补肾调肝法治疗高龄原发性骨质疏松症的理论、临床与实验研究
|
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。
Object ive
This 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.
Methods
The systemical research was made on elderly OP through three parts of theory inquiry, clinical observation and cell experiment.
1. Theoretical Studies
The 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 OP
In 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 disorder
Geriatric 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 OP
80osteoporotic 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 Studies
BMSCs 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.
Results
1. 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 patients
The 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 patients
The 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 patients
The 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 POP
The 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 POP
It 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 patients
Depressive 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 patients
There 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 syndrome
32cases 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 syndrome
Among31cases 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 syndrome
Among31cases 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 syndrome
After4weeks 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 BMSCs
The 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 microscopy
Compared 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 staining
After 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 BMSCs
After 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 recipes
After 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 microscope
SAHF 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 BMSCs
Immunohisto-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 BMSCs
Immunohistochemistry 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 BMSCs
Compared 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.
Conclusion
1. 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.
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。
Object ive
This 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.
Methods
The systemical research was made on elderly OP through three parts of theory inquiry, clinical observation and cell experiment.
1. Theoretical Studies
The 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 OP
In 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 disorder
Geriatric 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 OP
80osteoporotic 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 Studies
BMSCs 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.
Results
1. 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 patients
The 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 patients
The 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 patients
The 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 POP
The 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 POP
It 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 patients
Depressive 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 patients
There 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 syndrome
32cases 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 syndrome
Among31cases 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 syndrome
Among31cases 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 syndrome
After4weeks 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 BMSCs
The 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 microscopy
Compared 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 staining
After 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 BMSCs
After 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 recipes
After 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 microscope
SAHF 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 BMSCs
Immunohisto-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 BMSCs
Immunohistochemistry 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 BMSCs
Compared 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.
Conclusion
1. 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.
引文
[1]郭世绂,罗先正,邱贵兴.骨质疏松基础与临床[M].天津：天津科学技术出版社,2001,第版：1-3.
[2]Tilman D Rachner, Sundeep Khosla, Lorenz C Hofbauer. Osteoporosis:now and the future [J].The Lancet,2011,377 (9773):1276-1287.
[3]R Rizzoli. Management of the oldest old with osteoporosis[J].European Geriatric Medicine,2010,1 (1):15-21.
[4]石作荣.关于衰老与抗衰老的中医理论探析[J].时珍国医国药,2009,20(12)：3173-3175.
[5]陈晚娇,雷静.从肝论治绝经后骨质疏松症探讨[J].陕西中医,2011,32(3)：304-306.
[6]王攀攀,张荣华,朱晓峰,等.肝与骨质疏松症关系探析[J].时珍国医国药,2009,20(10)：2632-2633.
[7]Majumdar MK, Thiede MA, Mosca JD, et al. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells[J].J Cell Physiol, 1998,176:57-66.
[8]Weixi Zhang, Mark Hamrick, Carlos Isales, et al. Age-related changes in the osteogenic differentiation potential of BMSC[J]. Bone,2008,23 (7):1118-1128.
[9]舒晓春,刘君静,朱丹华,等.不同浓度的骨碎补总黄酮对大鼠骨髓间充质干细胞向成骨细胞分化的影响[J].中国病理生理杂志,2010,26(7)：1261-1264.
[10]谭峰,樊巧玲,王明艳,等.左归丸对SD大鼠骨髓间充质干细胞增殖的影响[J].中国实验实验方剂学杂志,2011,17(7)：145-148.
[11]David GM, Meghan EM, Merry JO, et al. Update on Wnt signaling in bone cell biology and bone disease[J]. Gene,2012,492 (1):1-18.
[12]Jia-Ling Teo, Michael Kahn. The Wnt signaling pathway in cellular proliferation and differentiation:A tale of two coactivators[J]. Advanced Drug Delivery Reviews,2010, 62 (13):1149-1155.
[13]Jennifer JW, Rachel AK, Tania MS. Wnt signaling in osteoblasts and bone diseases[J].Gene,2004,341 (27),19-39.
[14]Tetsu Akiyama. Wnt/3-catenin signaling[J]. Cytokine & Growth Factor Reviews,2000, 11 (4):273-282.
[15]George SJ. Wnt pathway:a new role in regulation of inflammation[J]. Arterioscler Thromb Vase Biol,2008; 28(3):400-402.
[16]Martina Rauner, Wolfgang Sipos, Peter Pietschmann. Age-dependent Wnt gene expression in bone and during the course of osteoblast differentiation[J]. AGE,2008; 30:273-282.
[17]任路,李浩.关于肝郁证动物模型研究现状及存在问题的思考[J].中医药学报,2006,34(4)：44-47.
[18]潘其民,陈国林,赵玉秋,等.中医肝病流行病学调查[J].中西医结合杂志,1991,11(3)：153-155.
[19]滕晶,王玉来,刘子旺.肝气郁结证的研究总结与未来发展探讨[J].中医药学刊,2006,24(12)：23-24.
[20]王睿琦,赵丽慧.以肝为中心的肝郁辨证临床意义及方法探讨[J].江苏中医药,2011,43(6)：1-3.
[21]王惠英.肝郁证治[J].河南中医,2009,29(9)：252-254.
[22]邓洋洋,郑洪新.人之衰老肾为其本-试论肾虚衰老说[J].中华中医药学刊,2007,25(12)2593-2594.
[23]黄信初.《内经》对“脑与神”的认识及后世的发挥[J].贵阳中医学院学报,2000,22(3)：5-7.
[24]李瀚旻.“肝肾同源”的理论体系[J].中医药管理杂志,2007,3：203-206.
[25]李瀚,张六通,邱幸凡.“肝肾同源于脑”与肝肾本质研究[J].中医杂志,2000,41(2)：69-71.
[26]李健伟.中医肝脏“三证”有了诊治规范[J].世界华人消化杂志,1999,7(12)：1035-1037.
[27]李瀚旻,张六通,邱幸凡,等.左归丸改善MSG-肝再生-大鼠肝肾精血亏虚证的作用机制研究[J].湖北中医学院学报,2001,3(4)：30-33.
[28]车志英,何建成,马利庄,等.肝肾阴虚证与病毒性肝炎、肝炎后肝硬化关系之探讨[J].辽宁中医杂志,2011,38(4)：632-633.
[29]丁伟伟,周富明,韦先进,等.慢性肾衰患者肝脏酶谱变化与肝肾同源的临床研究[J].浙江中医杂志,2011,46(2)：95-96.
[30]廖长秀,汪晖.从大鼠正常及肾损伤状态下肝肾氧化应激角度探讨“肝肾同源”[J].广东医学,2010,31(5)：537-539.
[31]曾民德,萧树东.肝脏与内分泌[M].北京：人民卫生出版社,1997,第一版：64-68.
[32]张喜芬,梅晓云.肝肾同源论[J].辽宁中医学院学报,2005,7(6)：578-580.
[33]雷长国,何仙童.肝郁证实质研究进展[J].湖南中医杂志,2006,22(5)：88-89.
[34]徐舒,颜贤忠,吕志平,等.肝郁证大鼠模型的建立及代谢组学的初步研究[J].中华中医药杂志,2009,24(6)：54-56.
[35]岳利峰,陈家旭,王大伟,等.逍遥散对肝郁脾虚证模型大鼠海马和杏仁核AMPA受体亚基基因表达的影响[J].北京中医药大学学报,2009,32(12)：810-814.
[36]刘建鸿,姚凝,王淳,等.肝郁证与下丘脑-腺垂体-肾上腺皮质轴和肝组织过氧化损伤的实验研究[J].中国中西医结合消化杂志,2008,16(5)：302-304.
[37]乔明琦,张惠云,陈雨振,等.肝郁证动物模型研究的理论思考[J].中国医药学报,1997,12(5)：42-44.
[38]刘文娟,张虹,高萧枫.柴胡对肝郁证中枢神经递质作用的实验研究[J].实用医药杂志,2009,26(1)：50-51.
