当归多糖、黄芪多糖及其配伍对化疗性骨髓抑制小鼠骨髓干细胞凋亡和脱核因子的影响
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摘要
目的:通过实验研究当归多糖、黄芪多糖及其配伍对化疗性骨髓抑制小鼠骨髓干细胞细胞凋亡因子BCL-2、Caspase9、Bax,脱核因子C-myc、HDAC2的影响。方法:C57BL/6小鼠20只,雌雄各半,28～32 g。给模型组小鼠每日腹腔注射一次环磷酰胺380 mg·kg-1,正常小鼠腹腔注射等量生理盐水,连续3 d。造模后,以脱颈椎法处死各组小鼠,取出股骨,剔除肌肉和结缔组织,剪开股骨两端,用6号针头以生理盐水反复冲洗骨髓腔,并将冲出的骨髓打碎,再用4号针头过滤制成单个细胞悬液,在离心机上2000 r/min,离心10 min,去上清液,加入4 mL冷70%乙醇固定,上振荡器摇匀制成骨髓干细胞样本。细胞实验分为6组,包括正常组、模型组、"EPO"组、"EPO+(G-CSF)"组、当归多糖组、黄芪多糖组、两药配伍组。直接给药当归多糖0.2 mg/mL,黄芪多糖0.2 mg/mL,EPO50 U/mL,G-CSF 50 U/mL于骨髓干细胞体外培养体系中,培养3 d,室内18～20℃,20%相对湿度,5%CO2,37℃孵育箱。采用RT-qPCR法检测骨髓干细胞中BCL-2、Caspase9、Bax、C-myc、HDAC2的mRNA表达。结果:(1)造模后模型组小鼠骨髓干细胞BCL-2 mRNA表达下降,Caspase9、BaxmRNA表达升高,与正常组比较差异均有统计学意义(P<0.05)。①各给药组与模型组BCL-2 mRNA均有明显差异,当归多糖有提升BCL-2 mRNA作用,黄芪多糖作用不明显,两药间有交互作用,合用不如单一用药。②各给药组与模型组Caspase9 mRNA均有明显差异,当归多糖与黄芪多糖均有降低Caspase9 mRNA作用,两药交互作用明显,合用不如单用黄芪多糖。③各给药组与模型组Bax mRNA均有明显差异,黄芪多糖有降低Bax mRNA作用,当归糖作用不明显,两药交互作用无统计学意义。(2)造模后模型组小鼠骨髓干细胞C-myc、HDAC2 mRNA表达升高,与正常组比较差异均有统计学意义(P<0.05)。①各给药组与模型组C-myc mRNA均有明显差异,当归多糖与黄芪多糖均有降低C-myc mRNA作用,两药交互作用明显,合用不如单一用药;②各给药组与模型组HDAC2 mRNA均有明显差异,当归多糖与黄芪多糖均有降HDAC2 mRNA作用,两药交互作用明显,合用不如单一用药。结论:当归多糖、黄芪多糖及其配伍对化疗性骨髓抑制小鼠骨髓干细胞能促进BCL-2 mRNA表达、降低Bax、Caspase9 mRNA表达,同时也能降低C-myc、HDAC2mRNA表达,对细胞凋亡因子和细胞脱核因子具有一定的调控作用。并且当归多糖、黄芪多糖两药交互作用明显,合用不如单一用药。
Objective:To study the effects of Angelica polysaccharides,Astragalus polysaccharides and their compatibility on apoptotic factors BCL-2,Caspase9,Bax,denucleation factors C-myc and HDAC2 of bone marrow stem cells in mice with chemotherapy-induced bone marrow depression. Methods:Twenty C57 BL/6 mice,half male and half female,28-32 g. The mice in the model group were given intraperitoneal injection of cyclophosphamide 380 mg kg-1 once a day,and the normal mice were given intraperitoneal injection of normal saline for 3 consecutive days. After modeling,the mice in each group were killed by cervical decortication. The femur was taken out,the muscles and connective tissue were removed,the ends of femur were cut off,the marrow cavity was washed repeatedly with No. 6 needle with normal saline,and the washed marrow was broken. Then the washed marrow was filtered with No. 4 needle to form a single cell suspension. The cell suspension was centrifuged for 2000 r/min,centrifuged for 10 min,the supernatant was removed,fixed with 4 mL cold 70% ethanol,and shaken by an oscillator to make the bone marr cell samples. Cell experiments were divided into six groups,including normal group,model group,EPO group,EPO+(G-CSF)group,Angelica polysaccharide group,Astragalus polysaccharide group,two drug compatibility group. Directly administered Angelica polysaccharide 0.2 mg/mL,Astragalus polysaccharide0.2 mg/mL,EPO 50 U/mL,and(G-CSF)50 U/mL in vitro culture