【作者】 梁祖建；

【导师】 庄洪；

【作者基本信息】 广州中医药大学 ， 中医骨伤科学， 2012， 博士

【摘要】 目的：在中医理论指导下,深入分析高龄原发性骨质疏松症(Primary Osteoporosis, POP)的中医临床症候特征,结合文献研究,探讨高龄POP的基本病机、治则及方药组成,研究从肝郁肾虚论治高龄POP的可行性与合理性,观察补肾调肝方治疗高龄POP的临床效果,并基于衰老理论,结合“肾藏精主骨生髓”、“肝肾同源”理论,立足Wnt/β-catenin信号转导通路,深入研究补肾调肝方对衰老骨髓间充质干细胞(Bone Mesenehymal Stem Cells,BMSCs)的影响及诱导其成骨分化的效应,探讨补肾调肝方抗骨质疏松的机制,为临床应用补肾调肝法治疗高龄POP提供新的理论和实验依据。方法：分三部分研究：一、中医基本理论研究方面从中医对POP的认识、中医对肝郁肾虚的认识、POP与肝郁肾虚的关系、POP从肝肾论治的途径、补肾调肝方的组方依据等方面,分析、探讨从肝郁肾虚论治高龄POP的可行性与合理性。二、临床研究方面(一)高龄POP患者的中医证候特征研究选取166例在广州中医药大学附属骨伤科医院门诊或住院治疗的POP患者,收集临床信息,观察高龄POP的临床主要症状,分析中医证候特征。采用全身双能X线骨密度仪测量患者腰椎前后位骨密度(Bone Mineral Density,BMD)值,研究高龄POP患者病性、病位证素与BMD水平的相关性。(二)高龄POP患者抑郁障碍的临床研究采用老年抑郁量表(Geriatric Depression Scale,GDS)进行抑郁障碍评分,调查高龄POP患者并发抑郁障碍情况,分析高龄POP患者抑郁障碍发生的原因,研究高龄POP患者中医证候与抑郁障碍的相关性。(三)补肾调肝方治疗高龄肝郁肾虚型POP的临床研究随机选取在广州中医药大学附属骨伤科医院门诊就诊的POP患者80例,随机分为治疗组和对照组,治疗组给予口服补肾调肝方；对照组给予口服福善美。两组治疗时间均为4周。观察临床治疗效果、肝郁肾虚中医症候及疼痛改善情况。三、实验研究方面(一)大鼠BMSCs的分离、培养与鉴定贴壁培养法分离纯化大鼠BMSCs,体外培养和连续传代,在倒置显微镜下连续观察细胞的形态变化,流式细胞仪检测BMSCs CD29、 CD44、CD34抗原表型。(二)D-半乳糖诱导BMSCs衰老的研究用D-半乳糖诱导制作BMSCs衰老模型,培养11天、21天做β-半乳糖苷酶染色,镜下观察细胞的形态和结构变化,确定衰老染色阳性衰老细胞数的百分比。(三)补肾调肝方对BMSCs细胞增殖的影响将BMSCs分为空白组及不同浓度补肾调肝方水提液中药组,MTT法检测不同浓度补肾调肝方水提液对BMSCs增殖的情况。(四)补肾调肝方诱导衰老BMSCs成骨分化的研究将培养成功的衰老BMSCs分为空白组、成骨诱导组和中药组,培养7d、14d后行矿化结节茜素红染色(Alizarin Red S,ARS),倒置显微镜下观察矿化骨结节形成情况。(五)补肾调肝方对衰老BMSCs SAHF结构形成的影响将培养成功的衰老BMSCs分为空白组、正常组和中药组,14天后行DAPI染色,激光共聚焦显微镜观察BMSCs SAHF结构形成情况。免疫组织化学及Western blot法检测各组BMSCs HIRA、 ASFla表达情况。(六)补肾调肝方对衰老BMSCs Wnt/β-catenin信号转导通路的影响将培养成功的衰老BMSCs随机分为空白组、成骨诱导组和中药组,7天后免疫组织化学及Western blot法检测BMSCs Wnt2、 β-catenin、 GSK3β表达情况,Western blot法检测BMSCs Phospho-S9-GSK3β (pGSK3β)表达情况。结果：一、中医基本理论研究方面1.基于“肾主骨”、“肝肾同源”理论,提出“肝郁肾虚”是高龄POP的关键病机：(1)肾虚是高龄POP核心病机。(2)肝郁是高龄POP重要病机。(3)肝郁、肾虚相互影响,互为因果。(4)筋骨并重是确立高龄POP肝郁肾虚型的基本体现。(5)肝肾同源、肝肾同治是确立高龄POP肝郁肾虚证型的内在要求。2.高龄POP属本虚标实,提出治疗的主要原则是补肾调肝,重要途径是“治用、治体、治少阴”,治用即疏肝气,治体即补肝血和养肝阴,治少阴就是填肾精、补肾阳、滋肾阴。3.根据中医理论,参考专家经验,结合临床实践,筛选中药,确立补肾调肝方(碎补15g、狗脊15g、白芍30g、柴胡12g、郁金15g、当归15g、玫瑰花12g、川楝子12g、川芎10g、白术15g、合欢皮15g、菊花15g、菖蒲15g、甘草10g)。二、临床研究方面1.高龄POP患者的主要证候特点166例高龄POP患者临床出现频率较高的中医症状是腰酸背痛、胸胁胀痛、精神抑郁、善太息、烦躁易怒、视物模糊、肢酸软痛、倦怠乏力、畏寒喜温、口苦、夜尿频数、耳鸣耳聋、面色晄白或萎黄。舌质淡或暗,舌苔少、苔白,脉沉细或沉弦。2.高龄POP患者的病位证素分布特点166例高龄POP患者病位证素属肾151例,属肝140例,属脾26例,属心10例,属肺5例。高龄POP患者组合脏腑定位在肝肾128例、脾肾15例、肝脾8例、肺肾5例、心肾3例、心脾3例、心肝4例。病位证素分布结果提示高龄POP病位主要在肝肾。3.高龄POP患者的病性证素分布特点166例高龄POP患者病性证素主要是气虚、气郁、血瘀、阴虚、阳虚。病性证素组合常见气郁血瘀、阳虚血瘀、气虚血瘀、阴阳两虚,血瘀多因气郁所致。4.高龄POP患者的证候分析综合分析高龄POP患者临床证候特点、病性、病位证素分布规律,判定高龄POP患者与肝肾密切相关,病性属本虚标实,以肝郁肾虚型最为多见。5.高龄POP患者病性、病位证素与BMD水平的相关性研究166例高龄POP患者病性、病位证素与BMD水平比较,差异无统计学意义(P>0.05),提示高龄POP患者病性、病位证素与BMD水平没有直接相关性。6.高龄POP患者抑郁障碍的发生情况166例高龄POP患者发生抑郁障碍81例,发生率为48.80%,其中,轻度抑郁31例、中度抑郁22例、重度抑郁28例。7.高龄POP患者抑郁障碍的原因分析高龄POP患者抑郁障碍发生的原因主要有：躯体疼痛、心理对疾病的承受能力的下降、担心骨折的再次发生、担心生活不能自理、增加子女负担、医疗费用增加。8.高龄POP患者中医证候与抑郁障碍的相关性研究肝郁肾虚型与抑郁障碍关系最为密切,肝肾阴虚型易有抑郁障碍发病倾向。9.补肾调肝方与福善美治疗高龄肝郁肾虚型POP的效果治疗组32例,显效19例,有效9例,无效4例,总有效率87.50%,对照组31例,显效16例,有效10例,无效5例,总有效率83.87%,2组治疗总有效率比较,差异无统计学意义(P>0.05)。10.补肾调肝方与福善美治疗高龄肝郁肾虚型POP疼痛改善情况对照组31例,显效13例,有效10例,无效8例,总有效率74.19%；治疗组32例,显效13例,有效10例,无效9例,总有效率71.88%。2组有效率比较,差异无统计学意义(P>0.05)。11.补肾调肝方与福善美治疗高龄肝郁肾虚型POP中医证候改善情况对照组31例,痊愈11例,显效9例,有效6例,无效5例,总有效率83.87%；治疗组32例,痊愈16例,显效11例,有效3例,无效2例,总有效率93.75%。2组总有效率比较,差异有显著性意义(P<0.01),说明治疗组较对照组能更有效改善中医证候。12.补肾调肝方与福善美治疗高龄肝郁肾虚型POP中医证候积分变化情况2组治疗后与治疗前比较,中医证候积分降低,差异有统计学意义(P<0.05)；治疗后2周、3周、4周,中医证候积分治疗组较对照组显著降低,差异有统计学意义(P<0.05),说明治疗组较对照组能更有效改善中医证候。