【作者】 黄进；

【导师】 徐志伟；

【作者基本信息】 广州中医药大学 ， 中医基础理论， 2010， 博士

【摘要】 研究目的在“干细胞具先天之精的属性,是先天之精在细胞层次的存在形式”、“精血同源”等理论的指导下,本研究运用补精生血法、补精滋阴法、补精壮阳法、补气化髓生血法来探讨补肾益精法对骨髓间充质干细胞(MSCs)增殖的影响。把中医药与干细胞研究相结合,为体外培养、扩增MSCs提供新的思路,为中医药干预临床干细胞移植治疗疾病提供有力的实验依据。在药物筛选的基础上,观察对MSCs增殖确有促进作用的中药单体、有效成分及含药血清对细胞因子表达的影响,分析它们可能的作用途径,为阐释其促进MSCs增殖的作用机理提供实验依据,将补肾益精法对MSCs增殖影响的研究深入到分子水平,也为“精”的干细胞学说提供了更充分的科学依据。研究方法1.运用全骨髓贴壁法分离、培养MSCs;通过形态学、流式细胞仪检测表面标志物鉴定MSCs。2.运用MTT法筛选对MSCs增殖具有促进作用的益精中药的单体、有效成分及含药血清,并研究其最佳促增殖时间、浓度。3.用黄芪多糖(APS)及其含药血清、二苯乙烯苷(THSG)及何首乌含药血清诱导培养MSCs 72h后,RT-PCR检测不同细胞因子mRNA表达；Real-time PCR检测SCF mRNA表达；western blot检测SCF蛋白表达；ELISA法检测培养上清液SCF含量。4.用黄精含药血清诱导培养MSCs 72h后,RT-PCR检测不同细胞因子mRNA表达：Real-time PCR检测G-CSF mRNA表达；western blot检测G-CSF蛋白表达。5.用菟丝子含药血清诱导培养MSCs 72h后,RT-PCR检测不同细胞因子mRNA表达；Real-time PCR检测BMP-2 mRNA表达；western blot检测BMP-2蛋白表达。6.用巴戟天多糖诱导培养MSCs 72h后,RT-PCR检测不同细胞因子mRNA表达；Real-time PCR检测LIF. GM-CSF mRNA表达：western blot检测LIF、GM-CSF蛋白表达。7.用3月龄SD雌性大鼠双侧卵巢切除增龄3月法复制肾虚模型；选用补肾益精代表方左归丸对模型大鼠灌胃。8.观察证常组、模型组、左归丸组大鼠MSCs细胞形态、细胞表面标志物测定、生长曲线、β-半乳糖苷酶衰老细胞染色结果,比较各组大鼠MSCs增殖能力。运用RT-PCR检测正常组、模型组、左归丸组MSCs增殖过程中不同细胞因子mRNA表达。9.通过观察正常组、模型组、左归丸组大鼠MSCs在成骨细胞分化过程中ALP活性的表达、ALP染色阳性细胞率,比较各组大鼠MSCs的成骨细胞分化能力。10.通过观察正常组、模型组、左归丸组大鼠MSCs在成脂细胞分化过程中TG的表达、油红O染色阳性细胞率,比较各组大鼠MSCs的成脂细胞分化能力。结果1.建立了比较成熟的SD大鼠MSCs的分离、纯化、扩增技术平台,免疫荧光染色法和流式细胞仪检测大鼠MSCs表面标志抗原CD44、CD29表达阳性,CD45、CD34表达阴性。2.10-1mol/L THSG、10%何首乌含药血清诱导培养MSCs72h后,可促进MSCs增殖,在此过程中,均明显促进细胞SCF mRNA表达,与空白组、空白血清组相比有显著性差异。10mol/LTHSG明显促进膜结合型与可溶性两种SCF蛋白表达,10%何首乌含药血清明显促进可溶性SCF蛋白的分泌,与空白组、空白血清组相比有显著性差异。3.10%黄精含药血清诱导培养MSCs 72h后,可促进MSCs增殖,在此过程中,明显促进细胞G-CSF mRNA及G-CSF蛋白表达,与空白血清组相比有显著性差异。4. lmg/mL巴戟天多糖、10%菟丝子含药血清诱导培养MSCs 72h后,可促进MSCs增殖。在此过程中,lmg/mL巴戟天多糖明显促进细胞LIF、GM-CSF mRNA及LIF蛋白表达,10%菟丝子含药血清明显促进细胞BMP-2 mRNA表达,与空白组、空白血清组相比有显著性差异。5. lmg/mL APS、10%APS含药血清诱导培养MSCs 72h后,可促进MSCs增殖,在此过程中,均明显促进细胞SCF mRNA表达,与空白组、空白血清组相比有显著性差异。lmg/mL APS明显促进膜结合型与可溶性两种SCF蛋白表达,10%APS含药血清明显促进可溶性SCF蛋白的分泌,与空白组、空白血清组相比有显著性差异。6.正常组、模型组、左归丸组大鼠MSCs表面标志物测定的结果显示,单位体积内的原代骨髓单个核细胞中,模型组CD34阴性细胞比率明显高于正常组,有显著性差异：且CD105阳性细胞比率有低于正常组的趋势。从P3、P5代MSCs生长曲线图均可发现,模型组大鼠MSCs增殖能力较正常组、左归丸组明显降低,有显著性差异。β-半乳糖苷酶染色结果显示,模型组衰老细胞阳性率最高,明显高于正常组,有显著性差异。7.与正常组相比,模型组MSCs在增殖过程中,GM-CSF、LIF、CSF、BMP-2 mRNA表达明显降低,有显著性差异。8.骨向诱导18、21d时,模型组、左归丸组MSCs ALP表达量明显低于正常组,有显著性差异。14d开始,模型组、左归丸组大鼠MSCs的ALP染色阳性率明显低于正常组,有统计学意义。脂向诱导14d开始,模型组、左归丸组大鼠MSCs的脂肪细胞阳性率明显高于正常组,有显著性差异。结论1.采用全骨髓贴壁法可获得稳定、均质性良好的大鼠MSCs。2.10-1mol/L THSG可促进MSCs增殖,可能与其促进细胞SCF mRNA、膜结合型与可溶性两种SCF蛋白表达有关。10%何首乌含药血清可促进MSCs增殖,可能与其促进细胞SCF mRNA及可溶性SCF蛋白表达有关。3.10%黄精含药血清促进MSCs增殖的作用,可能与其促进细胞G-CSF mRNA及G-CSF蛋白表达有关。4.10%菟丝子含药血清促进MSCs增殖的作用,可能与其促进细胞BMP-2 mRNA表达有关。lmg/mL巴戟天多糖促进MSCs增殖的作用,可能与其促进细胞LIF、GM-CSF mRNA及LIF蛋白表达有关。5. lmg/mL APS可促进MSCs增殖,可能与其促进细胞SCF mRNA、膜结合型与可溶性两种SCF蛋白表达有关。10%APS含药血清可促进MSCs增殖,可能与其促进细胞SCFmRNA及可溶性SCF蛋白表达有关。6.去卵巢大鼠MSCs增殖能力减弱,可能与其表达GM-CSF、LIF、CSF、BMP-2 mRNA表达减少有关。补肾益精代表方左归丸对肾虚大鼠MSCs增殖能力的改善有一定的作用。7.去卵巢大鼠MSCs骨向分化能力降低,脂向分化能力增强。更多还原

【Abstract】 Research purpose and senseAccording to "essence and blood have the same source" and "stem cells have the same properties as congenital essence", we use these methods of nourishing essence and promoting blood, nourishing essence and fostering yin, nourishing essence and enriching yang, invigorating qi and manufacturing marrow to transforming blood, to study the effect of invigorating kidney and nourishing essence on mesenchymal stem cells (MSCs) proliferation in vitro. Combining traditional chinese medicine and stem cell research can provide new ideas for culture and amplification of MSCs in vitro, and provide strong experimental evidence for assisting clinical stem cell transplantation for treatment