珠子参皂苷诱导骨髓造血干细胞增殖分化的体外实验研究
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摘要
目的:观察珠子参皂苷对环磷酰胺干预后小鼠造血干细胞的增殖分化及细胞因子、转录因子、基质细胞衍生因子-1(SDF-1)及其对应的受体CXCR4表达的影响。方法:模型小鼠按照380mg/kg腹腔注射环磷酰胺,正常小鼠腹腔注射同体积的生理盐水,1次/d,连续3d。给药结束后分离小鼠骨髓单个核细胞(BMMNC),分成正常组、模型组、促红细胞生成素(EPO)+粒细胞集落刺激因子(G-CSF)组(50U/mL+250U/mL)和珠子参皂苷(50、100和200μg/mL)组。分别于给药d 4和d 8观察细胞增殖,计数粒-巨噬细胞集落形成单位(CFU-GM)、红系细胞集落形成单位(CFU-E)和红系爆式集落形成单位(BFU-E)数量,检测干细胞因子(SCF)、白细胞介素3(IL-3)、肿瘤坏死因子α(TNF-α)和干扰素γ(IFN-γ)分泌,检测SDF-1、CXCR4、GATA-1、PU.1基因mRNA和蛋白表达量。结果:珠子参皂苷可显著促进骨髓造血干细胞增殖分化,增加CFU-E、CFU-GM和BFU-E集落形成数量,促进细胞因子SCF和IL-3分泌、抑制细胞因子INF-γ和TNF-α分泌,上调SDF-1、CXCR4、GATA-1、PU.1基因mRNA和蛋白表达,与模型组比较差异具有统计学意义(P<0.05或P<0.01)。结论:珠子参皂苷可以显著促进骨髓抑制后小鼠造血干细胞增殖分化,其机制可能与上调SDF-1、CXCR4、GATA-1、PU.1的表达和调节细胞因子的分泌有关。
Objective:To observe the protective effects of saponins from Panacis majoris on proliferation and differentiation of hematopoietic stem cells and explore the potential mechanisms.Methods:The model mice were treated with cyclophosphamide(CP)380 mg/kg by intraperitoneal injection and normal mice were treated with saline,respectively once a day.After treatment with CP or saline,the bone marrow mononuclear cells(BMMNC)were isolated from mice.BMMNC were divided into normal group, model group,erythropoietin(EPO)+granulocyte colony-stimulating factor(G-CSF)(50 U/mL+250 U/mL)group and saponins from Panacis majoris(50,100 and 200μg/mL)groups.Cell proliferation,granulocyte macrophage colony forming unit(CFU-GM),erythroid colony forming unit(CFU-E),erythroid burst colony forming unit(BFU-E),secretions of stem cell factor(SCF),interleukin 3(IL-3),tumor necrosis factor alpha(TNF-α),interferon gamma(IFN-γ)were determined respectively on the fourth and eighth days after administrating corresponding drugs.The mRNA and protein expressions of stromal cell derived factor 1(SDF-1),cxc chemokin receptor 4(CXCR4),GATA-1 and PU.1 were detected by quantitative real-time PCR and western blot.Results:Compared with the model group,saponins from Panacis majoris(50,100 and200μg/mL)significantly promoted the proliferation and differentiation of bone marrow hematopoietic stem cells,increased colony formations of CFU-E,CFU-GM and BFU-E,elevated secretions of SCF and IL-3,inhibited secretions of INF-γand TNF-α,and upregulated mRNA and protein expressions of SDF-1,CXCR4,GATA-1 and PU.1(P <0.05 or P <0.01,respectively).Conclusion:The mechanisms of saponins from Panacis majoris involving in promoting proliferation and differentiation of bone marrow hematopoietic stem cells is related to regulate the mRNA and protein expressions of SDF-1,CXCR4,GATA-1 and PU.1 and secretions of various cytokines.
