圣愈汤传统煎剂和配方颗粒对骨髓抑制小鼠造血调控的实验研究
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摘要
目的：如何对抗放化疗所致的骨髓抑制,是肿瘤放化治疗成功的关键。寻找一些有效且副反应少的药物来缓解骨髓抑制也是众多医务工作者一直努力的方向。本研究拟选用经典的补气益血名方圣愈汤,采用现代血液学、细胞生物学技术和方法,观察其对骨髓抑制小鼠造血的调控作用及其可能的作用机理,并将其配方颗粒与传统煎剂作对比研究,为临床应用该方提供实验室依据。
方法：采用60钴(60Co)γ射线照射和注射环磷酰胺、氯霉素复合的方法制备骨髓抑制小鼠模型,使用血细胞计数、祖细胞集落分析方法观察圣愈汤传统煎剂和配方颗粒对骨髓抑制小鼠的外周血细胞数、各系祖细胞集落产率的影响；使用石蜡切片技术观察圣愈汤对模型小鼠骨髓和脾脏组织形态结构的影响；使用流式细胞术、免疫组织化学法观察圣愈汤对模型小鼠骨髓细胞增殖周期、凋亡率及凋亡相关蛋白表达的影响；使用半定量逆转录—聚合酶链反应(RT-PCR)和酶联免疫吸附(ELISA)法观察圣愈汤对模型小鼠部分重要的造血生长因子mRNA和血清中浓度的影响；使用基质细胞培养技术、免疫组织化学术观察圣愈汤对模型小鼠造血微环境的影响。从造血干/祖细胞、造血生长因子、造血微环境等多方面,系统地、多层次地研究圣愈汤对骨髓抑制小鼠造血功能的调控作用及其可能机制,并比较其配方颗粒和传统煎剂的作用有何差异。
结果：本研究发现：
1、圣愈汤传统煎剂和配方颗粒均可提高骨髓抑制小鼠外周血WBC、RBC、Hb、PLT数量、骨髓有核细胞数。
2、圣愈汤传统煎剂和配方颗粒均可有效提高骨髓抑制小鼠粒系、红系和巨核系体外造血祖细胞培养集落产率。
3、圣愈汤传统煎剂和配方颗粒均可改善骨髓抑制小鼠骨髓和脾脏组织形态学结构,增加骨髓造血组织面积和巨核细胞数。
4、圣愈汤传统煎剂和配方颗粒均可促使骨髓抑制小鼠骨髓细胞G0/G1期细胞向S期转化,S期细胞向G2/M期转化,促进骨髓细胞增殖。
5、圣愈汤传统煎剂和配方颗粒均可通过上调Bcl-2蛋白表达,减少Bax蛋白表达,而减少骨髓抑制小鼠的骨髓有核细胞凋亡
6、圣愈汤传统煎剂和配方颗粒均可上调骨髓抑制小鼠肾脏EPOmRNA、脾脏TPOmRNA、骨髓细胞EPO和GM-CSFmRNA的表达,增加血清EPO、TPO和GM-CSF的含量。
7、圣愈汤传统煎剂和配方颗粒均可增强骨髓抑制小鼠骨髓基质细胞增殖和其粘附力,并促进骨髓基质中FN、LN的表达,以改善造血微环境。
8、圣愈汤配方颗粒与传统煎剂对骨髓抑制小鼠造血功能的重建和作用机理相似,两者比较无明显差异。
结论：本研究显示圣愈汤传统煎剂和配方颗粒均可明显促进骨髓抑制小鼠造血功能的恢复。其机制可能是：通过调控骨髓细胞的细胞周期,促进骨髓细胞的增殖；抑制骨髓细胞的凋亡；改善骨髓造血微环境、促进造血生长因子的表达进而改善骨髓三系血细胞的造血；改善脾脏组织形态结构,而促进脾脏造血。圣愈汤配方颗粒与传统煎剂在改善骨髓抑制小鼠造血功能的重建和作用机理相似,二者间无明显差异。
Objective:How to cope with the myelosuppression induced by radiotherapy and chemotherapy is the successful key for radiochemical treatment of tumor. A large number of medical workers have been working for finding some effective drug with less side effects to alleviate myelosuppression. In the present study, our aim was to observe and compare the effects of Shengyu formula granula and Shengyu traditional decoction on hematopoietic regulation and its mechanisms of myelosuppression mice by using modern haematology techniques and methodology, and to provide experiment data for the improvement form of medication.