[39]徐舒.肝郁证大鼠模型血.清代谢组学研究.博士学位论文.南方医科大学,2009,62.
[40]崔海珍,陈家旭.亚健康肝郁证尿液代谢组学研究[J].山东中医杂志,2011,30(8)：537-539.
[41]杜雅薇.疏肝解郁法对肝郁证模型大鼠的生物学基础研究.博士学位论文.北京中医药大学,2010,46.
[42]李瀚.“肝。肾同源”现代研究进展、评述与展望[J].中国中医基础医学杂志,2002,8(12)：75-76.
[43]赵刚,蔡定芳.抑郁和骨质疏松症[J].国外医学：精神病学分册,2002,29(2)：104-107.
[44]许超,肖鲁伟,童培建,等.骨质疏松症与抑郁症关系的研究进展[J].中国骨质疏松杂志,2009,15(9)：693-696.
[45]Altindag 0. Altindag A, Asoglu M, et al. Relation of cortisol levels and bone mineral density among premenopausal women with major depression[J]. Int J Clin Pract,2007,61 (3):416-420.
[46]Yirmiya R, Goshen I, Bajayo A, et al. Depression induces bone loss through stimulation of the sympathetic nervous system[J]. Proc Natl Acad Sci USA,2006,103:16876-16881.
[47]Cizza G, Primma S, Csako G. Depression as a risk factor for osteoporosis[J]. Trends Endocrinol Metab,2009,20(8):367-373.
[48]赵治友,邬亚军.骨质疏松症的中医辨证思路与治法研究[J].浙江中医药大学学报,2007,31(3)：275-277.
[49]李瀚旻,高翔.“’肾生骨髓,髓生肝”的科学内涵[J].中医杂志,2006,47(1)：6-8.
[50]Tobias Johannes de Villiers. Bone health and osteoporosis in postmenopausal women [J].Best Practice & Research Clinical Obstetrics & Gynaecology,2009,23(1):73-85.
[51]叶日乔,刘道兵,贾经汉,等.绝经后骨质疏松症患者骨密度及性激素水平与肾虚证的关系[J].中医正骨,2005,2(2)：3-5.
[52]李泉玉,刘玉槐,徐文贵,等.肾虚者骨矿含量测定的意义.白求恩医科大学学报,1992,18(3)：296-298.
[53]赵玉堂,刘凯军,李金花,等.骨矿含量与肾虚、肾主骨关系的研究[J].中国骨质疏松杂志,1996,2(3)：19-21.
[54]金珉廷,郑洪新.中医肾藏精生髓主骨理论与骨质疏松症[J].辽宁中医药大学学报,2009,11(3)：35-36.
[55]朱辉,郑洪新.骨质疏松症“从肾论治”古今研究[J].中华中医药学刊,2011,29(8)：1741-1742.
[56]王昭洪.曾涛.补肾中药治疗骨质疏松的实验研究进展[J].环球中医药,2011,4(3)：235-238.
[57]彭文芳,赵文穗.绝经后患者焦虑、抑郁状态对骨质疏松和生活质量的影响[J].中国骨质疏桦杂志.2007,13(9)：642-644.
[58]Mark Rll. Carol AH, Kerr Whit field, et al. The nuclear vitamin D receptor controls the expression of genes encoding factors which feed the "Fountain of Youth" to mediate healthful aging[.jj. The Journal of Steroid Biochemistry and Molecular Biology, 2010, 121 (2): 88-97.
[59]Silvia Z, Delphino Firrincieli. Chantal llousset, et al. Vitamin D and Lhe vitamin D receptor in liver pathophysiology[J].Clinics and Research in llepatology and Gastroenten.logy. 2011, 35 (-\~): 295-302.
[60]Kazulomo Suzuki, Hiroshi Takada, llajime Kuwnyama . Clinical examination of osteoporosis in the chronic liver disease[J]. Gastroenterology, 2003, 1214(4):384.
[61]Loria 1, Milanese C, Giusto M> et al. Bone Disorders in Patients With Chronic Liver Disease Awaiting Liver Transplantation[JJ- Transplantation Proceedings, 2010. 42 (4): 1191-1193.
[62]Nuria Guanahens, Albert Pares. Liver and bone[J]. Archives of Biochemistry and Biophysics, 2010. 503 H *>: 84-94.
[63]Hanan MA, llala KH, Salwa SE et al. Lept in, osteocalein, and hone mineral density in post hepHtitic liver cirrhosis[J].Arab Journal of Gastroenierology, 2010, 11 (3) 130-135.
[64]Urayama S, Kawakam iA, Nakashima T, et al. Effect of vitam in K2 on osteoblastnapop tosis: vitamin K2 inhibits apop totic cell death of human osteoblasts induced by fas, proteasome inhibitor, etoposide, and stauosporine[J]. J Lab ClinMed, 2000, 136(3): 181-186.
[65]WE Duncan, AR Glass, HL W ray. Estrogen regulation of the nuclear 1, 25 - dihydroxyvitam in D3 recep tor in rat liver and kidney[J]. Endocrinology, 1991, 129: 2318-2324.
[66]师彬,孙国栋,王古荣.柔肝健脾方防治骨质疏松症的理论与临床可行研究[J].中华实用中西医杂志,2009,22(8)：443-446.
[67]张荣华,陈可冀,陆大祥,等.益骨胶囊治疗绝经后骨质疏松症的临床研究[J].中国中西医结合杂志,2004,24(8)：680-684.
[68]邓洋洋,郑洪新.人之衰老肾为其本-试论肾虚衰老说[J].中华中医药学刊杂志,2007,25(12)：2593-2595.
[69]马月香,乔明琦,张惠云.论肝主疏泄对人体生理功能的调节[J].河南中医学院学报,2006,21(3)：14-16.
[70]王京存,李伟,杨清峰.补肝气疏肝郁延缓衰老的探讨[J].中华实用中西医杂志,2004,5：712-713.
[71]赵延龙,闫润红,冯玉华.“虚-瘀-衰老”学说理论与实验研究进展[J].山西中医学院学报,2010,11(4)：70-72.
[72]颜德馨,胡泉林,王平平,等.气虚血瘀是人体衰老的主要机制[J].中国医药学报,1989, 4(2)：10-12.
[73]韩景献.“三焦气化失常-衰老”相关论[J].中医杂志,2008,49(3)：200-202.
[74]李国民,成海燕,于建春,等.“三焦气化失司-衰老”学说与“肾虚衰老学说”关系初探[J].江苏中医药,2010,42(8)：5-6.
[75]张一辉.衰老相关疾病及综合征[M].北京：人民卫生出版社,2011,第一版：7-13.
[76]Michael R. Ageing:life begins at 90[J]. The New Scientist,2011,211 (2824):42-45.
[77]Rebecca Bays. Why does life speed up? The impact of cognitive aging[J]. Procedia-Social and Behavioral Sciences,2010,2 (2):2657-2660.
[78]Charles AC. Theories and Mechanisms of Aging[J]. Clinics in Geriatric Medicine,2011, 27 (4):491-506.
[79]沈自尹,黄建华,林伟,等.从整体论到系统生物学进行肾虚和衰老的研究[J].中国中西医结合杂志,2009,29(6)：548-551.
[80]周安方,郭煜晖,舒劲松,等.肾脾虚损导致衰老的机理探析[J].湖北中医杂志,2011,33(8)：23-24.
[81]王霞灵,周大桥,李延福.肝郁患者雌激素水平研究[J].湖北中医杂志,1996,18(4)：38-39.
[82]王青,王雪梅,李倩,等.痰瘀互结理论基础渊流探赜[J].云南中医学院学报,2011,34(2)：5-6,10.
[83]王春霞,许善锦,王夔.自由基在大骨节病病理过程中的作用[J].北京医科大学学报,1989,21(4)：307-310.
[84]Saeed H, Abdallah BM, Ditzel N, et al. Telomerase-deficiency-related bone loss is caused by intrinsic impairment of mesenchymal stem cell (MSC) functions and increased osteoclastogenesis due to pro-inflammatory micro-environment[J]. Bone,2010,47 (S1): S39.