三、实验研究方面1.大鼠BMSCs的分离、培养与鉴定BMSCs流式细胞仪免疫表型检测结果显示：CD29、 CD44高表达,阳性细胞比率分别为96.59%和93.86%,CD34低表达,阳性细胞比率分别为0.23%,符合BMSCs表型特征。2.D-半乳糖诱导BMSCs衰老透视电镜观察诱导组BMSCs呈现典型的衰老形态和结构变化,细胞体积稍大,细胞扁平、胀大、颗粒增多,核增大、染色深,核膜内陷,核染色质凝聚、固缩、碎裂,形成不同形状的块状；细胞质色素积聚,存在大量空泡,线粒体数量减少,体积增大或肿胀,内质网扩张；但细胞膜保持完整。正常组BMSCs核膜平整光滑,染色质分布均匀,胞浆内可见丰富的线粒体。3.衰老染色检测D-半乳糖诱导BMSCs衰老的情况BMSCs培养11天,正常组衰老率(23.97±7.89)%,诱导组衰老率(61.19±18.36)%,培养21天正常组衰老率(27.52±10.26)%,诱导组衰老率(78.81±21.73)%。D-半乳糖诱导培养11天、21天,诱导组BMSCs衰老明显,与正常组比较,差异有显著性意义(P<0.01)；D-半乳糖诱导培养21天较培养11天,BMSCs衰老显著,差异有显著性意义(P<0.01)；正常组培养21天较培养11天,BMSCs衰老不明显,差异无统计学意义(P>0.05)。说明采用D-半乳糖诱导培养能建立良好的BMSCs衰老模型。4.补肾调肝方对BMSCs细胞增殖的影响补肾调肝方水提液干预24h、48h后,与空白组比较,浓度为10mg/ml、 lmg/ml的补肾调肝方水提液明显促进BMSCs增殖,浓度为100mg/ml的补肾调肝方水提液明显抑制BMSCs增殖,浓度在0.01mg/ml-0.1mg/ml范围内的补肾调肝方水提液对BMSCs生长无明显影响。干预24h后,浓度为10mg/m1与lmg/ml的补肾调肝方水提液对BMSCs增殖的影响差异无统计学意义(P>0.05),但干预48h与干预24h比较,lOmg/ml与lmg/m浓度的补肾调肝方水提液能明显促进BMSCs增殖,且10mg/m1较lmg/ml浓度的补肾调肝方水提液更能明显促进BMSCs增殖,差异有统计学意义(P<0.05)。虽然浓度为10mg/ml的补肾调肝方水提液更能促进BMSCs增殖,但该浓度在体内较难以达到,推测其作用可能为假阳性,而浓度为lmg/ml的补肾调肝方水提液能明显促进BMSCs的增殖,因此确定将浓度为lmg/ml的补肾调肝方水提液用于下一步的实验研究。5.补肾调肝方诱导衰老BMSCs成骨分化的研究在成骨诱导剂、补肾调肝方水提液的作用下,衰老的BMSCs变宽、成多角形、多个细胞逐渐向中间聚拢,形成矿化结节。成骨诱导7d后行茜素红染色,与空白组比较,中药组、成骨诱导组钙化结节数量明显增多,差异有非常显著的统计学意义(P<0.01)。14d后,空白组少见大量橘红色矿化结节形成,而中药组、成骨诱导组可见大量橘红色矿化结节形成,但中药组的矿化结节明显多于成骨诱导组。6.激光共聚焦显微镜观察衰老BMSCs的SAHF结构形成情况空白组见大量BMSCs的SAHF结构形成,而中药组、正常组少见有SAHF的形成,3组比较,差异有统计学意义(P<0.05),说明补肾调肝方能减少衰老BMSCs的SAHF结构形成。7. HIRA、 ASFla免疫组织化学染色及Western blot法检测空白组衰老BMSCs HIRA、 ASFla的表达明显增强,而中药组及正常组表达较弱,表明HIRA、ASFla可能介导了BMSCs衰老进程,补肾调肝方可能通过下调HIRA、 ASFla的表达延缓BMSCs的衰老。8. Wnt2、 β-catenin、 GSK3β免疫组织化学染色及Western blot法检测空白组衰老BMSCs Wnt2、β-catenin的表达明显减弱,GSK3β表达明显增强,而成骨诱导组、补肾调肝方水提液中药组Wnt2、 β-catenin表达增强,GSK3β表达明显减弱,表明Wnt2、 β-catenin、 GSK3β在衰老的BMSCs成骨分化中起着重要作用,成骨诱导剂及补肾调肝方水提液通过上调Wnt2、 β-catenin表达、下调GSK3β表达促进衰老BMSCs成骨分化。9. pGSK3β表达Western blot法检测空白组衰老BMSCs pGSK3β的表达较低,而成骨诱导组及中药组表达较高,说明成骨诱导剂及补肾调肝方水提液能增加GSK3β蛋白磷酸化水平,抑制GSK3β活性,表明GSK3β磷酸化水平在衰老BMSCs成骨分化中起着重要作用。成骨诱导剂及补肾调肝方通过调控GSK3β磷酸化水平介导Wnt/β-catenin通路促进衰老BMSCs成骨分化。结论：1.高龄POP患者以肝郁肾虚证型为主,从肝肾论治高龄POP符合临床实际。2.肝郁、肾虚与衰老、POP密切相关。高龄POP患者与衰老、抑郁障碍、BMSCs老化密切相关。3.D-半乳糖具有诱导SD大鼠BMSCs衰老的作用,采用D-半乳糖诱导培养可建立BMSCs衰老模型。4.补肾调肝方能有效减轻高龄肝郁肾虚型POP患者疼痛,明显改善中医症状,显著改善功能,疗效确切,无明显毒副作用,值得临床推广应用。5.补肾调肝方促进衰老BMSCs的增殖效应与其下调HIRA.ASFla表达,减少SAHF结构形成,延缓BMSCs衰老有关。6.补肾调肝方抗骨质疏松的机制之一是通过上调Wnt2. β-Catenin表达,下调GSK3β表达,增加GSK3β蛋白磷酸化水平,抑制GSK3β活性,活化Wnt/β-catenin通路促进衰老BMSCs成骨分化。更多还原

【Abstract】 Object iveThis study aimed to (1) investigate the basic pathogenesis and therapeutic principle of elderly osteoporosis (OP);and (2) analyse the feasibility and rationality of the treatment of elderly OP of kidney deficiency and hepatic stagnation syndrome based on not only analysing the characteristics of traditional Chinese medical (TCM) syndrome but also reviewing literature related to elderly OP under the guidance of traditional Chinese medical theory; and (3) observe the clinical effect of kidney-tonifying and liver-regulating recipes;and (4) explore the mechanism of osteogenic differentiation of aging bone mesenehymal stem cells (BMSCs) induced by the recipes through regulating Wnt signaling pathway according to the theory of aging, homogeny of liver and kidney, and the latest study on the