Objective:To observe the protective effects of saponins from Panacis majoris on proliferation and differentiation of hematopoietic stem cells and explore the potential mechanisms.Methods:The model mice were treated with cyclophosphamide(CP)380 mg/kg by intraperitoneal injection and normal mice were treated with saline,respectively once a day.After treatment with CP or saline,the bone marrow mononuclear cells(BMMNC)were isolated from mice.BMMNC were divided into normal group, model group,erythropoietin(EPO)+granulocyte colony-stimulating factor(G-CSF)(50 U/mL+250 U/mL)group and saponins from Panacis majoris(50,100 and 200μg/mL)groups.Cell proliferation,granulocyte macrophage colony forming unit(CFU-GM),erythroid colony forming unit(CFU-E),erythroid burst colony forming unit(BFU-E),secretions of stem cell factor(SCF),interleukin 3(IL-3),tumor necrosis factor alpha(TNF-α),interferon gamma(IFN-γ)were determined respectively on the fourth and eighth days after administrating corresponding drugs.The mRNA and protein expressions of stromal cell derived factor 1(SDF-1),cxc chemokin receptor 4(CXCR4),GATA-1 and PU.1 were detected by quantitative real-time PCR and western blot.Results:Compared with the model group,saponins from Panacis majoris(50,100 and200μg/mL)significantly promoted the proliferation and differentiation of bone marrow hematopoietic stem cells,increased colony formations of CFU-E,CFU-GM and BFU-E,elevated secretions of SCF and IL-3,inhibited secretions of INF-γand TNF-α,and upregulated mRNA and protein expressions of SDF-1,CXCR4,GATA-1 and PU.1(P <0.05 or P <0.01,respectively).Conclusion:The mechanisms of saponins from Panacis majoris involving in promoting proliferation and differentiation of bone marrow hematopoietic stem cells is related to regulate the mRNA and protein expressions of SDF-1,CXCR4,GATA-1 and PU.1 and secretions of various cytokines.
引文
[1]赵仁,赵毅,李东明.珠子参研究进展[J].中国现代中药,2008,10(7):3-6.
[2]熊尚林.珠子参治疗30例白细胞减少症临床观察[J].中医药研究,1996,5:22.
[3]段径云,陈瑞明.珠子参对血液和造血功能的影响[J].西北药学杂志,1996,11(2):72-73.
[4]张继红,王心怡,石孟琼,等.珠子参皂苷诱导骨髓干细胞分化为肝样细胞的体外实验研究[J].中国现代中药,2008,10(7):3-6.
[5]张继红,王海燕,石孟琼,等.珠子参皂苷与BMSCs联用治疗大鼠肝纤维化作用研究[J].三峡大学学报(自然科学版),2016,38(3):104-108.
[6]李秀军,周凌云.补益药在血液病治疗中的应用[J].贵阳中医学院学报,2004,26(3):54-56.
[7]张金生.活血化瘀与干细胞循环[J].中医杂志,2012,53(6):451-453.
[8]张涓,张恩户.重组人粒细胞集落刺激因子联合丹参酮ⅡA与丹皮酚配伍对脑缺血大鼠骨髓干细胞动员的影响[J].中国医院药学杂志,2014,34(2):90-94.
[9]田晨,张新雪,张丰丰,等.补肾益髓生血法再障大鼠含药血清对大鼠造血祖细胞增殖分化及其机制的影响[J].世界科学技术—中医药现代化,2014,16(5):1076-1082.
[10]Zhang J,Ren X L,Shi W,et al.Small molecule Me6TREN mobilizes hematopoietic stem/progenitor cells by activating MMP-9 expression and disrupting SDF-1/CXCR4axis[J].Blood,2014,123(3):428-441.
[11]Lataillade J J,Clay D,Dupuy C,et al.Chemokine SDF-1enhances circulating CD34(+)cell proliferation in synergy with cytokines:possible role in progenitor survival[J].Blood,2000,95(3):756-768.
[12]Van O S,Beguin Y,Gothot A.Role of stromal-derived factor-1in the hematopoietic supporting activity of human mesenchymal stem cells[J].Eur J Haematol,2006,76(6):488-493.
[13]Sugiyama T,Kohara H,Noda M,et al.Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell niches[J].Immunity,2006,25(6):977-988.
[14]董玢,刘涓,娄利霞,等.补肾生血解毒方对再生障碍性贫血小鼠红细胞生成相关细胞因子表达的影响[J].北京中医药大学学报,2014,37(6):397-401.
[15]李影迪,初杰,范颖,等.当归多糖与黄芪多糖配伍对骨髓造血干细胞增殖分化的影响[J].辽宁中医药大学,2016,18(6):31-34.