Methods:the model of myelosuppressed mice was prepared by 60Coγirradiation, cyclophosphamide injection and chloramphenicol injection. Shengyu formula granula and Shengyu traditional decoction on peripheralblood cells, bone marrow nucleated cells, hematopoiesis progenitors colonies counts of myelosuppressed mice were investigated by blood cell count, hematopoietic progenitor cells culture; Paraffin sectioning are used to study the tissue structure of bone marrow and spleen;Flow cytometry, immunohistochemistry (IHC), semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), as well as bone marrow stromal cells culture were used to observe bone marrow cell cycle and apoptosis, bcl-2, bax, the kidney EPO mRNA, the spleen TPO mRNA, BMC GM-CSF and EPO mRNA quantities, serum EPO, GM-CSF, TPO concentrations as well as bone marrow stromal cells, extracellular matrix of bone marrow hematopoietic microenvironment and other aspects. We explored and compared systematically and deeply the effect of Shengyu formula granula and Shengyu traditional decoction on hematopoietic system of myelosuppression mice and its Mechanisms.
Results:The present research shows:
1 Shengyu traditional decoction and Shengyu formula granula could significantly increase the number of white blood cell(WBC), red blood cell(RBC), hemoglobin(Hb) and platelet (PLT) of myelosuppressed mice; increase the number of bone marrow nucleated cells.
2 Shengyu traditional decoction and Shengyu formula granula could effectively improve the colony forming-unit -erythroid(CFU-E), burst colony-forming-unit-erythroid(BFU-E), colony-forming-unit-granulocyte macrophage(CFU-GM), colony-forming-unit-megakaryocyte(CFU-Meg) of myelosuppressed mice.
3 Shengyu traditional decoction and Shengyu formula granula may improve the bone marrow and spleen of myelosuppressed mice morphological structure, increase bone marrow hematopoietic tissue area and the number of megakaryocytes.
4 Shengyu traditional decoction and Shengyu formula granula could promote bone marrow cells into the cell cycle, promote the G0/G1 phase cells into S phase, S phase cells into G2/M phase transformation, thereby promoting the proliferation of bone marrow cells.
5 Shengyu traditional decoction and Shengyu formula granula could increased expression of Bcl-2 protein, reduce the Bax protein expression, decrease the bone marrow cells apoptosis of myelosuppressed mice.
6 Shengyu traditional decoction and Shengyu formula granula could promote gene expression of Erythropoietin(EPO)mRAN in Kidney and BMC, Thrombopoietin(TPO)mRNA in spleen, Granulocyte macrophage-colony stimulating factor (GM-CSF)mRNA in BMC, increase the level of serum EPO, TPO and GM-CSF.
7 Shengyu traditional decoction and Shengyu formula granula could enhance the proliferation of bone marrow stromal cells and their adhesion, and to promote the expression of fibronectin (FN), laminin (LN) in extracellular matrix of bone marrow, to improve the bone marrow hematopoietic microenvironment.
8 There has no significant difference between the Shengyu traditional decoction and Shengyu formula granula in reconstructing the bone marrow hematopoietic function and mechanism of myelosuppressed mice. Conclusion:This study demonstrated that Shengyu traditional decoction and Shengyu formula granula could significantly promote the myelosuppressed mice of bone marrow hematopoietic function recovery. The mechanism may be:regulate bone marrow cell cycle, promote the proliferation of bone marrow cells; inhibition of apoptosis of bone marrow cells; improve the bone marrow hematopoietic microenvironment; and promote the expression of hematopoietic growth factors and thus improve the three lines blood cells of bone marrow hematopoiesis; to improve spleen morphology, thus contributing to spleen hematopoiesis. It was no significant difference that Shengyu traditional decoction and Shengyu formula granula improved the bone marrow hematopoietic function on myelosuppressed mice and reconstruction mechanism of drug.