kidneys’physiological functions of storing essence, taking charge of the bone and manufacturing marrow. The assignment served for the purpose of providing new ideas and measures to prevent and cure the eldely OP.MethodsThe systemical research was made on elderly OP through three parts of theory inquiry, clinical observation and cell experiment.1. Theoretical StudiesThe literatures of elderly OP were overviewed, generalized and summarized to analysis the feasibility and rationality of the treatment of elderly OP based on diagnosis of kidney deficiency and hepatic stagnation syndrome.2. Clinical Studies (1) The study of the characteristics of TCM syndrome of elder OPIn this study we chosed the osteoporotic patients as experimental subjects.166cases of out-and in-patients suffering from OP were observed by using dual-functional X-ray bone mineral density apparatus to measure BMD, and judging the syndrome according to the TCM standard. The syndrome database of the patients were established to study the syndrome elements and syndrome combination laws.(2) The clinical study of elderly OP with depressive disorderGeriatric depression scale (GDS) was applyed to assess depressive disorder and analyze the reasons. The syndrome was judged according to the TCM standard. The scores of TCM syndrome and GDS were calculated and analysed to assess the correlativity between TCM syndrome and depressive disorder.(3) The clinical study of the effect of kidney-tonifying and liver-regulating recipes on elderly OP80osteoporotic subjects enrolled in this clinical study were randomly divided into the treatment group and the control group. The clinical symptom improvement and the scores of TCM syndrome were observed, the pain intensities were recorded, and analgesic efficiency was calculated.3. Experimental StudiesBMSCs of SD rats were isolated with adherent separation method and primarily cultured in vitro, morphologically observed under an inverted microscope, and identified by flow cytometry. BMSCs senescence were induced by D-galactose and were detected by β-gal dye staining. The aging BMSCs cultured with conditioned media (Osteo-induced), different concentrations of water extract of kidney-tonifying and liver-regulating recipes and there was nothing as a control. The SAHF of aging BMSCs pretreated with different concentrations of water extract of the recipes were imaged and analyzed by confocal laser scanning microscope.The proliferation of aging BMSCs were detected with MTT assay. The osteogenic differentiation potential of aging BMSCs, which were effected by Chinese herbs, was determined by using standard osteogenic differentiation procedures. Alizarin bordeaux staining method was used to assay the function of cell mineralization. Immunohisto-chemistry and western blot were applied to detect the expression of HIRA, ASFla, Wnt2,β-catenin, GSK33, pGSK3β of the cells. Bands were semi-quantitatively analyzed by Bio-Rad Quantity one Gel-Pro Analyzer software. Results1. Theoretical Studies(1) According to the theory of homogeny of liver and kidney and "kidney controlling bone", kidney deficiency and hepatic stagnation are the key of pathogenesis of elderly OP. Kidney deficiency is the nuclear pathogenesis. Hepatic stagnation is a major disease pathogenesis. Kidney deficiency and hepatic stagnation influence each other and interact as both cause and effect. It is essential to put a great deal of emphasis on both sinews and bones, and to supply the theory homogeny of liver and kidney in the prophylaxis and treatment of elderly osteoporosis. The conclusion establishes a theoretical framework to study the cause and the pathogenesis of POP.