of diseases by Chinese medicine.On the basis of drug screening, we observed the influence of traditional Chinese medicine monomer, active ingredients and medicated serum which is useful for the promotion of MSCs on cytokine expression, to analyze their possible pathway. It can provide experimental basis for explaining its mechanism of promoting proliferation of MSCs, furnish more adequate scientific basis for the "essence" of the stem cell theory, and this study will be improved to the level of molecular.Research methods1. MSCs was isolated and purified by whole bone marrow adherent method; MSCs was identified by morphologic characteristics, surface antigens detected with flow cytometer.2. The effect of nourishing essence herbs-induced proliferation on MSCs was detected by MTT,it help us to judge which traditional Chinese medicine can promote proliferation of MSCs and to study its best time, concentration for promoting proliferation. 3. To cultivate MSCs 72h by medium of APS and its medicated serum, medium of THSG and polygonum multiflorum medicated serum, different cytokine mRNA expression were detected by RT-PCR, SCF mRNA expression was detected by Real-time PCR;SCF protein expression was detected by western blot;SCF content of supernatant was detected by ELISA.4. To cultivate MSCs 72h by polygonatum medicated serum, different cytokine mRNA expression were detected by RT-PCR, G-CSF mRNA expression was detected by Real-time PCR;G-CSF protein expression was detected by western blot.5. To cultivate MSCs 72h by dodder medicated serum,different cytokine mRNA expression were detected by RT-PCR, BMP-2 mRNA expression was detected by Real-time PCR;BMP-2 protein expression was detected by western blot.6. To cultivate MSCs 72h by medium of morinda officinalis polysaccharide, different cytokine mRNA expression were detected by RT-PCR, LIF、GM-CSF mRNA expression were detected by Real-time PCR;LIF、GM-CSF protein expression were detected by western blot.7. The weak kidney animal model was established by ovariectomy on female Sprague-Dawley (SD) rats aged 3 month;some model animal were fed with zuo gui pill.8. To investigate MSCs proliferation capacity of normal group, model group, and zuo gui pill group by comparing results of morphologic characteristics, surface antigens andβ-galactosidase staining senescent cells of differernt groups;different cytokine mRNA expression in the course of MSCs proliferation of differernt groups were detected by RT-PCR.9. To investigate differentiation capacity into osteoblasts of normal group, model group, and zuo gui pill group by compairing the expression of alkaline phosphatase (ALP) activity and the number of ALP stained positive cells.10. To investigate differentiation capacity into steatoblast of normal group, model group, and zuo gui pill group by compairing the expression of triglyeride (TG) and the number of oil red stained positive cells.Results1. We established a mature separation, purification, amplification technology platform of SD rat MSCs. The results of the surface antigens of MSCs detected by immunofluorescence and flow cytometer showed the positive expressions of CD29、CD44 and the negative expressions of CD34、CD45.2.10-1mol/L THSG、10% polygonum multiflorum medicated serum can significantly promote the proliferation of MSCs, and significantly promote SCF mRNA expression.10-1mol/L THSG significantly promote membrane binding and soluble SCF protein expression,10% polygonum multiflorum medicated serum significantly promote soluble