system of bone marrow stem cells for 3 days,incubation chamber at 18-20 g,20% relative humidity,5% CO2,37 ℃. The expression of BCL-2,Caspase9,Bax,C-myc and HDAC2 in bone marrow stem cells was detected by RT-q PCR. Results:(1)The expression of BCL-2 and Caspase9 and Bax in bone marrow stem cells of mice in the model group decreased and increased,with significant difference compared with the normal group(P<0.05). ①There were significant differences in BCL-2 mRNA between each drug group and model group. Angelica polysaccharides could enhance BCL-2 mRNA,but astragalus polysaccharides had no obvious effect,and there was interaction between the two drugs. It is better to use combined drugs than single drugs.②There were significant differences in Caspase9 mRNA between each drug group and model group.Angelica polysaccharide and Astragalus polysaccharide both had the effect of reducing Caspase9 mRNA.The interaction between the two drugs was obvious,and the combination of Angelica polysaccharide and Astragalus polysaccharide was better than that of Astragalus polysaccharide alone. ③There were significant differences in Bax mRNA between each administration group and model group. Astragalus polysaccharide had the effect of reducing Bax mRNA,Angelica sinensis sugar had no significant effect,and there was no significant interaction between the two drugs.(2)The expression of C-myc and HDAC2 in bone marrow stem cells of model group was higher than that of normal group(P<0.05). ① There were significant differences in C-myc gene expression between each administration group and model group. Angelica polysaccharides and Astragalus polysaccharides both had the effect of lowering C-myc gene expression.The interaction between the two drugs was obvious,and the combination of the two drugs was not as good as that of single administration.②There were significant differences in HDAC2 gene expression between each administration group and model group. Angelica polysaccharides and Astragalus both had the effect of lowering HDAC2 gene expression. The interaction between the two drugs was obvious,and the combination of the two drugs Conclusion:Angelica polysaccharides,Astragalus polysaccharides and their compatibility can promote the expression of BCL-2 and reduce the expression of Bax and Caspase-9 in bone marrow stem cells of mice with chemotherapy-induced bone marrow depression. They can also reduce the expression of C-myc and HDAC2,and regulate apoptotic factors and cell denucleation factors. The interaction between Angelica polysaccharides and Astragalus polysaccharides is obvious,and the combination of Angelica polysaccharides and Astragalus polysaccharides is not as good as the single drug.