(2) The basic therapeutic principle for elderly OP, which attributes to primary deficiency and secondary excess, is to tonify the kidney and regulate the liver. It is extremely important to disperse liver stagnation, fill with liver blood, nourish liver-yin, add up essence to kidney, encourage kidney yang and nourish kidney-yin. This conclusion is the same as the theory framework that we have built.(3) According to TCM theory, the reference of some theories from specialists and by trial and error, we screened Chinese crude drugs to establish kidney-tonifying and liver-regulating recipes, meanwhile, instructed clinical application.2. Clinical Research(1) The main characteristics of syndrome in eldly POP patientsThe high frequency of TCM symptoms of166cases OP patients were back pain, the chest coerces bloated pain, depression, preference for sighing, irritability, blurred vision, limb weakness in pain, fatigue, chills, bitter taste, nocturia increased, tinnitus and deafness, pale or sallow complexion, pale or dark tongue, little and white tongue coating, deep and thin or deep and string pulse.(2) The distribution characteristics of the symptom factor of disease location in eldly POP patientsThe symptom factors of disease location were related to kidney in151cases, liver in140cases, spleen in26cases, heart in10cases, and lung in5cases. The location complex were1iver and kidney in128cases,1iver and spleen in15cases, spleen and kidney in8cases, lung and kidney in5cases, heart and kidney in3cases, heart and spleen in3cases, heart and liver in4cases. The finding suggested that the symptom factor of disease location in eldly POP patients were mainly related to liver and kidney.(3) The distribution characteristics of the syndrome factors of disease nature in eldly POP patientsThe syndrome factors of disease nature mainly included deficiency of qi, qi stagnation, blood stasis, deficiency of Yin, deficiency of Yang. The common syndrome combination nature of eldly POP were tagnation and blood stasis, Yin deficiency and blood stasis, Qi deficiency and blood stasis, Yin and Yang deficiency. The blood stasis was due to qi stagnation commonly. The combination of syndrome factors of disease nature in eldly POP patients mainly include hepatic stagnation and kidney deficiency, Yin deficiency of liver and kidney, Yang deficiency of spleen and kidney.(4) The syndrome combination law of eldly POPThe syndrome combination law of eldly POP was mainly featured as:hepatic stagnation and kidney deficiency.(5) Correlation study among the syndrome factors of disease nature, location and BMD level in eldly POPIt showed no statistically significant difference in the comparison of the syndrome factors of disease nature, location and BMD level(P>0.05). The finding suggested that there were no obvious correlation among the syndrome factors of disease nature, location and BMD level in eldly POP.