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1、成都中医药大学 博士学位论文 十全大补汤配方颗粒对骨髓抑制小鼠造血调控的实验研究 姓名：石娅萍 申请学位级别：博士 专业：中西医结合基础 指导教师：祝彼得 2009-05 成都中医药大学博士学位论文 1 摘 要 摘 要 目的：目的：骨髓抑制是肿瘤放、化疗的主要毒副作用，表现为外周血单系或多血细胞 的减少。临床上，中医对放、化疗所致骨髓抑制的基本治疗原则是以“扶正”为 主。 本课题拟研究十全大补汤配方颗粒对骨髓抑制小鼠造血功能的调控作用及其 可能机制。 方法：方法：本研究从造血效应、造血干/祖细胞增殖力、细胞周期、细胞周期素、细 胞周期依赖性蛋白激酶、 骨髓基质细胞、 细胞外基质、 造血生长因子
2、等方面着手， 应用细胞培养术、流式细胞术、免疫组织化学术、蛋白免疫印迹法、酶联免疫吸 附法以及半定量聚合酶链反应等技术， 较系统地研究十全大补汤配方颗粒对骨 髓抑制小鼠造血功能的调控作用及相关机制。 结果： 结果： 1 在一定剂量范围内，十全大补汤配方颗粒可明显提高骨髓抑制小鼠外周血 WBC、RBC、Hb、PLT 数量，增加骨髓有核细胞数，促进骨髓显微组织结构损伤的 修复,增加骨髓造血面积； 2 十全大补汤配方颗粒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 从造血效应方面来考察，十全大补汤配方颗粒与标准汤剂对骨髓抑制小鼠 造血功能的恢复均有促进作用，两者比较无明
3、显统计学差异； 4 十全大补汤配方颗粒可使骨髓抑制小鼠 G0/G1期细胞减少, S 期和 G2/M 期 细胞增多，增殖指数增加，同时降低骨髓细胞凋亡率。 5 十全大补汤配方颗粒可促进细胞周期调控蛋白细胞周期素 CyclinD1、 CyclinEmRNA 和细胞周期依赖性蛋白激酶 CDK2、CDK4mRNA 基因的表达，进而调控 细胞周期时程； 6 十全大补汤配方颗粒可上调肾脏 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，促进骨髓三系血 细胞造血； 7 十全大补汤配方颗粒可促进骨髓基质中 FN、LN
4、 蛋白表达，抑制 MMP-2、 MMP-7、MMP-9 蛋白表达，增强骨髓基质细胞粘附力； 成都中医药大学博士学位论文 2 结论：结论：在一定剂量范围内，十全大补汤配方颗粒能有效促进骨髓抑制小鼠造血 功能的恢复。其机制可能是：通过解除骨髓细胞的细胞周期阻滞，促进骨髓细胞 增殖；改善骨髓造血微环境，促进造血干/祖细胞的黏附和归巢；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功能。从总体造血效应方面考察，十 全大补汤配方颗粒与标准汤剂在改善骨髓抑制小鼠造血功能方面无明显差异。 关键词 关键词 十全大补汤配方颗粒 骨髓抑制小鼠 细胞周期 骨髓造血微环境 造血生长因子 成都中医药大学博士学位论
5、文 3 Abstract Objective: Myelosuppression is followed, when the cancer patients undergo radiotherapy and chemotherapy.The basic principle of treatment for myelosuppression is based on “Fuzheng“ mainly. In this study, our aim was to explore the effect of Shiquandabu Tang Granules on hematopoietic regu
6、lation and its mechanisms, and provide experiment data for the improvement form of medication. Methods: In the present study, the model of myelosuppressed mice was prepared by 60Co irradiation , cyclophosphamide injection and chloramphenicol injection. Hematopoietic progenitor cells culture, flow cy
7、tometry, immunohistochemistry(IHC), western blot method(WB), Enzyme-linked immuno-sorbent assay（ELISA）, as well as semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technologies are used to study the hematopoietic effects， proliferation of bone marrow nuclear cells, and cell
8、 cycle, cyclin, cyclin-dependent kinase, as well as bone marrow stromal cells, extracellular matrix of bone marrow hematopoietic microenvironment and other aspects. We explored systematically and deeply the effect of Shiquandabu Tang Granules on hematopoietic system and its mechanisms. Results: 1 Wi
9、thin a certain dose range, Shiquandabu Tang Granules could significantly increase the number of white blood cell(WBC), red blood cell(RBC), hemoglobin(Hb) and platelet (PLT); increase the number of bone marrow nucleated cells; increase hematopoietic tissue area in bone marrow; 2 Shiquandabu Tang Gra
10、nules could effectively improve the colony forming-unit-erythroid(CFU-E),burst colony forming-unit-erythroid (BFU-E), colony forming-unit-granulocytemacrophage(CFU-GM), colony forming-unit-megakaryocyte(CFU-Meg); 3 There has no significant difference between the Shiquandabu Tang Granules and the tra
11、ditional decoction in improving the bone marrow hematopoietic function of myelosuppressed mice; 成都中医药大学博士学位论文 4 4 Shiquandabu Tang Granules 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
12、 the proliferation of bone marrow cells. 5 Shiquandabu Tang Granules could promote gene expression of cell cycle regulatory protein (CyclinD1, CyclinE) and cyclin-dependent kinase (CDK2, CDK4); 6 Shiquandabu Tang Granules could promote gene expression of EPOmRAN in Kidney and BMC, TPOmRNA in spleen,
13、 GM-CSFmRNA in BMC, increase the level of serum EPO, TPO and GM-CSF;. 7 Shiquandabu Tang Granules could promote early hematopoietic stem / progenitor cells adhesion and homing by increasing the adhesion of bone marrow stromal cells, promoting the expression of FN, LN, and inhibiting the expression o
14、f MMP-2, MMP-7, MMP-9 in extracellular matrix of bone marrow. Conclusion: Within a certain dose range, Shiquandabu Tang Granules could promote effectively the recovery of impaired hematopoietic function in myelosuppressed mice . The main channels may be regulating cell cycle of BMC, improving the bo
15、ne marrow micro-environment, promoting the gene expression of hematopoietic growth factors. There has no significant difference between the Shiquandabu Tang Granules and the traditional decoction in improving the bone marrow hematopoietic function of myelosuppressed mice. Key words: Shiquandabu Tang
16、 Granules Myelosuppressed mice Cell cycle Hematopoietic microenvironment Hematopoietic growth factors 成都中医药大学博士学位论文 缩略词表 缩略词表 英文缩写 英 文 英文缩写 英 文 中 文 AP Ammonium persulfate AP Ammonium persulfate 过硫酸铵 Acr acrylamide Acr acrylamide 丙烯酰胺 BMC Bone marrow cell BMC Bone marrow cell 骨髓细胞 BMNC Bone marrow nucleated cell BMNC Bone marrow nucleated cell 骨髓有核细胞 BMSCs Bone marrow stromal cells BMSCs Bone marrow stromal cells 骨髓基质细胞 BFU-E Burst forming unit-erythroid BFU-E Burst for