Objective:To study the effects of Angelica polysaccharides,Astragalus polysaccharides and their compatibility on apoptotic factors BCL-2,Caspase9,Bax,denucleation factors C-myc and HDAC2 of bone marrow stem cells in mice with chemotherapy-induced bone marrow depression. Methods:Twenty C57 BL/6 mice,half male and half female,28-32 g. The mice in the model group were given intraperitoneal injection of cyclophosphamide 380 mg kg-1 once a day,and the normal mice were given intraperitoneal injection of normal saline for 3 consecutive days. After modeling,the mice in each group were killed by cervical decortication. The femur was taken out,the muscles and connective tissue were removed,the ends of femur were cut off,the marrow cavity was washed repeatedly with No. 6 needle with normal saline,and the washed marrow was broken. Then the washed marrow was filtered with No. 4 needle to form a single cell suspension. The cell suspension was centrifuged for 2000 r/min,centrifuged for 10 min,the supernatant was removed,fixed with 4 mL cold 70% ethanol,and shaken by an oscillator to make the bone marr cell samples. Cell experiments were divided into six groups,including normal group,model group,EPO group,EPO+(G-CSF)group,Angelica polysaccharide group,Astragalus polysaccharide group,two drug compatibility group. Directly administered Angelica polysaccharide 0.2 mg/mL,Astragalus polysaccharide0.2 mg/mL,EPO 50 U/mL,and(G-CSF)50 U/mL in vitro culture system of bone marrow stem cells for 3 days,incubation chamber at 18-20 g,20% relative humidity,5% CO2,37 ℃. The expression of BCL-2,Caspase9,Bax,C-myc and HDAC2 in bone marrow stem cells was detected by RT-q PCR. Results:(1)The expression of BCL-2 and Caspase9 and Bax in bone marrow stem cells of mice in the model group decreased and increased,with significant difference compared with the normal group(P<0.05). ①There were significant differences in BCL-2 mRNA between each drug group and model group. Angelica polysaccharides could enhance BCL-2 mRNA,but astragalus polysaccharides had no obvious effect,and there was interaction between the two drugs. It is better to use combined drugs than single drugs.②There were significant differences in Caspase9 mRNA between each drug group and model group.Angelica polysaccharide and Astragalus polysaccharide both had the effect of reducing Caspase9 mRNA.The interaction between the two drugs was obvious,and the combination of Angelica polysaccharide and Astragalus polysaccharide was better than that of Astragalus polysaccharide alone. ③There were significant differences in Bax mRNA between each administration group and model group. Astragalus polysaccharide had the effect of reducing Bax mRNA,Angelica sinensis sugar had no significant effect,and there was no significant interaction between the two drugs.(2)The expression of C-myc and HDAC2 in bone marrow stem cells of model group was higher than that of normal group(P<0.05). ① There were significant differences in C-myc gene expression between each administration group and model group. Angelica polysaccharides and Astragalus polysaccharides both had the effect of lowering C-myc gene expression.The interaction between the two drugs was obvious,and the combination of the two drugs was not as good as that of single administration.②There were significant differences in HDAC2 gene expression between each administration group and model group. Angelica polysaccharides and Astragalus both had the effect of lowering HDAC2 gene expression. The interaction between the two drugs was obvious,and the combination of the two drugs Conclusion:Angelica polysaccharides,Astragalus polysaccharides and their compatibility can promote the expression of BCL-2 and reduce the expression of Bax and Caspase-9 in bone marrow stem cells of mice with chemotherapy-induced bone marrow depression. They can also reduce the expression of C-myc and HDAC2,and regulate apoptotic factors and cell denucleation factors. The interaction between Angelica polysaccharides and Astragalus polysaccharides is obvious,and the combination of Angelica polysaccharides and Astragalus polysaccharides is not as good as the single drug.
引文
[1]徐宏贵,方建培,黄绍良,等.环磷酰胺对小鼠骨髓造血干/祖细胞作用及机制研究[J].中国小儿血液与肿瘤杂志,2007,12(3):106-110.
[2]HaslingerB,Mandl-WeberS,SitterT. Thrombin suppresses metalloprotei-nase 2 activity and increases tissue inhibitor of metalloproteinase 1syn-thesis in cultured hunman peritoneal mesothlial cells[J]. peritDialhit,2000,20(6):775-783.
[3]WongB,LummaWC,SmithAM,et al. Statins suppress THP-1cellm migration and secretion of matrix metalloproteinase by iinhibiting geranylgeranylation[J]. J Leukoc Biol,2001,69(6):959-962.
[4]NagaseH,woessnerJF. Matrix metalloProteinases[J]. J Biol Chem,1999,274:21491-21494.
[5]Pender SLF,MacDonald TT. Matrix metalloproteinases and the gut-new roles for old enzymes[J].Currrent Opinion in Pharmacology,2004,4:546-550.