[85]邢小平,王鸥,韩桂艳.代谢性骨病与免疫[J].临床内科杂志,2007,24(3)：149-152.
[86]张为杰,刘锡仪.骨代谢活动与神经内分泌系统[J].中华医学研究杂志,2005,5(4)：30-32.
[87]Meinrad Peterlik. Aging, neuroendocrine function, and osteoporosis [J]. Experimental Gerontology,1997,32 (5):577-586.
[88]Adrienne B, Matthew J, Phyllis M. The morphometry of astrocytes in the rostral preoptic area exhibits a diurnal rhythm on proestrus:relationship to the luteinizing hormone surge and effects of age[J]. Endocrinology,2003,144 (1):274-280.
[89]Hou JW, Li B, Yang ZH, et al. Functional integrity of ErbB-4/2 tyrosine kinase receptor complex in the hypothalamus is required for maintaining normal reproduction in young adult female rats[J]. Endocrinology,2002,143 (5):1901-1912.
[90]林雁龙,李钿,王伟森,等.损毁弓状核导致移植骨骨质疏松[J].中国骨质疏松杂志,2005,11(2)：178-180.
[91]蔡维望,王世立.间充质干细胞的成骨分化及对骨质疏松的影响[J].中国生物工程杂志,2012,32(4)：89-95.
[92]eixi Zhang, Mark Hamrick, Carlos Isales, et al. Age-related changes in the osteogenic differentiation potential of BMSC[J]. Bone,2008,43 (S1):S50.
[93]Mohamadreza BE, Soura M, Marzieh E. Growth Kinetics and in Vitro Aging of Mesenchymal Stem Cells Isolated From Rat Adipose Versus Bone Marrow Tissues[J]. Iranian Journal of Veterinary Surgery,2008,3 (2):9-20.
[94]Ali Mirsaidi, Karin NK, Markus Rimann, et al. Telomere length, telomerase activity and osteogenic differentiation are maintained in adipose-derived stromal cells from senile osteoporotic SAMP6 mice[J]. Cell Therapies & Tissue Engineering,2011,5 (6): 378-390.
[95]Baxter MA, Wynn RF, Jowitt SN, et al. Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion[J]. Stem Cells,2004,22 (5): 675-682.
[96]Shi S, GronthosS, Chen S, et al. Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression[J]. Nat Biotechnol,2002,20(6):587-91.
[97]陈槐卿,药蓉,韩君,等.增龄对大鼠骨髓基质细胞生长分化的影响[J].中国医学科学院学报,2003,3：244-249.
[98]杨丽,王攀攀,张荣华.去卵巢对SD大鼠MSCs多向分化能力的影响[J].中国细胞生物学学报,2012,34(1)：61-66.
[99]Nishikawa K, Nakashima T, Takeda S, et al. Maf promotes osteoblast differentiation in mice by mediating the age-related switch in mesenchymal cell differentiation[J]. Clin Invest,2010,120(10):3455-3465.
[100]Clifford JR, Julie Glowacki, John PB. The Aging Skeleton[M]. Academic Press,1999: 642.
[101]Kim KW, Chung Ha-Na, Ha Kee-Yong S, et al. Senescence of nucleus pulposus chondrocytes in human intervertebral discs. Asian Spine J,2008,2(1):1-8.
[102]Hye Bin Noh, Hee-Jin Ahn, Woo-Jung Lee, et al.The molecular signature of in vitro senescence in human mesenchymal stem cells[J]. Genes & Genomics,2010,32 (1):87-93.
[103]Patricia AM, Kristi CT, Colleen Jackson-Cook, et al. Telomerase Resets the Homeostatic Telomere Length and Prevents Telomere Dysfunction in Immortalized Human Cells[J]. DNA and Cell Biology,2004,23(5):293-300.
[104]Maria A Blasco. Telomere length, stem cells and aging[J]. Nature Chemical Biology, 2007,3 (10):640-649.
[105]Ryu E, Hong S, Kang J, et al. Identification of senescene-associated genes in human bone marrow marrow mesenchymal stem cells[J]. Biochem Biophys Res Commun,2008,371 (3):431-436.
[106]Molofsky A, Slutsky SG, Joseph NM, etal. Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during aging [J]. Nature,2006,443(7110):448-452.
[107]Offenstadt G, Moreau A, Ponsot P, etal. Hypoxemia and aging [J]. Poumon Coeur,1980, 36(1):49-53.
[108]Michael Fossel. Cells, Aging, and Human Disease[M]. Oxford University Press,2004, 51.
[109]Hung SC, Yang DM, Chang CF, et al. Immortalization without neoplastic transformation of human mesenchymal stem cells by transduction with HPV16 E6/E7 genes[J].Int J Cancer, 2004,110(3):313-319.
[110]Tsai CC, Chen CL, Liu HC, et al. Overexpression of hTERT increases stem-like properties and decreases spontaneous differentiation in human mesenchymal stem cell lines[J].J Biomed Sci,2010,17 (1):64-70.
[111]赵擎,孙颖,胡燕,等.构建永生化人肝细胞系人端粒酶反转录酶真核表达质粒[J].中国临床康得,2006,10(45)：47-51.
[112]Warren L, Manos PD, Ahfeldt T, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA[J]. Cell Stem Cell,2010,7 (5):618-630.
[113]Sungwoo Kim, Yunqing Kang, Chad A, et al. Sequential delivery of BMP-2 and IGF-1 using a chitosan gel with gelatin microspheres enhances early osteoblastic differentiation[J]. Acta Biomaterialia,2012,8(5):1768-1777.
[114]王运涛,郑启新,吴小涛,等Smad3选择性调节TGF-β 1促进大鼠骨髓间充质干细胞成骨分化的实验研究[J].华中科技大学学报(医学版),2007,5：625-629.
[115]T Naganawa, L Xiao, E Abogunde, et al. In vivo and in vitro comparison of the effects of FGF-2 null and haplo-insufficiency on bone formation in mice[J]. Biochemical and Biophysical Research Communications,2006,339 (2):490-498.
[116]John CS. A woman's journey through the reproductive, transitional and postmenopausal periods of life:Impact on cardiovascular and musculo-skeletal risk and the role of estrogen replacement[J].Maturitas,2011,70 (2):197-205.
[117]Dzmitry Shcharbin, Elzbieta Pedziwiatr, Janusz Blasiak, et al. How to study dendriplexes II:Transfection and cytotoxicity[J]. Journal of Controlled Release,2010, 141 (2):110-127.
[118]Goodwin HS,Bicknese AR,Chien SN, et al. Multilineage differentiation activity by cells isolated from umbilical cord blood:expression of bone, fat, and neural markers [J]. Biol Blood Marrow Trans plant,2001,7 (11):581-588.
[119]Walsh S, Jordan GR, Jefferiss C, et al. High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro:relevance to glucocorticoid-induced osteoporosis[J]. Rheumatology (Oxford),2001,40 (1):74-83.
[120]Otsuka E, Yamaguchi A, Hirose S, et al. Characterization of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid[J]. Am J Physiol, 1999,277(1):132-138.
[121]林建华,修忠标,吴朝阳,等.鹿茸多肽对兔骨髓间质干细胞体外增殖的影响[J].中华实验外科杂志,2005,7(7)：827-830.
[122]刘钰瑜,姚卫民,艾春媚,等.大黄素对体外大鼠骨髓基质细胞向成骨细胞方向分化的影响[J].中国临床药理学与治疗学,2005,10(2)：191-195.
[123]马慧萍贾正平张汝学,等.淫羊藿总黄酮含药血清促进骨髓间充质干细胞增殖与成骨性分化[J].中国骨质疏松杂志,2004,10(4)：420-422,428.
[124]翟远坤,葛宝丰,陈克明,等.淫羊藿苷与其代谢产物淫羊藿次苷Ⅱ对骨髓间充质干细胞成骨性分化影响的比较研究[J].中药材,2010,33(12)：1896-1900.