方法：采用60钴(60Co)γ射线照射和注射环磷酰胺、氯霉素复合的方法制备骨髓抑制小鼠模型,使用血细胞计数、祖细胞集落分析方法观察圣愈汤传统煎剂和配方颗粒对骨髓抑制小鼠的外周血细胞数、各系祖细胞集落产率的影响；使用石蜡切片技术观察圣愈汤对模型小鼠骨髓和脾脏组织形态结构的影响；使用流式细胞术、免疫组织化学法观察圣愈汤对模型小鼠骨髓细胞增殖周期、凋亡率及凋亡相关蛋白表达的影响；使用半定量逆转录—聚合酶链反应(RT-PCR)和酶联免疫吸附(ELISA)法观察圣愈汤对模型小鼠部分重要的造血生长因子mRNA和血清中浓度的影响；使用基质细胞培养技术、免疫组织化学术观察圣愈汤对模型小鼠造血微环境的影响。从造血干/祖细胞、造血生长因子、造血微环境等多方面,系统地、多层次地研究圣愈汤对骨髓抑制小鼠造血功能的调控作用及其可能机制,并比较其配方颗粒和传统煎剂的作用有何差异。
结果：本研究发现：
1、圣愈汤传统煎剂和配方颗粒均可提高骨髓抑制小鼠外周血WBC、RBC、Hb、PLT数量、骨髓有核细胞数。
2、圣愈汤传统煎剂和配方颗粒均可有效提高骨髓抑制小鼠粒系、红系和巨核系体外造血祖细胞培养集落产率。
3、圣愈汤传统煎剂和配方颗粒均可改善骨髓抑制小鼠骨髓和脾脏组织形态学结构,增加骨髓造血组织面积和巨核细胞数。
4、圣愈汤传统煎剂和配方颗粒均可促使骨髓抑制小鼠骨髓细胞G0/G1期细胞向S期转化,S期细胞向G2/M期转化,促进骨髓细胞增殖。
5、圣愈汤传统煎剂和配方颗粒均可通过上调Bcl-2蛋白表达,减少Bax蛋白表达,而减少骨髓抑制小鼠的骨髓有核细胞凋亡
6、圣愈汤传统煎剂和配方颗粒均可上调骨髓抑制小鼠肾脏EPOmRNA、脾脏TPOmRNA、骨髓细胞EPO和GM-CSFmRNA的表达,增加血清EPO、TPO和GM-CSF的含量。
7、圣愈汤传统煎剂和配方颗粒均可增强骨髓抑制小鼠骨髓基质细胞增殖和其粘附力,并促进骨髓基质中FN、LN的表达,以改善造血微环境。
8、圣愈汤配方颗粒与传统煎剂对骨髓抑制小鼠造血功能的重建和作用机理相似,两者比较无明显差异。
结论：本研究显示圣愈汤传统煎剂和配方颗粒均可明显促进骨髓抑制小鼠造血功能的恢复。其机制可能是：通过调控骨髓细胞的细胞周期,促进骨髓细胞的增殖；抑制骨髓细胞的凋亡；改善骨髓造血微环境、促进造血生长因子的表达进而改善骨髓三系血细胞的造血；改善脾脏组织形态结构,而促进脾脏造血。圣愈汤配方颗粒与传统煎剂在改善骨髓抑制小鼠造血功能的重建和作用机理相似,二者间无明显差异。
Objective:How to cope with the myelosuppression induced by radiotherapy and chemotherapy is the successful key for radiochemical treatment of tumor. A large number of medical workers have been working for finding some effective drug with less side effects to alleviate myelosuppression. In the present study, our aim was to observe and compare the effects of Shengyu formula granula and Shengyu traditional decoction on hematopoietic regulation and its mechanisms of myelosuppression mice by using modern haematology techniques and methodology, and to provide experiment data for the improvement form of medication.