[6]Szabo KA,Ablin RJ,Singh G. Matrix metalloProteinases and the immune response[J]. Clinical&Applied Lmmunology Reviews,2004,4:295-319.
[7]Van Wart HE,Birkedal-Hansen H. The cysteine switch:a principle of regulation of metalloproteinase activity with potential applicability to the entire Matrix metallo Proteinase gene family[J].Proc Natl Acad Sci USA,1990,87:5578-5582.
[8]Sheen Y,Shenk T. Relief of P53-Mediated Transcriptional Repre ssion by the Adeno-virus EIB 19-kDa Protein or the Cellular Bcl-2 Protein[J].PNAS,1994,91(19):8940-8944.
[9]OgilvyS,Metcalf D,Print Chq,et al. Constitutive Bcl-2expression through outhemato poietic compartment affects multiple lineages and enhances progenitor survival[J].Proc Natl Acad Sci,1999,96(26):14943-14948.
[10]Vairo Q,Innes KM,Adams JM,et al. Bcl-2 has a cell cycle inhibitory functionsenarahle from its enhancement of cell survival[J]. OncoQene,1996,13(7):1511-1519.
[11]Kasid U,Suy S,Dent P,et al. Activation of Raf by ionizing radiation[J]. Nature,1996,382(6594):813-816.
[12]Yang J,Liu X,Bhalla K,et al. Prevention of apoptosis by Bcl-2:release of cyto-chromec from mitochondria blocked[J].Scienee,1997,275:1129-1132.
[13]Kluck RM,Boss-Wetzel E,Green DR,et al. The release of cytochrome c from mitochondria:a primary site for Bcl-2regulation of apoptosis[J]. Science,1997,275:1132-1136.
[14]SusanSA,LorenzoHlK,Zamzami,etal.Molecular characterization of Mitochondrial apoptosis-inducing factor[J].Nature,1999,397:441-446.
[15]D Josefsen. Differential Expresoion of Bcl-2 Homologs in Human CD34+Hematopoieitc Progenitor Cells Induced to Differentiate into Erythroid or Granulocytic Cells[J].Stem Cells,2000,18(4):261.
[16]Sanz C. The expression of Bcl-x is downregulated during differentiation of human hematopoietic progenitor Cells along the granulocyte but not the monocyte/macroPhage lineage[J].Blood,1997,89(8):3199.
[17]DeliaD,Aiello A,Soligo D,et al. Bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells[J].Blood,1992,79(5):1291-1298.
[18]刘文励,孙汉英,周剑锋.复方活血汤对放射性损伤小鼠骨髓细胞Bcl-2、bax、Bcl-XL基因表达的作用[J].中华放射医学与防护杂志,1998,18:256-258.
[19]崔玉芳,丁彦青,徐菡,等.致死剂量的γ线照射后小鼠胸腺淋巴细胞的凋亡规律与bax、Bcl-2和Bcl-xl表达的关系[J].细胞与分子免疫杂志,2004,20(6):750-753.
[20]Kurokawa M,Kornbluth S. Caspases and kinases in a death grip[J].Cell,2009,138(5):838-854.
[21]杨静,王明娟,程玉,等.胃癌组织中Caspase 3、Caspase 9的表达及其临床意义[J].临床与实验病理学杂志,2016,32(10):1159-1161.
[22]Lai GH,Zhang Z,Sirica AE. Celecoxib acts in a cyclooxy genase-2 independent manner and in synergy with emodin to suppress rat cholangiocarcinoma growth in vitro through a mechanism involving enhanced Akt inactivation and increased activation of caspases-9 and-3[J].Mol Cancer Ther,2003,2(3):265-271.
[23]徐丁洁,徐洪,张碧溦,等.金匮温经汤对围绝经期大鼠卵巢凋亡因子Caspase3、Caspase9、Apaf-1蛋白表达的影响[J].中华中医药学刊,2018,36(10):2502-2504.
[24]Bratton S B,Salvesen G S. Regulation of the Apaf-1-caspase-9apoptosome[J]. J Cell Sci,2010,123(19):3209-3214.