[125]廖清船,肖洲生,秦艳芳,等.植物雌激素金雀异黄酮通过P38MAPK通路促进骨髓间充质干细胞向成骨细胞分化[J].中国药理学通报,2006,22(6)：683-687.
[126]Tetsu Akiyama. Wnt/β-catenin signal ing [J]. Cytokine & Growth Factor Reviews,2000, 11 (4):273-282.
[127]Akira Kikuchi, Hideki Yamamoto, Akira Sato. Selective activation mechanisms of Wnt signaling pathways [J]. Trends in Cell Biology,2009,19 (3):119-129.
[128]Jia-Ling Teo, Michael Kahn. The Wnt signaling pathway in cellular proliferation and differentiation:A tale of two coactivators[J]. Advanced Drug Delivery Reviews,2010, 62 (12):1149-1155.
[129]Ivan IPacheco, John MacLeod 385 Extracellular Calcium-Sensing Receptor (CaSR) Mediated Production of DKK-1 and Low Density Lipoprotein Receptor-Related Protein 6 (LRP6) Inhibits Defective Wnt Signaling[J]. Gastroenterology,2008,134 (4):51-52.
[130]卞琴,沈自尹,王拥军.骨髓间充质干细胞在中医理论中的归属[J].中国中医基础医学杂志,2011,17(7)：794-797.
[131]Gaur TLengner CJ, Hovhannisyan H, et al. Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression[J].J Biol Chem,2005,280(39):33132-33140.
[132]Bennett CN, Longo KA, Wright WS, et al. Regulation of osteoblastogenesis and bone mass by Wntl0b[J]. Proc NatI Acad Scj USA,2005,102(9):3324-3329.
[133]Tripti Gaur, Christopher JL, Hayk Hovhannisyan, et al. Canonical WNT Signaling Promotes Osteogenesis by Directly Stimulating Runx2 Gene Expression[J]. Journal of Biological Chemistry,2005,280 (39):33132-33140.
[134]Chi Zhang, Kyucheol Cho, Yehong Huang, et al. Inhibition of Wnt signaling by the osteoblast-specific transcription factor Osterix[J]. PNAS,2008,105 (19):6936-6941.
[135]Komori T. Regulation of osteoblast differentiation by transcription factors[J]. J Cell Biochem,2006,99(5):1233-1239.
[136]David GM, Meghan McGee-Lawrence, Merry JO, et al. Update on Wnt signaling in bone cell biology and bone disease[J].Gene,2012,492 (1):1-18.
[137]Maria Almeida, Li Han, Teresita Bellido, et al. Wnt Proteins Prevent Apoptosis of Both Uncommitted Osteoblast Progenitors and Differentiated Osteoblasts by Catenin-dependentand-independentSignalingCascades Involving Src/ERK and Phosphatidylinositol 3-Kinase/AKT[J]. Journal of Biological Chemistry,2006,280 (50): 41342-41351.
[138]Dianqing Wu, Weijun Pan. GSK3:a multifaceted kinase in Wnt signaling[J]. Trends in Biochemical Sciences,2010,35 (3):161-168.
[139]Kang S, Bennett CN, Gerin I, et al. Wnt Signaling Stimulates Osteoblastogenesis of Mesenchymal Precursors by Suppressing CCAAT/Enhancer-binding Protein a and Peroxisome Proliferator-activated Receptor γ [J].J Biol Chem,2007,282(19):14515-14524.
[140]Bennett CN, Ouyang H, Ma YL, et al. Wnt10b increases postnatal bone formation by enhancing osteoblast differentiation[J]. J Bone Miner Res,2007,22(12):1924-32.
[141]Gonzalez-Sancho JM, Brennan KR, Castelo-Soccio LA, et al. Wnt Proteins Induce Dishevelled Phosphorylation via an LRP5/6-Independent Mechanism, Irrespective of Their Ability To Stabilize β-Catenin[J]. Mol Cell Biol,2004,24(11):4757-4768.
[142]李萍华,刘钰瑜,崔燎.骨髓间充质干细胞成脂和成骨分化过程中Wnt信号通路的调控效应[J].中国组织丁程研究与临床,2010,14(10)：1749-1754.
[143]侯秋科,吴静,易香华,等.龟板提取物上调维生素D受体表达促骨髓间充质干细胞向成骨分化[J].中草药,2010,41(4)：607-609.
[144]Tang DZ, Hou W, Zhou Q, et al. Osthole Stimulates Osteoblast Differentiation and Bone Formation by Activation of β-Catenin-BMP Signaling[J]. J Bone Miner Res,2010, 25(6):1234-1245.
[145]卞琴,刘书芬,黄建华,等.3种补肾中药有效成分对去卵巢骨质疏松大鼠骨髓间充质干细胞的调控作用[J].中华中医药杂志,2011,5：889-893.
[146]Zhao J, Ohba S, Shinkai M, et al. Icariin induces osteogenic differentiation in vitro in a BMP-and Runx2-dependent manner [J]. Biochem Biophys Res Commun,2008,369(2):444-448.
[147]刘海江,王小平,林娟,等.淫羊藿苷和黄芪苷Ⅰ对骨髓基质细胞增殖及成骨能力的影响[J]. 中药材,2006,29(10)：1062-1065.
[148]田李军,苏健,杨慧,等.补肾柔肝法防治卵巢早衰患者骨质疏松症的临床观察[J].河北中医,2006,28(12)：888-890.
[149]张洪,魏之玉,崔德芝.补肾疏肝健脾方调节性腺轴治疗老年骨质疏松症[J].中国中医药信息杂志,1999,6(4)：43-44.
[150]刘忠厚,杨定焯,朱汉民,等.中国人骨质疏松症建议诊断标准(第2稿)[J].中国骨质疏松杂志,2000,6(1)：1-4.
[151]国家技术监督局.中华人民共和国国家标准(GB/16751.2-1997)中医临床诊疗术语证候部分[M].北京：中国标准出版社,1997,第一版：10-41.
[152]Lenore Kurlowicz. The geriatric depression scale[J]. Home Care Provider,2000,5 (2):76-77.
[153]郑筱萸.中药新药临床研究指导原则[M].北京：中国医药科技出版社,2002,第一版：210-296.
[154]Bird SB, Dickson EW. Clinically significant changes in pain along the visual analog scale Original[J]. Annals of Emergency Medicine,2001,38 (6):639-643.
[155]国家中医药管理局.中华人民共和国中医药行业标准·中医病症诊断疗效标准[M].南京：南京大学出版社,1994,第一版：10.
[156]徐国柱,蔡志基.镇痛药临床评价方法研究[J].中国新药杂志,1995,4(4)：20-22.
[157]徐进秀,焦安钦.中医证候规范化研究的思路与设想[J].中国中医基础医学杂志,2005,11(4)：261-265.
[158]盛彤,杨惠民,田金洲,等.老年性骨量减少的证候特征及其与BMD的相关性研究[J].北京中医药大学学报(中医临床版),2006,13(6)：15-18.
[159]孙江波,李智雄,刘绪银,等.骨质疏松症中医证候与骨密度水平的相关性研究[J].中医临床研究,2011,3(5)：12-14.
[160]廖怀章,孙江波,刘绪银.骨质疏松症中医定性证候与骨密度水平的相关性研究[J].湖南中医杂志,2010,26(5)：51-53.
[16l]Copeland JR.Beekman T,Dewey ME, et al. Depression in Europe Geographical Distribution Among Older People[J]. Brit J Psychiatry,1999,174:312-321.
[162]Irwin MR, Miller AH. Depressive disorders and immunity:20 years of progress and discovery[J]. Brain Behav Immun,2007,21:374-383.
[163]蔡焯基.抑郁症临床与基础[M].北京：科学出版社,1997,第一版：1.