(6) The incidence of depressive disorder in eldly POP patientsDepressive disorder occurred in81of the osteoporotic cases (48.8per cent), including31cases of mild depression,22cases of moderate depression, and28cases of severe depression.(7) Cause analysis on depressive disorder in elderly POP patientsThere were several major causes of depressive disorder in elderly POP patients. These included body pain, decreased mental and physical endurance, worried about fracture happening again, worried that they could not take care of themselves, increasing the burden to their children, and increasing medical expenses.(8) The correlation analysis between TCM syndrome and depressive disorder in elderly POP patients.Hepatic stagnation and kidney deficiency syndrome was closely related to depressive disorder. Yin deficiency of liver and kidne syndrome tended to depressive disorder easily.(9)The comparison of the clinical efficacy between kidney-tonifying and liver-regulating recipes and Fosamax treatment in eldly POP patients with hepatic stagnation and kidney deficiency syndrome32cases insisted on taking kidney-tonifying and liver-regulating recipes, among them,19cases had obvious improvement, and9certain improvement,4cases no response. The total effective rate was87.50%. In control group,31cases insisted on treatment,16cases have obvious improvement, and10certain improvement,5no response. The total effective rate was83.87%. The total effective rate had no significant difference between two groups (P>0.05).(10) The comparison of pain relief between kidney-tonifying and liver-regulating recipes and Fosamax treatment in eldly POP patients with hepatic stagnation and kidney deficiency syndromeAmong31cases in the control group,13cases had obvious improvement, and10certain improvement,8no response. The effective rate was74.19%. Among32cases in treatment group,13cases have obvious improvement, and10certain improvement,9no response. The effective rate was71.88%. The effective rate had no significant difference between two groups (P>0.05).(11) The comparison of TCM symptom between kidney-tonifying and liver-regulating recipes and Fosamax treatment in eldly POP patients with hepatic stagnation and kidney deficiency syndromeAmong31cases in the control group,11cases were cured, and9obvious improvement,6certain improvement,5no response. The total effective rate was83.87%. Among32cases in treatment group,16cases were cured, and11obvious improvement,3certain improvement,2no response. The total effective rate was93.75%. There was significant difference between two groups (P<0.05).It suggested that kidney-tonifying and liver-regulating recipes could improve TCM symptom more effectively than Fosamax.