[25]潘景轩,朱振宇,李树浓,等.亚致死量60Coγ射线照射后小鼠骨髓造血细胞程序性死亡特性研究[J].实验血液学杂志,1996,4(2):195-199.
[26]彭瑞云,王德文,熊呈琦,等.bax及bcl-2基因表达在γ线照后小鼠骨髓造血细胞凋亡及修复中的定量变化及意义探讨[J].中国体视学与图像分析,1996,1(3):63-68.
[27]彭瑞云,王德文,熊呈琦,等.辐射诱导骨髓细胞凋亡及相关基因表达研究[J].中华放射医学与防护杂志,2001,21(1):19-20.
[28]Radford IR,Murphy TK,Radley JM,et al. Radiation response of mouse lymPhoid and myeloid cell lines PartⅡApoptotic death is shown by all lines examined[J].Int J Radiat Biol,1994,65(2):217-227.
[29]宋俊玲,杨岚,田琼,等.血小板因子4对环磷酞胺诱导化学损伤小鼠骨髓细胞的保护作用[J].第四军医大学学报,2002;23(9):803-806.
[30]Sherr CJ. The Pezcoller lecture cancer cell cycles revisited[J].Cancer Res,2001,60(14):3685-3695.
[31]Sherr CJ. Cancer cell cycles[J].Science,1996,274(5293):1672-1677.
[32]房芳,谢小燕,岳文,等.红细胞成熟脱核机制的研究[J].生物化学与生物物理进展,2013,40(8):703-710.
[2]HaslingerB,Mandl-WeberS,SitterT. Thrombin suppresses metalloprotei-nase 2 activity and increases tissue inhibitor of metalloproteinase 1syn-thesis in cultured hunman peritoneal mesothlial cells[J]. peritDialhit,2000,20(6):775-783.
[3]WongB,LummaWC,SmithAM,et al. Statins suppress THP-1cellm migration and secretion of matrix metalloproteinase by iinhibiting geranylgeranylation[J]. J Leukoc Biol,2001,69(6):959-962.
[4]NagaseH,woessnerJF. Matrix metalloProteinases[J]. J Biol Chem,1999,274:21491-21494.
[5]Pender SLF,MacDonald TT. Matrix metalloproteinases and the gut-new roles for old enzymes[J].Currrent Opinion in Pharmacology,2004,4:546-550.
[6]Szabo KA,Ablin RJ,Singh G. Matrix metalloProteinases and the immune response[J]. Clinical&Applied Lmmunology Reviews,2004,4:295-319.
[7]Van Wart HE,Birkedal-Hansen H. The cysteine switch:a principle of regulation of metalloproteinase activity with potential applicability to the entire Matrix metallo Proteinase gene family[J].Proc Natl Acad Sci USA,1990,87:5578-5582.
[8]Sheen Y,Shenk T. Relief of P53-Mediated Transcriptional Repre ssion by the Adeno-virus EIB 19-kDa Protein or the Cellular Bcl-2 Protein[J].PNAS,1994,91(19):8940-8944.
[9]OgilvyS,Metcalf D,Print Chq,et al. Constitutive Bcl-2expression through outhemato poietic compartment affects multiple lineages and enhances progenitor survival[J].Proc Natl Acad Sci,1999,96(26):14943-14948.
[10]Vairo Q,Innes KM,Adams JM,et al. Bcl-2 has a cell cycle inhibitory functionsenarahle from its enhancement of cell survival[J]. OncoQene,1996,13(7):1511-1519.
[11]Kasid U,Suy S,Dent P,et al. Activation of Raf by ionizing radiation[J]. Nature,1996,382(6594):813-816.
[12]Yang J,Liu X,Bhalla K,et al. Prevention of apoptosis by Bcl-2:release of cyto-chromec from mitochondria blocked[J].Scienee,1997,275:1129-1132.