[164]Wong SY, Lau EM, Lynn H, et al. Depression and bone mineral density.is there a relationship in elderly Asian men? Results from Mr. Os (Hong Kong)[J]. Osteoporos Int, 2005,16:610-615.
[165]Konstantynow icz J, KadzielaOlech H, Kaczmarski M, et al. Depression in anorexia nervosa:a risk factor for osteoporosis [J]. J Clin Endocrinol Metab,2005,90:5382-5385.
[166]齐璇.绝经后骨质疏松症的抑郁状态及心理干预治疗[J].中国临床康复,2002,6(23)：1950-1951.
[167]彭文芳,赵文穗.绝经后患者焦虑、抑郁状态对骨质疏松和生活质量的影响[J].中国骨质疏松杂志,2007,13(9)：642-644.
[168]Giovanni Cizza. Svetlana Primma, Gyorgy Csako. Depression as a risk factor for osteoporosis Original[J]. Trends in Endocrinology & Metabolism,2009,20 (8):367-373.
[169]Reginster JY, Deroisy R, Paul I, et al. Depressive vulnerabil ity is not an independent risk factor for osteoporosis in postmenopausal women[J]. Maturitas,1999,33(2):133-137.
[170]Williams LJ, Pasco JA, Jacka FN, et al. Depression and bone metabolism. Psychother Psychosom,2009,78(1):16-25.
[171]Yirmiya R, Inbal Goshen, Alon Bajayo, et al. Depression induces bone loss through stimulation of the sympathetic nervous system[J]. PNAS,2006,103 (45):16876-16881.
[172]Yirmiya R, Bab I. Major depression is a risk factor for low bone mineral density: A meta-analysis[J]. Biological Psychiatry,2009,66:423-32.
[173]Massimo Cocchi, Lucio Tonello, Fabio Gabrielli, et al. Depression, osteoporosis, serotonin and cell membrane viscosity between biology and philosophical anthropology [J]. Ann Gen Psychiatry,2011,10 (9):1-7.
[174]邵大清.绝经后骨质疏松症患者应重视抑郁症状的评估.浙江省医学会-浙江省医学会麻醉学术年会,2007,274-275.
[175]Coelho R, Silva C, Maia A, et al. Bone mineral density and depression:a community study in women[J]. Psychosom Res,1999,46(1):29-35.
[176]刘存斌.骨质疏松症中医研究进展[J].中医临床研究,2011,3(7)：16-17.
[177]黄平,王会仍.骨质疏松症骨密度测定与中医辨证分型[J].浙江中西医杂志,1999,9(1)：21-22.
[178]魏之玉,张洪,朱振铎,等.196例原发性骨质疏松症辨证分析[J].山东中医学院学报,1996,1：30-31.
[179]张学娅,许东云,张颖,等.从“肝肾同源”论探讨骨质疏松症病因病机及其治疗原则[J].辽宁中医杂志,2011,38(12)：2362-2363.
[180]张荣华,丘和明.中医防治退行性骨质疏松症用药分析[J].中医药学报,1997,25(4)：30-31.
[181]杨帆,徐妙容,杨妙馥,等.中药治疗骨质疏松症的用药规律[J].中国临床康复,2005,9(31)：203-205.
[182]黄连芳,吴铁,谢华,等.何首鸟煎剂对去卵巢大鼠骨质丢失的防治作用[J].中国老年学杂志,2005,25(6)：709-710.
[183]陈象青,王钦茂,方华武,等.白芍总苷和当归提取物对小鼠免疫性肝损伤的保护作用[J]. 承德医学院学报,2004,21(1)：8-10.
[184]郝习,赵明耀.枸杞多糖对树突状细胞的成熟及免疫学功能的影响[J].中医研究,2012,23(17)：24-27.
[185]高成芬.培补肝肾为主治疗老年骨质疏松症临床观察[J].现代康复,2001,5(6)：91.
[186]章薇,金华,袁静.养血.调肝方防治绝经后骨质疏松症的研究思路[J].中国中医骨伤科杂志,2005,13(3)：52-53.
[187]Rugang Zhang, Maxim V, Xiaofen Ye, et al. Formation of MacroH2A-Containing Senescence-Associated Heterochromatin Foci and Senescence Driven by ASFla and HIRA[J]. Development Cell,2005,8:19-30.
[188]肖建德.实用骨质疏松学[M].北京：科学出版社,2004,第一版：3-5.
[189]江军亮.骨质疏松症的中医药治疗进展[J].陕西中医,2012,33(1)：119-121.
[190]代平,詹瑞森,王卫国.骨质疏松患者体外培养BM SCs的生物学特性[J].医学临床研究,2007,12(24)：2019-2023.
[191]Bei Chen, Yi Zhong, Wei Peng, et al. Age-related changes in the central auditory system:Comparison of d-galactose-induced aging rats and naturally aging rats Original [J]. Brain Research,2010,1344 (16):43-53.
[192]裴凌鹏,董福慧,惠伯棣.D-半乳糖致衰老作用对大鼠骨质的影响[J].中医正骨,2007,19(3)：1-3.
[193]邢玉芝,杨玲麟,林洪,等.D-半乳糖对大鼠骨髓间充质干细胞拟衰老作用的实验研究[J].中国修复重建外科杂志,2006,20(4)：475-479.
[194]张进,徐志伟,丁富平.“肾藏精”的现代实质新理论[J].世界科学技术-中医药现代化,2010,12(4)：550-552.
[195]邓元江,易受乡,严洁.细胞信号转导理论在中医药研究中的应用[J].中国中医药信息杂志,2004；11(9)：836-839.
[196]陈希,梁祖建,邵敏,等.补肾健脾活血方对骨质疏松症信号转导基因表达的调控作用[J].新中医,2008,40(3)：60-62.
[2]Tilman D Rachner, Sundeep Khosla, Lorenz C Hofbauer. Osteoporosis:now and the future [J].The Lancet,2011,377 (9773):1276-1287.
[3]R Rizzoli. Management of the oldest old with osteoporosis[J].European Geriatric Medicine,2010,1 (1):15-21.
[4]石作荣.关于衰老与抗衰老的中医理论探析[J].时珍国医国药,2009,20(12)：3173-3175.
[5]陈晚娇,雷静.从肝论治绝经后骨质疏松症探讨[J].陕西中医,2011,32(3)：304-306.
[6]王攀攀,张荣华,朱晓峰,等.肝与骨质疏松症关系探析[J].时珍国医国药,2009,20(10)：2632-2633.
[7]Majumdar MK, Thiede MA, Mosca JD, et al. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells[J].J Cell Physiol, 1998,176:57-66.
[8]Weixi Zhang, Mark Hamrick, Carlos Isales, et al. Age-related changes in the osteogenic differentiation potential of BMSC[J]. Bone,2008,23 (7):1118-1128.
[9]舒晓春,刘君静,朱丹华,等.不同浓度的骨碎补总黄酮对大鼠骨髓间充质干细胞向成骨细胞分化的影响[J].中国病理生理杂志,2010,26(7)：1261-1264.
[10]谭峰,樊巧玲,王明艳,等.左归丸对SD大鼠骨髓间充质干细胞增殖的影响[J].中国实验实验方剂学杂志,2011,17(7)：145-148.
[11]David GM, Meghan EM, Merry JO, et al. Update on Wnt signaling in bone cell biology and bone disease[J]. Gene,2012,492 (1):1-18.
[12]Jia-Ling Teo, Michael Kahn. The Wnt signaling pathway in cellular proliferation and differentiation:A tale of two coactivators[J]. Advanced Drug Delivery Reviews,2010, 62 (13):1149-1155.
[13]Jennifer JW, Rachel AK, Tania MS. Wnt signaling in osteoblasts and bone diseases[J].Gene,2004,341 (27),19-39.
[14]Tetsu Akiyama. Wnt/3-catenin signaling[J]. Cytokine & Growth Factor Reviews,2000, 11 (4):273-282.
[15]George SJ. Wnt pathway:a new role in regulation of inflammation[J]. Arterioscler Thromb Vase Biol,2008; 28(3):400-402.