(12) The comparison of TCM syndrome score between kidney-tonifying and liver-regulating recipes and Fosamax treatment in eldly POP patients with hepatic stagnation and kidney deficiency syndromeAfter4weeks of treatment, TCM syndrome scores decreased remarkablely in two groups. There were remarkable differences in TCM syndrome score between at4,3,2weeks post-treatment in two groups (P<0.05).It showed that kidney-tonifying and liver-regulating recipes could improve TCM symptoms more effectively than Fosamax.3. Experimental Studies(1) Isolation, culture, identification of BMSCsThe morphology of cultured BMSCs was consistent with the biological characteristics of BMSCs. The flow cytometry results showed that high expression of CD44was seen with positive rates of96.59(%), and CD2993.86(%), while the low expression of CD34was found with positive rates of0.23%. The results revealed that the cultured BMSCs could meet experimental requirements.(2) BMSCs induced by D-galactose were detected by transmission electron microscopyCompared with control cells, BMSCs induced by D-galactose displayed morphological and biological changes in the cell senescence with the senescent characteristic morphological markers. There were significant difference between the two groups in cellular structure and morphology.(3) BMSCs senescence induced by D-galactose were detected by β-gal stainingAfter cultured with D-galactose for lldays,23.97±7.89%of the cells were β-gal dye masculine in the normal group, and as were significantly higher in the induced group (p<0.01). After cultured for21days,27.52±10.26%of the cells were β-gal dye masculine in the normal group, while78.81±21.73%in the induced group(p<0.01).But there were no difference between at11days and at21days in the normal group (P>0.05), to the contrary there were significant differencein the induced group(p<0.01).(4) Effect of water extract of kidney-tonifying and liver-regulating recipes on proliferation of aging BMSCsAfter cultured for24h、48h, MTT assay showed that absorbance of BMSCs treated with water extract of kidney-tonifying and liver-regulating recipes at the concentration range of lmg/ml-10mg/ml were significantly higher than that of intervented with no disposal in the blank group (p<0.01). The water extract at the concentration of100mg/ml inhibited the proliferation of BMSCs obviously. However the water extract at the concentration range of0.01mg/ml-0. lmg/ml had no significant effect on the proliferation. There were no difference between the concentration of lmg/mland10mg/ml after24hours (P>0.05). In contrast, the the concentration of10mg/ml increased BMSCs proliferation more significantly than that of lmg/ml after48hours. The above observation demonstrated that the water extract had the effect on promoting proliferation of BMSCs. But it was difficult to reach the concentration of10mg/ml in vivo, thus we concentrated on further confirmation of its effects on BMSCs proliferation at the concentration of lmg/ml.(5) The study of the osteogenic differentiation potential of aging BMSCs induced by water extract of kidney-tonifying and liver-regulating recipesAfter cultured for7d and14d, osteogenic inducer and water extract of kidney-tonifying and liver-regulating recipes promoted formation of mineralization nodes more notebally than the placebo. Significant differences were observed(P<0.01). But after cultured for14d the same were exhibited between the osteo-induced group and the water extract group (P<0.01). A strong increase of formation of mineralization nodes in the water extract groups at all examined time points showed that the water extract had the effect on promoting proliferation of BMSCs.