞增殖,并阻止半胱天冬酶(caspase)连锁链的活化而抑制造血细胞凋亡,这为PNS应用于临床治疗凋亡过度的疾病如再生障碍性贫血提供了可能性。

The study was aimed to investigate the action of Panax Notoginosides （PNS, extracted from notoginseng herb） on the expression of the apoptosis-related proteins （Daxx, Fas） and transcription factors （NFkB,c-Rel） in the hematopoietic cells and to explore the mechanisms of supporting cells to survive. The colony formation of CFU-GM and CFU-E in human bone marrow was assayed in the presence of various concentrations of PNS. The viability of cells was assayed by trypan blue and the changes of cell morphology were observed with microscope. The Annexin-V positive cells were detected by FCM. Three lineages of human myeloid HL-60, erythroid K562, megakaryoid CHRF-288 and Meg-01 cells were incubated in addition of PNS（ 10 mg/L） for 14 days. The nuclear or cytoplasm protein of cells was extracted and analyzed by Western blot with monoclonal antibodies against Daxx, Fas or NFkB, c-Rel. The results showed： （ 1 ） the proliferation on hematopoietic progenitor cells （ CFU-GM and CFU-E） and four cell lines was promoted by PNS; （ 2 ） after the four cell lines were promoted by PNS and hungered through wiping off the sera, the viability of the four cell lines was high without significant morphological change and neither the detection of Annexin-V positive cells; （3） the expression of Daxx and Fas protein could be inhibited by PNS. Western Blot showed that Daxx in four cell lines treated by PNS were 33.3 -61.5% lower than that in untreated controls. The Fas protein was also descended in three cell lines of K562, CHRF-288 and Meg-01 by 33.3 -71.4% respectively, while Fas protein in HL-60 cells was no detectable difference after PNS treatment. （4） The Wanscription factors NFkB and c-Rel protein could be increased by PNS. The NFkB ,c-Rel protein were also enhanced in three cell lines of K562, CHRF-288 and Meg-01 by （2.0 - 2.7 ） and （ 1.5 - 2.3 ） -fold respectively, while there were also no detectable difference in HL-60 cells after PNS treatment. It is concluded that PNS inhibites the expression of Daxx and Fas proteins, may decrease the apoptosis of the hematopoietic cells. The level of NFkB and c-Rel proteins can be enhanced by PNS, which not only stimulates the proliferation of cells, but also inhibits the activity of the waterfall of caspase and apoptosis of the hematopoietic cells. PNS may treat the disease with over-apoptosis of hematopoietic cells, as aplastic anemia.