[16]Martina Rauner, Wolfgang Sipos, Peter Pietschmann. Age-dependent Wnt gene expression in bone and during the course of osteoblast differentiation[J]. AGE,2008; 30:273-282.
[17]任路,李浩.关于肝郁证动物模型研究现状及存在问题的思考[J].中医药学报,2006,34(4)：44-47.
[18]潘其民,陈国林,赵玉秋,等.中医肝病流行病学调查[J].中西医结合杂志,1991,11(3)：153-155.
[19]滕晶,王玉来,刘子旺.肝气郁结证的研究总结与未来发展探讨[J].中医药学刊,2006,24(12)：23-24.
[20]王睿琦,赵丽慧.以肝为中心的肝郁辨证临床意义及方法探讨[J].江苏中医药,2011,43(6)：1-3.
[21]王惠英.肝郁证治[J].河南中医,2009,29(9)：252-254.
[22]邓洋洋,郑洪新.人之衰老肾为其本-试论肾虚衰老说[J].中华中医药学刊,2007,25(12)2593-2594.
[23]黄信初.《内经》对“脑与神”的认识及后世的发挥[J].贵阳中医学院学报,2000,22(3)：5-7.
[24]李瀚旻.“肝肾同源”的理论体系[J].中医药管理杂志,2007,3：203-206.
[25]李瀚,张六通,邱幸凡.“肝肾同源于脑”与肝肾本质研究[J].中医杂志,2000,41(2)：69-71.
[26]李健伟.中医肝脏“三证”有了诊治规范[J].世界华人消化杂志,1999,7(12)：1035-1037.
[27]李瀚旻,张六通,邱幸凡,等.左归丸改善MSG-肝再生-大鼠肝肾精血亏虚证的作用机制研究[J].湖北中医学院学报,2001,3(4)：30-33.
[28]车志英,何建成,马利庄,等.肝肾阴虚证与病毒性肝炎、肝炎后肝硬化关系之探讨[J].辽宁中医杂志,2011,38(4)：632-633.
[29]丁伟伟,周富明,韦先进,等.慢性肾衰患者肝脏酶谱变化与肝肾同源的临床研究[J].浙江中医杂志,2011,46(2)：95-96.
[30]廖长秀,汪晖.从大鼠正常及肾损伤状态下肝肾氧化应激角度探讨“肝肾同源”[J].广东医学,2010,31(5)：537-539.
[31]曾民德,萧树东.肝脏与内分泌[M].北京：人民卫生出版社,1997,第一版：64-68.
[32]张喜芬,梅晓云.肝肾同源论[J].辽宁中医学院学报,2005,7(6)：578-580.
[33]雷长国,何仙童.肝郁证实质研究进展[J].湖南中医杂志,2006,22(5)：88-89.
[34]徐舒,颜贤忠,吕志平,等.肝郁证大鼠模型的建立及代谢组学的初步研究[J].中华中医药杂志,2009,24(6)：54-56.
[35]岳利峰,陈家旭,王大伟,等.逍遥散对肝郁脾虚证模型大鼠海马和杏仁核AMPA受体亚基基因表达的影响[J].北京中医药大学学报,2009,32(12)：810-814.
[36]刘建鸿,姚凝,王淳,等.肝郁证与下丘脑-腺垂体-肾上腺皮质轴和肝组织过氧化损伤的实验研究[J].中国中西医结合消化杂志,2008,16(5)：302-304.
[37]乔明琦,张惠云,陈雨振,等.肝郁证动物模型研究的理论思考[J].中国医药学报,1997,12(5)：42-44.
[38]刘文娟,张虹,高萧枫.柴胡对肝郁证中枢神经递质作用的实验研究[J].实用医药杂志,2009,26(1)：50-51.
[39]徐舒.肝郁证大鼠模型血.清代谢组学研究.博士学位论文.南方医科大学,2009,62.
[40]崔海珍,陈家旭.亚健康肝郁证尿液代谢组学研究[J].山东中医杂志,2011,30(8)：537-539.
[41]杜雅薇.疏肝解郁法对肝郁证模型大鼠的生物学基础研究.博士学位论文.北京中医药大学,2010,46.
[42]李瀚.“肝。肾同源”现代研究进展、评述与展望[J].中国中医基础医学杂志,2002,8(12)：75-76.
[43]赵刚,蔡定芳.抑郁和骨质疏松症[J].国外医学：精神病学分册,2002,29(2)：104-107.
[44]许超,肖鲁伟,童培建,等.骨质疏松症与抑郁症关系的研究进展[J].中国骨质疏松杂志,2009,15(9)：693-696.
[45]Altindag 0. Altindag A, Asoglu M, et al. Relation of cortisol levels and bone mineral density among premenopausal women with major depression[J]. Int J Clin Pract,2007,61 (3):416-420.
[46]Yirmiya R, Goshen I, Bajayo A, et al. Depression induces bone loss through stimulation of the sympathetic nervous system[J]. Proc Natl Acad Sci USA,2006,103:16876-16881.
[47]Cizza G, Primma S, Csako G. Depression as a risk factor for osteoporosis[J]. Trends Endocrinol Metab,2009,20(8):367-373.
[48]赵治友,邬亚军.骨质疏松症的中医辨证思路与治法研究[J].浙江中医药大学学报,2007,31(3)：275-277.
[49]李瀚旻,高翔.“’肾生骨髓,髓生肝”的科学内涵[J].中医杂志,2006,47(1)：6-8.
[50]Tobias Johannes de Villiers. Bone health and osteoporosis in postmenopausal women [J].Best Practice & Research Clinical Obstetrics & Gynaecology,2009,23(1):73-85.
[51]叶日乔,刘道兵,贾经汉,等.绝经后骨质疏松症患者骨密度及性激素水平与肾虚证的关系[J].中医正骨,2005,2(2)：3-5.
[52]李泉玉,刘玉槐,徐文贵,等.肾虚者骨矿含量测定的意义.白求恩医科大学学报,1992,18(3)：296-298.
[53]赵玉堂,刘凯军,李金花,等.骨矿含量与肾虚、肾主骨关系的研究[J].中国骨质疏松杂志,1996,2(3)：19-21.
[54]金珉廷,郑洪新.中医肾藏精生髓主骨理论与骨质疏松症[J].辽宁中医药大学学报,2009,11(3)：35-36.
[55]朱辉,郑洪新.骨质疏松症“从肾论治”古今研究[J].中华中医药学刊,2011,29(8)：1741-1742.
[56]王昭洪.曾涛.补肾中药治疗骨质疏松的实验研究进展[J].环球中医药,2011,4(3)：235-238.
[57]彭文芳,赵文穗.绝经后患者焦虑、抑郁状态对骨质疏松和生活质量的影响[J].中国骨质疏桦杂志.2007,13(9)：642-644.
[58]Mark Rll. Carol AH, Kerr Whit field, et al. The nuclear vitamin D receptor controls the expression of genes encoding factors which feed the "Fountain of Youth" to mediate healthful aging[.jj. The Journal of Steroid Biochemistry and Molecular Biology, 2010, 121 (2): 88-97.
[59]Silvia Z, Delphino Firrincieli. Chantal llousset, et al. Vitamin D and Lhe vitamin D receptor in liver pathophysiology[J].Clinics and Research in llepatology and Gastroenten.logy. 2011, 35 (-\~): 295-302.
[60]Kazulomo Suzuki, Hiroshi Takada, llajime Kuwnyama . Clinical examination of osteoporosis in the chronic liver disease[J]. Gastroenterology, 2003, 1214(4):384.
[61]Loria 1, Milanese C, Giusto M> et al. Bone Disorders in Patients With Chronic Liver Disease Awaiting Liver Transplantation[JJ- Transplantation Proceedings, 2010. 42 (4): 1191-1193.
[62]Nuria Guanahens, Albert Pares. Liver and bone[J]. Archives of Biochemistry and Biophysics, 2010. 503 H *>: 84-94.