(6) Effect of SAHF formation during BMSCs senescence on different concentration of the water extract of kidney-tonifying and liver-regulating recipes by confocal laser scanning microscopeSAHF formation was universal among BMSCs senescence, while the water extract could reduce SAHF formation. There was significant difference.(7) Effect of the water extract of kidney-tonifying and liver-regulating recipes on the HIRA、ASFla expression of BMSCsImmunohisto-chemistry and western blot measurements revealed that high expression of HIRA、ASFla were observed among aging BMSCs, while low expression among the nomal BMSCs. The water extract reduced HIRA、 ASFla expression to postpone BMSCs senescence process.(8) Effect of the water extract of kidney-tonifying and liver-regulating recipes on the Wnt2, β-catenin, GSK3β expression of BMSCsImmunohistochemistry and western blot measurements revealed that expression of Wnt2、 β-catenin in the blank group were lower than those in the osteo-induced group and in the water extract group, and the expression of GSK3β in the blank group was higher than that in the osteo-induced group and in the water extract group. It suggested the water extract of kidney-tonifying and liver-regulating recipes and the osteogenic inducer could promote proliferation, differentiation, maturation and mineralization of BMSCs in vitro by increasing the expression of Wnt2、β-catenin and reducing the expression of GSK3β.(9) Effect of the water extract of kidney-tonifying and liver-regulating recipes on the phosphorylated GSK3β protein expression of BMSCsCompared with treated BMSCs, phosphorylated GSK3β protein were reduced in the aging BMSCs, which demonstrated that the water extract of kidney-tonifying and liver-regulating recipes and the osteogenic inducer could increase phosphorylated GSK3β protein.Conclusion1. Kidney deficiency and hepatic stagnation is the main syndrome type of elderly osteoporosis. The method of tonifying the kidney and regulating the liver to treat elderly osteoporosis is logical, rational and clinically practical.2. Kidney deficiency and hepatic stagnation is closely correlated with aging and POP. The pathogenesis of elderly osteoporosis is closely correlated with aging, depression and BMSCs senescence.3. D-galactose can induce BMSCs senescence. The D-galactose induced aging model is a good one for the research of BMSCs senescence.4. Kidney-tonifying and liver-regulating recipes, with their proven efficacy and safety, could be the prospective medicine to treat elderly osteoporosis.5. Kidney-tonifying and liver-regulating recipes treat elderly osteoporosis through reducing SAHF formation via downregulating HIRA、 ASFla to postpone BMSCs senescence process.6. The cellular and molecular mechanisms of kidney-tonifying and liver-regulating recipes in treatment of elderly osteoporosis is to promote proliferation, differentiation, maturation and mineralization of BMSCs by upregulating Wnt2、β-catenin expression, downregulating GSK3β expression and increasing phosphorylated GSK3β protein via the activation of Wnt/β-catenin signaling pathway.更多还原