Methods:the model of myelosuppressed mice was prepared by 60Coγirradiation, cyclophosphamide injection and chloramphenicol injection. Shengyu formula granula and Shengyu traditional decoction on peripheralblood cells, bone marrow nucleated cells, hematopoiesis progenitors colonies counts of myelosuppressed mice were investigated by blood cell count, hematopoietic progenitor cells culture; Paraffin sectioning are used to study the tissue structure of bone marrow and spleen;Flow cytometry, immunohistochemistry (IHC), semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), as well as bone marrow stromal cells culture were used to observe bone marrow cell cycle and apoptosis, bcl-2, bax, the kidney EPO mRNA, the spleen TPO mRNA, BMC GM-CSF and EPO mRNA quantities, serum EPO, GM-CSF, TPO concentrations as well as bone marrow stromal cells, extracellular matrix of bone marrow hematopoietic microenvironment and other aspects. We explored and compared systematically and deeply the effect of Shengyu formula granula and Shengyu traditional decoction on hematopoietic system of myelosuppression mice and its Mechanisms.
Results:The present research shows:
1 Shengyu traditional decoction and Shengyu formula granula could significantly increase the number of white blood cell(WBC), red blood cell(RBC), hemoglobin(Hb) and platelet (PLT) of myelosuppressed mice; increase the number of bone marrow nucleated cells.
2 Shengyu traditional decoction and Shengyu formula granula could effectively improve the colony forming-unit -erythroid(CFU-E), burst colony-forming-unit-erythroid(BFU-E), colony-forming-unit-granulocyte macrophage(CFU-GM), colony-forming-unit-megakaryocyte(CFU-Meg) of myelosuppressed mice.
3 Shengyu traditional decoction and Shengyu formula granula may improve the bone marrow and spleen of myelosuppressed mice morphological structure, increase bone marrow hematopoietic tissue area and the number of megakaryocytes.
4 Shengyu traditional decoction and Shengyu formula granula could promote bone marrow cells into the cell cycle, promote the G0/G1 phase cells into S phase, S phase cells into G2/M phase transformation, thereby promoting the proliferation of bone marrow cells.
5 Shengyu traditional decoction and Shengyu formula granula could increased expression of Bcl-2 protein, reduce the Bax protein expression, decrease the bone marrow cells apoptosis of myelosuppressed mice.
6 Shengyu traditional decoction and Shengyu formula granula could promote gene expression of Erythropoietin(EPO)mRAN in Kidney and BMC, Thrombopoietin(TPO)mRNA in spleen, Granulocyte macrophage-colony stimulating factor (GM-CSF)mRNA in BMC, increase the level of serum EPO, TPO and GM-CSF.
7 Shengyu traditional decoction and Shengyu formula granula could enhance the proliferation of bone marrow stromal cells and their adhesion, and to promote the expression of fibronectin (FN), laminin (LN) in extracellular matrix of bone marrow, to improve the bone marrow hematopoietic microenvironment.
8 There has no significant difference between the Shengyu traditional decoction and Shengyu formula granula in reconstructing the bone marrow hematopoietic function and mechanism of myelosuppressed mice. Conclusion:This study demonstrated that Shengyu traditional decoction and Shengyu formula granula could significantly promote the myelosuppressed mice of bone marrow hematopoietic function recovery. The mechanism may be:regulate bone marrow cell cycle, promote the proliferation of bone marrow cells; inhibition of apoptosis of bone marrow cells; improve the bone marrow hematopoietic microenvironment; and promote the expression of hematopoietic growth factors and thus improve the three lines blood cells of bone marrow hematopoiesis; to improve spleen morphology, thus contributing to spleen hematopoiesis. It was no significant difference that Shengyu traditional decoction and Shengyu formula granula improved the bone marrow hematopoietic function on myelosuppressed mice and reconstruction mechanism of drug.
引文
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