[63]Hanan MA, llala KH, Salwa SE et al. Lept in, osteocalein, and hone mineral density in post hepHtitic liver cirrhosis[J].Arab Journal of Gastroenierology, 2010, 11 (3) 130-135.
[64]Urayama S, Kawakam iA, Nakashima T, et al. Effect of vitam in K2 on osteoblastnapop tosis: vitamin K2 inhibits apop totic cell death of human osteoblasts induced by fas, proteasome inhibitor, etoposide, and stauosporine[J]. J Lab ClinMed, 2000, 136(3): 181-186.
[65]WE Duncan, AR Glass, HL W ray. Estrogen regulation of the nuclear 1, 25 - dihydroxyvitam in D3 recep tor in rat liver and kidney[J]. Endocrinology, 1991, 129: 2318-2324.
[66]师彬,孙国栋,王古荣.柔肝健脾方防治骨质疏松症的理论与临床可行研究[J].中华实用中西医杂志,2009,22(8)：443-446.
[67]张荣华,陈可冀,陆大祥,等.益骨胶囊治疗绝经后骨质疏松症的临床研究[J].中国中西医结合杂志,2004,24(8)：680-684.
[68]邓洋洋,郑洪新.人之衰老肾为其本-试论肾虚衰老说[J].中华中医药学刊杂志,2007,25(12)：2593-2595.
[69]马月香,乔明琦,张惠云.论肝主疏泄对人体生理功能的调节[J].河南中医学院学报,2006,21(3)：14-16.
[70]王京存,李伟,杨清峰.补肝气疏肝郁延缓衰老的探讨[J].中华实用中西医杂志,2004,5：712-713.
[71]赵延龙,闫润红,冯玉华.“虚-瘀-衰老”学说理论与实验研究进展[J].山西中医学院学报,2010,11(4)：70-72.
[72]颜德馨,胡泉林,王平平,等.气虚血瘀是人体衰老的主要机制[J].中国医药学报,1989, 4(2)：10-12.
[73]韩景献.“三焦气化失常-衰老”相关论[J].中医杂志,2008,49(3)：200-202.
[74]李国民,成海燕,于建春,等.“三焦气化失司-衰老”学说与“肾虚衰老学说”关系初探[J].江苏中医药,2010,42(8)：5-6.
[75]张一辉.衰老相关疾病及综合征[M].北京：人民卫生出版社,2011,第一版：7-13.
[76]Michael R. Ageing:life begins at 90[J]. The New Scientist,2011,211 (2824):42-45.
[77]Rebecca Bays. Why does life speed up? The impact of cognitive aging[J]. Procedia-Social and Behavioral Sciences,2010,2 (2):2657-2660.
[78]Charles AC. Theories and Mechanisms of Aging[J]. Clinics in Geriatric Medicine,2011, 27 (4):491-506.
[79]沈自尹,黄建华,林伟,等.从整体论到系统生物学进行肾虚和衰老的研究[J].中国中西医结合杂志,2009,29(6)：548-551.
[80]周安方,郭煜晖,舒劲松,等.肾脾虚损导致衰老的机理探析[J].湖北中医杂志,2011,33(8)：23-24.
[81]王霞灵,周大桥,李延福.肝郁患者雌激素水平研究[J].湖北中医杂志,1996,18(4)：38-39.
[82]王青,王雪梅,李倩,等.痰瘀互结理论基础渊流探赜[J].云南中医学院学报,2011,34(2)：5-6,10.
[83]王春霞,许善锦,王夔.自由基在大骨节病病理过程中的作用[J].北京医科大学学报,1989,21(4)：307-310.
[84]Saeed H, Abdallah BM, Ditzel N, et al. Telomerase-deficiency-related bone loss is caused by intrinsic impairment of mesenchymal stem cell (MSC) functions and increased osteoclastogenesis due to pro-inflammatory micro-environment[J]. Bone,2010,47 (S1): S39.
[85]邢小平,王鸥,韩桂艳.代谢性骨病与免疫[J].临床内科杂志,2007,24(3)：149-152.
[86]张为杰,刘锡仪.骨代谢活动与神经内分泌系统[J].中华医学研究杂志,2005,5(4)：30-32.
[87]Meinrad Peterlik. Aging, neuroendocrine function, and osteoporosis [J]. Experimental Gerontology,1997,32 (5):577-586.
[88]Adrienne B, Matthew J, Phyllis M. The morphometry of astrocytes in the rostral preoptic area exhibits a diurnal rhythm on proestrus:relationship to the luteinizing hormone surge and effects of age[J]. Endocrinology,2003,144 (1):274-280.
[89]Hou JW, Li B, Yang ZH, et al. Functional integrity of ErbB-4/2 tyrosine kinase receptor complex in the hypothalamus is required for maintaining normal reproduction in young adult female rats[J]. Endocrinology,2002,143 (5):1901-1912.
[90]林雁龙,李钿,王伟森,等.损毁弓状核导致移植骨骨质疏松[J].中国骨质疏松杂志,2005,11(2)：178-180.
[91]蔡维望,王世立.间充质干细胞的成骨分化及对骨质疏松的影响[J].中国生物工程杂志,2012,32(4)：89-95.
[92]eixi Zhang, Mark Hamrick, Carlos Isales, et al. Age-related changes in the osteogenic differentiation potential of BMSC[J]. Bone,2008,43 (S1):S50.
[93]Mohamadreza BE, Soura M, Marzieh E. Growth Kinetics and in Vitro Aging of Mesenchymal Stem Cells Isolated From Rat Adipose Versus Bone Marrow Tissues[J]. Iranian Journal of Veterinary Surgery,2008,3 (2):9-20.
[94]Ali Mirsaidi, Karin NK, Markus Rimann, et al. Telomere length, telomerase activity and osteogenic differentiation are maintained in adipose-derived stromal cells from senile osteoporotic SAMP6 mice[J]. Cell Therapies & Tissue Engineering,2011,5 (6): 378-390.
[95]Baxter MA, Wynn RF, Jowitt SN, et al. Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion[J]. Stem Cells,2004,22 (5): 675-682.
[96]Shi S, GronthosS, Chen S, et al. Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression[J]. Nat Biotechnol,2002,20(6):587-91.
[97]陈槐卿,药蓉,韩君,等.增龄对大鼠骨髓基质细胞生长分化的影响[J].中国医学科学院学报,2003,3：244-249.
[98]杨丽,王攀攀,张荣华.去卵巢对SD大鼠MSCs多向分化能力的影响[J].中国细胞生物学学报,2012,34(1)：61-66.
[99]Nishikawa K, Nakashima T, Takeda S, et al. Maf promotes osteoblast differentiation in mice by mediating the age-related switch in mesenchymal cell differentiation[J]. Clin Invest,2010,120(10):3455-3465.
[100]Clifford JR, Julie Glowacki, John PB. The Aging Skeleton[M]. Academic Press,1999: 642.
[101]Kim KW, Chung Ha-Na, Ha Kee-Yong S, et al. Senescence of nucleus pulposus chondrocytes in human intervertebral discs. Asian Spine J,2008,2(1):1-8.
[102]Hye Bin Noh, Hee-Jin Ahn, Woo-Jung Lee, et al.The molecular signature of in vitro senescence in human mesenchymal stem cells[J]. Genes & Genomics,2010,32 (1):87-93.
[103]Patricia AM, Kristi CT, Colleen Jackson-Cook, et al. Telomerase Resets the Homeostatic Telomere Length and Prevents Telomere Dysfunction in Immortalized Human Cells[J]. DNA and Cell Biology,2004,23(5):293-300.
[104]Maria A Blasco. Telomere length, stem cells and aging[J]. Nature Chemical Biology, 2007,3 (10):640-649.
[105]Ryu E, Hong S, Kang J, et al. Identification of senescene-associated genes in human bone marrow marrow mesenchymal stem cells[J]. Biochem Biophys Res Commun,2008,371 (3):431-436.
[106]Molofsky A, Slutsky SG, Joseph NM, etal. Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during aging [J]. Nature,2006,443(7110):448-452.