[13]Kluck RM,Boss-Wetzel E,Green DR,et al. The release of cytochrome c from mitochondria:a primary site for Bcl-2regulation of apoptosis[J]. Science,1997,275:1132-1136.
[14]SusanSA,LorenzoHlK,Zamzami,etal.Molecular characterization of Mitochondrial apoptosis-inducing factor[J].Nature,1999,397:441-446.
[15]D Josefsen. Differential Expresoion of Bcl-2 Homologs in Human CD34+Hematopoieitc Progenitor Cells Induced to Differentiate into Erythroid or Granulocytic Cells[J].Stem Cells,2000,18(4):261.
[16]Sanz C. The expression of Bcl-x is downregulated during differentiation of human hematopoietic progenitor Cells along the granulocyte but not the monocyte/macroPhage lineage[J].Blood,1997,89(8):3199.
[17]DeliaD,Aiello A,Soligo D,et al. Bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells[J].Blood,1992,79(5):1291-1298.
[18]刘文励,孙汉英,周剑锋.复方活血汤对放射性损伤小鼠骨髓细胞Bcl-2、bax、Bcl-XL基因表达的作用[J].中华放射医学与防护杂志,1998,18:256-258.
[19]崔玉芳,丁彦青,徐菡,等.致死剂量的γ线照射后小鼠胸腺淋巴细胞的凋亡规律与bax、Bcl-2和Bcl-xl表达的关系[J].细胞与分子免疫杂志,2004,20(6):750-753.
[20]Kurokawa M,Kornbluth S. Caspases and kinases in a death grip[J].Cell,2009,138(5):838-854.
[21]杨静,王明娟,程玉,等.胃癌组织中Caspase 3、Caspase 9的表达及其临床意义[J].临床与实验病理学杂志,2016,32(10):1159-1161.
[22]Lai GH,Zhang Z,Sirica AE. Celecoxib acts in a cyclooxy genase-2 independent manner and in synergy with emodin to suppress rat cholangiocarcinoma growth in vitro through a mechanism involving enhanced Akt inactivation and increased activation of caspases-9 and-3[J].Mol Cancer Ther,2003,2(3):265-271.
[23]徐丁洁,徐洪,张碧溦,等.金匮温经汤对围绝经期大鼠卵巢凋亡因子Caspase3、Caspase9、Apaf-1蛋白表达的影响[J].中华中医药学刊,2018,36(10):2502-2504.
[24]Bratton S B,Salvesen G S. Regulation of the Apaf-1-caspase-9apoptosome[J]. J Cell Sci,2010,123(19):3209-3214.
[25]潘景轩,朱振宇,李树浓,等.亚致死量60Coγ射线照射后小鼠骨髓造血细胞程序性死亡特性研究[J].实验血液学杂志,1996,4(2):195-199.
[26]彭瑞云,王德文,熊呈琦,等.bax及bcl-2基因表达在γ线照后小鼠骨髓造血细胞凋亡及修复中的定量变化及意义探讨[J].中国体视学与图像分析,1996,1(3):63-68.
[27]彭瑞云,王德文,熊呈琦,等.辐射诱导骨髓细胞凋亡及相关基因表达研究[J].中华放射医学与防护杂志,2001,21(1):19-20.
[28]Radford IR,Murphy TK,Radley JM,et al. Radiation response of mouse lymPhoid and myeloid cell lines PartⅡApoptotic death is shown by all lines examined[J].Int J Radiat Biol,1994,65(2):217-227.
[29]宋俊玲,杨岚,田琼,等.血小板因子4对环磷酞胺诱导化学损伤小鼠骨髓细胞的保护作用[J].第四军医大学学报,2002;23(9):803-806.
[30]Sherr CJ. The Pezcoller lecture cancer cell cycles revisited[J].Cancer Res,2001,60(14):3685-3695.
[31]Sherr CJ. Cancer cell cycles[J].Science,1996,274(5293):1672-1677.
[32]房芳,谢小燕,岳文,等.红细胞成熟脱核机制的研究[J].生物化学与生物物理进展,2013,40(8):703-710.