[16]田晨,赵宗江,张新雪,等.补肾益髓生血法AA大鼠含药血清对大鼠造血干细胞红系分化JAK2/STAT5信号通路的影响[J].世界中医药,2014,9(6):713-716.
[17]张小丽,朱道银,唐恩洁.干细胞因子研究进展[J].川北医学院学报,2006,21(6):91-95.
[18]赵燕娜,高瑞兰,汪丽佩,等.白藜芦醇对K562白血病细胞抑制增殖和诱导分化的作用[J].中国药理学通报,2014,30(6):853-856.
[19]何丽,肖扬,蒋祖军,等.荧光定量聚合酶链反应检测GATA-1、GATA-2、过氧化物酶体增殖物激活受体γ和干细胞因子基因在再生障碍性贫血患者及正常人骨髓间充质干细胞中的表达[J].中国组织工程研究与临床康复,2011,15(19):3451-3454.
[2]熊尚林.珠子参治疗30例白细胞减少症临床观察[J].中医药研究,1996,5:22.
[3]段径云,陈瑞明.珠子参对血液和造血功能的影响[J].西北药学杂志,1996,11(2):72-73.
[4]张继红,王心怡,石孟琼,等.珠子参皂苷诱导骨髓干细胞分化为肝样细胞的体外实验研究[J].中国现代中药,2008,10(7):3-6.
[5]张继红,王海燕,石孟琼,等.珠子参皂苷与BMSCs联用治疗大鼠肝纤维化作用研究[J].三峡大学学报(自然科学版),2016,38(3):104-108.
[6]李秀军,周凌云.补益药在血液病治疗中的应用[J].贵阳中医学院学报,2004,26(3):54-56.
[7]张金生.活血化瘀与干细胞循环[J].中医杂志,2012,53(6):451-453.
[8]张涓,张恩户.重组人粒细胞集落刺激因子联合丹参酮ⅡA与丹皮酚配伍对脑缺血大鼠骨髓干细胞动员的影响[J].中国医院药学杂志,2014,34(2):90-94.
[9]田晨,张新雪,张丰丰,等.补肾益髓生血法再障大鼠含药血清对大鼠造血祖细胞增殖分化及其机制的影响[J].世界科学技术—中医药现代化,2014,16(5):1076-1082.
[10]Zhang J,Ren X L,Shi W,et al.Small molecule Me6TREN mobilizes hematopoietic stem/progenitor cells by activating MMP-9 expression and disrupting SDF-1/CXCR4axis[J].Blood,2014,123(3):428-441.
[11]Lataillade J J,Clay D,Dupuy C,et al.Chemokine SDF-1enhances circulating CD34(+)cell proliferation in synergy with cytokines:possible role in progenitor survival[J].Blood,2000,95(3):756-768.
[12]Van O S,Beguin Y,Gothot A.Role of stromal-derived factor-1in the hematopoietic supporting activity of human mesenchymal stem cells[J].Eur J Haematol,2006,76(6):488-493.
[13]Sugiyama T,Kohara H,Noda M,et al.Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell niches[J].Immunity,2006,25(6):977-988.
[14]董玢,刘涓,娄利霞,等.补肾生血解毒方对再生障碍性贫血小鼠红细胞生成相关细胞因子表达的影响[J].北京中医药大学学报,2014,37(6):397-401.
[15]李影迪,初杰,范颖,等.当归多糖与黄芪多糖配伍对骨髓造血干细胞增殖分化的影响[J].辽宁中医药大学,2016,18(6):31-34.
[16]田晨,赵宗江,张新雪,等.补肾益髓生血法AA大鼠含药血清对大鼠造血干细胞红系分化JAK2/STAT5信号通路的影响[J].世界中医药,2014,9(6):713-716.
[17]张小丽,朱道银,唐恩洁.干细胞因子研究进展[J].川北医学院学报,2006,21(6):91-95.
[18]赵燕娜,高瑞兰,汪丽佩,等.白藜芦醇对K562白血病细胞抑制增殖和诱导分化的作用[J].中国药理学通报,2014,30(6):853-856.
[19]何丽,肖扬,蒋祖军,等.荧光定量聚合酶链反应检测GATA-1、GATA-2、过氧化物酶体增殖物激活受体γ和干细胞因子基因在再生障碍性贫血患者及正常人骨髓间充质干细胞中的表达[J].中国组织工程研究与临床康复,2011,15(19):3451-3454.