Objective： To investigate the effects of panax notoginseng saponins （PNS） on expression, regulation and phosphorylation of multiple protein kinases in mitogen activated protein kinase （MAPK） intracellular signal ...Objective： To investigate the effects of panax notoginseng saponins （PNS） on expression, regulation and phosphorylation of multiple protein kinases in mitogen activated protein kinase （MAPK） intracellular signal pathway and GATA transcription factors in hematopoietic cells, so as to explore its mechanism of proliferation and differentiation activity on hematopoiesis. Methods： The human granulocytic HL-60, erythrocytic K562, megakaryocytic CHRF-288 and Meg-01 cell lines were treated by PNS, the positive control of K562, CHRF-288 ceils treated by recombination human erythropoietin （Epo） and thrombopoietin （Tpo） respectively. The total cell lysate and nuclei protein were extracted after being treated by PNS, subsequently, analyzed by both Western blot and immune-precipitation. Meanwhile, the nuclei extract was performed for electrophoretic mobility shift assay （EMSA） by using 32p radio labeled double-stranded GATA consensus oligonucleotide. Results： The expression levels of kinase MEK-1, MEK-2, ERK-1, ERK-2, AKT-1, AKT-2 and PI- 3K were increased by PNS treatment to different extent in four cell lines, depending on cellular heterogeneity and sensitivity to PNS, also phosphorylation of MEK-1, ERK-1 was differentially promoted by PNS respectively （P〈0.05, 0.01, 0.001）. The expression levels of transcription factors GATA-1 and GATA-2 were increased, moreover, their DNA binding activities were raised dramatically in PNS treated K562, CHRF-288 and Meg-01 cells compared with the controls respectively （P〈0.05, 0.01, 0.001）. The positive control of K562, CHRF-288 cells treated by Epo or Tpo respectively also displayed up-regulation of protein kinases and GATA transcription factors respectively （P〈0.05, 0.01, 0.001）. Conclusion： The results indicated that intracellular signal pathway initiated by PNS was involved in MAPK pathway and transcription factors of GATA family in hematopoietic cells. PNS displayed the role to promote proliferation and differentiation, by means of increas展开更多
目的研究衰老骨髓基质细胞对骨髓造血细胞增殖分化能力的影响,为阐述机体造血微环境衰老对造血干/祖细胞增殖的影响提供实验依据。方法全骨髓贴壁法体外培养大鼠骨髓基质细胞,分为对照组和衰老组。衰老组:常规培养基内加入30 mg/m L D-...目的研究衰老骨髓基质细胞对骨髓造血细胞增殖分化能力的影响,为阐述机体造血微环境衰老对造血干/祖细胞增殖的影响提供实验依据。方法全骨髓贴壁法体外培养大鼠骨髓基质细胞,分为对照组和衰老组。衰老组:常规培养基内加入30 mg/m L D-半乳糖作用48 h。CCK-8法测定BMSCs增殖;流式细胞术分析细胞周期;β-半乳糖苷酶(SA-β-Gal)染色观察衰老BMSCs百分率;Western blot检测P16、P21和P53蛋白表达。骨髓造血细胞与BMSCs共培养,集落计数检测髓系多向性造血祖细胞(CFU-Mix)增殖分化。ELISA检测BMSCs培养上清液中IL-1β、GM-CSF和SCF含量;DCFH-DA流式荧光检测BMSC活性氧簇(ROS)水平;酶学法检测BMSCs内过氧化物丙二醛(MDA)含量和总超氧化物歧化酶(SOD)活性。结果与对照组相比,D-半乳糖诱导BMSCs衰老,细胞阻滞于G0/G1期(P<0.01),增殖能力显著下降,SA-β-Gal染色阳性率升高(P<0.01);衰老相关蛋白P16、P21和P53表达明显上调(P<0.01)。与衰老BMSCs共培养的骨髓造血细胞增殖分化能力减弱。衰老BMSCs内ROS、MDA氧化损伤指标上升,SOD抗氧化指标下降(P<0.01);BMSCs培养上清液IL-1β、GM-CSF和SCF含量明显下降(P<0.01)。结论衰老骨髓基质细胞抑制造血细胞增殖、分化能力,其机制可能与骨髓基质细胞氧化损伤,分泌活性因子改变有关。展开更多
基金Supported by National Natural Science Foundation of China (No.30070933)
文摘Objective： To investigate the effects of panax notoginseng saponins （PNS） on expression, regulation and phosphorylation of multiple protein kinases in mitogen activated protein kinase （MAPK） intracellular signal pathway and GATA transcription factors in hematopoietic cells, so as to explore its mechanism of proliferation and differentiation activity on hematopoiesis. Methods： The human granulocytic HL-60, erythrocytic K562, megakaryocytic CHRF-288 and Meg-01 cell lines were treated by PNS, the positive control of K562, CHRF-288 ceils treated by recombination human erythropoietin （Epo） and thrombopoietin （Tpo） respectively. The total cell lysate and nuclei protein were extracted after being treated by PNS, subsequently, analyzed by both Western blot and immune-precipitation. Meanwhile, the nuclei extract was performed for electrophoretic mobility shift assay （EMSA） by using 32p radio labeled double-stranded GATA consensus oligonucleotide. Results： The expression levels of kinase MEK-1, MEK-2, ERK-1, ERK-2, AKT-1, AKT-2 and PI- 3K were increased by PNS treatment to different extent in four cell lines, depending on cellular heterogeneity and sensitivity to PNS, also phosphorylation of MEK-1, ERK-1 was differentially promoted by PNS respectively （P〈0.05, 0.01, 0.001）. The expression levels of transcription factors GATA-1 and GATA-2 were increased, moreover, their DNA binding activities were raised dramatically in PNS treated K562, CHRF-288 and Meg-01 cells compared with the controls respectively （P〈0.05, 0.01, 0.001）. The positive control of K562, CHRF-288 cells treated by Epo or Tpo respectively also displayed up-regulation of protein kinases and GATA transcription factors respectively （P〈0.05, 0.01, 0.001）. Conclusion： The results indicated that intracellular signal pathway initiated by PNS was involved in MAPK pathway and transcription factors of GATA family in hematopoietic cells. PNS displayed the role to promote proliferation and differentiation, by means of increas