SCF protein secretion.3.10% polygonatum medicated serum can significantly promote the proliferation of MSCs, and significantly promote G-CSF mRNA and G-CSF protein expression.4.1mg/mL morinda officinalis polysaccharide and 10% dodder medicated serum can significantly promote the proliferation of MSCs. 1mg/mL Morinda officinalis polysaccharide significantly promote LIF、GM-CSF mRNA and LIF protein expression.10% dodder medicated serum significantly promote BMP-2 mRNA expression.5.1mg/mL APS、10% APS medicated serum can significantly promote the proliferation of MSCs, and significantly promote SCF mRNA expression. 1mg/mL APS significantly promote membrane binding and soluble SCF protein expression, 10% APS medicated serum significantly promote soluble SCF protein secretion.6. Unit volume of primary bone marrow mononuclear cells, the number of CD34 negative cells in model group was significantly higher than normal group, and there was a trend that the number of CD105 positive cells in model group lower than normal group. From growth curve of P3、P5 MSCs, the proliferation capacity of MSCs in model group was significantly lower than normal group and Zuo gui pill group;β-galactosidase staining showed that the model group had the highest positive rate of aging cells, significantly higher than the normal group.7. In the proliferation process of MSCs, GM-CSF, LIF, CSF, BMP-2 mRNA expression of model group was significantly lower than normal group.8. On 18、21d of differentiating into osteoblasts, MSCs ALP expression of normal group was significantly higher than model group,Zuo gui pill group. From 14d, the rate of ALP stained positive cells of model group and Zuo gui pill group MSCs was significantly lower than normal group. From 14d of differentiating into steatoblast, the rate of oil red stained positive cells of model group and Zuo gui pill group MSCs was significantly higher than normal group.Conclusion1. The stable and uniformal MSCs could be obtained by whole bone marrow adherent method.2. 10-4mol/L THSG can promote the proliferation of MSCs, and it may be related to the promotion of SCF mRNA、membrane binding and soluble SCF protein expression.10% polygonum multiflorum medicated serum can promote the proliferation of MSCs, and it may be related to the promotion of SCF mRNA and soluble SCF protein expression.3.10% polygonatum medicated serum can promote the proliferation of MSCs, and it may be related to the promotion of G-CSF mRNA and G-CSF protein expression.4.10% dodder medicated serum can promote the proliferation of MSCs, and it may be related to the promotion of BMP-2 mRNA expression. lmg/mL morinda officinalis polysaccharide can promote the proliferation of MSCs may be related to the promotion of LIF、GM-CSF mRNA and LIF protein expression.5. lmg/mL APS can promote the proliferation of MSCs, and it may be related to the promotion of SCF mRNA、membrane binding and soluble SCF protein expression.10% APS medicated serum can promote the proliferation of MSCs, and it may be related to the promotion of SCF mRNA and soluble SCF protein expression.6. The decreased proliferation capacity of MSCs from ovariectomized rats might be associated with the decreased expression of GM-CSF、LIF、CSF、BMP-2 mRNA. To some extent, Zuo gui pill can improve proliferation capacity of weak kidney rats MSCs.7. Compared with normal rats MSCs, the ability of differentiation into osteoblast of MSCs from ovariectomized rats increased, and the ability of differentiation into adipocytes of MSCs reduced.更多还原