[107]Offenstadt G, Moreau A, Ponsot P, etal. Hypoxemia and aging [J]. Poumon Coeur,1980, 36(1):49-53.
[108]Michael Fossel. Cells, Aging, and Human Disease[M]. Oxford University Press,2004, 51.
[109]Hung SC, Yang DM, Chang CF, et al. Immortalization without neoplastic transformation of human mesenchymal stem cells by transduction with HPV16 E6/E7 genes[J].Int J Cancer, 2004,110(3):313-319.
[110]Tsai CC, Chen CL, Liu HC, et al. Overexpression of hTERT increases stem-like properties and decreases spontaneous differentiation in human mesenchymal stem cell lines[J].J Biomed Sci,2010,17 (1):64-70.
[111]赵擎,孙颖,胡燕,等.构建永生化人肝细胞系人端粒酶反转录酶真核表达质粒[J].中国临床康得,2006,10(45)：47-51.
[112]Warren L, Manos PD, Ahfeldt T, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA[J]. Cell Stem Cell,2010,7 (5):618-630.
[113]Sungwoo Kim, Yunqing Kang, Chad A, et al. Sequential delivery of BMP-2 and IGF-1 using a chitosan gel with gelatin microspheres enhances early osteoblastic differentiation[J]. Acta Biomaterialia,2012,8(5):1768-1777.
[114]王运涛,郑启新,吴小涛,等Smad3选择性调节TGF-β 1促进大鼠骨髓间充质干细胞成骨分化的实验研究[J].华中科技大学学报(医学版),2007,5：625-629.
[115]T Naganawa, L Xiao, E Abogunde, et al. In vivo and in vitro comparison of the effects of FGF-2 null and haplo-insufficiency on bone formation in mice[J]. Biochemical and Biophysical Research Communications,2006,339 (2):490-498.
[116]John CS. A woman's journey through the reproductive, transitional and postmenopausal periods of life:Impact on cardiovascular and musculo-skeletal risk and the role of estrogen replacement[J].Maturitas,2011,70 (2):197-205.
[117]Dzmitry Shcharbin, Elzbieta Pedziwiatr, Janusz Blasiak, et al. How to study dendriplexes II:Transfection and cytotoxicity[J]. Journal of Controlled Release,2010, 141 (2):110-127.
[118]Goodwin HS,Bicknese AR,Chien SN, et al. Multilineage differentiation activity by cells isolated from umbilical cord blood:expression of bone, fat, and neural markers [J]. Biol Blood Marrow Trans plant,2001,7 (11):581-588.
[119]Walsh S, Jordan GR, Jefferiss C, et al. High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro:relevance to glucocorticoid-induced osteoporosis[J]. Rheumatology (Oxford),2001,40 (1):74-83.
[120]Otsuka E, Yamaguchi A, Hirose S, et al. Characterization of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid[J]. Am J Physiol, 1999,277(1):132-138.
[121]林建华,修忠标,吴朝阳,等.鹿茸多肽对兔骨髓间质干细胞体外增殖的影响[J].中华实验外科杂志,2005,7(7)：827-830.
[122]刘钰瑜,姚卫民,艾春媚,等.大黄素对体外大鼠骨髓基质细胞向成骨细胞方向分化的影响[J].中国临床药理学与治疗学,2005,10(2)：191-195.
[123]马慧萍贾正平张汝学,等.淫羊藿总黄酮含药血清促进骨髓间充质干细胞增殖与成骨性分化[J].中国骨质疏松杂志,2004,10(4)：420-422,428.
[124]翟远坤,葛宝丰,陈克明,等.淫羊藿苷与其代谢产物淫羊藿次苷Ⅱ对骨髓间充质干细胞成骨性分化影响的比较研究[J].中药材,2010,33(12)：1896-1900.
[125]廖清船,肖洲生,秦艳芳,等.植物雌激素金雀异黄酮通过P38MAPK通路促进骨髓间充质干细胞向成骨细胞分化[J].中国药理学通报,2006,22(6)：683-687.
[126]Tetsu Akiyama. Wnt/β-catenin signal ing [J]. Cytokine & Growth Factor Reviews,2000, 11 (4):273-282.
[127]Akira Kikuchi, Hideki Yamamoto, Akira Sato. Selective activation mechanisms of Wnt signaling pathways [J]. Trends in Cell Biology,2009,19 (3):119-129.
[128]Jia-Ling Teo, Michael Kahn. The Wnt signaling pathway in cellular proliferation and differentiation:A tale of two coactivators[J]. Advanced Drug Delivery Reviews,2010, 62 (12):1149-1155.
[129]Ivan IPacheco, John MacLeod 385 Extracellular Calcium-Sensing Receptor (CaSR) Mediated Production of DKK-1 and Low Density Lipoprotein Receptor-Related Protein 6 (LRP6) Inhibits Defective Wnt Signaling[J]. Gastroenterology,2008,134 (4):51-52.
[130]卞琴,沈自尹,王拥军.骨髓间充质干细胞在中医理论中的归属[J].中国中医基础医学杂志,2011,17(7)：794-797.
[131]Gaur TLengner CJ, Hovhannisyan H, et al. Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression[J].J Biol Chem,2005,280(39):33132-33140.
[132]Bennett CN, Longo KA, Wright WS, et al. Regulation of osteoblastogenesis and bone mass by Wntl0b[J]. Proc NatI Acad Scj USA,2005,102(9):3324-3329.
[133]Tripti Gaur, Christopher JL, Hayk Hovhannisyan, et al. Canonical WNT Signaling Promotes Osteogenesis by Directly Stimulating Runx2 Gene Expression[J]. Journal of Biological Chemistry,2005,280 (39):33132-33140.
[134]Chi Zhang, Kyucheol Cho, Yehong Huang, et al. Inhibition of Wnt signaling by the osteoblast-specific transcription factor Osterix[J]. PNAS,2008,105 (19):6936-6941.
[135]Komori T. Regulation of osteoblast differentiation by transcription factors[J]. J Cell Biochem,2006,99(5):1233-1239.
[136]David GM, Meghan McGee-Lawrence, Merry JO, et al. Update on Wnt signaling in bone cell biology and bone disease[J].Gene,2012,492 (1):1-18.
[137]Maria Almeida, Li Han, Teresita Bellido, et al. Wnt Proteins Prevent Apoptosis of Both Uncommitted Osteoblast Progenitors and Differentiated Osteoblasts by Catenin-dependentand-independentSignalingCascades Involving Src/ERK and Phosphatidylinositol 3-Kinase/AKT[J]. Journal of Biological Chemistry,2006,280 (50): 41342-41351.
[138]Dianqing Wu, Weijun Pan. GSK3:a multifaceted kinase in Wnt signaling[J]. Trends in Biochemical Sciences,2010,35 (3):161-168.
[139]Kang S, Bennett CN, Gerin I, et al. Wnt Signaling Stimulates Osteoblastogenesis of Mesenchymal Precursors by Suppressing CCAAT/Enhancer-binding Protein a and Peroxisome Proliferator-activated Receptor γ [J].J Biol Chem,2007,282(19):14515-14524.
[140]Bennett CN, Ouyang H, Ma YL, et al. Wnt10b increases postnatal bone formation by enhancing osteoblast differentiation[J]. J Bone Miner Res,2007,22(12):1924-32.
[141]Gonzalez-Sancho JM, Brennan KR, Castelo-Soccio LA, et al. Wnt Proteins Induce Dishevelled Phosphorylation via an LRP5/6-Independent Mechanism, Irrespective of Their Ability To Stabilize β-Catenin[J]. Mol Cell Biol,2004,24(11):4757-4768.
[142]李萍华,刘钰瑜,崔燎.骨髓间充质干细胞成脂和成骨分化过程中Wnt信号通路的调控效应[J].中国组织丁程研究与临床,2010,14(10)：1749-1754.
[143]侯秋科,吴静,易香华,等.龟板提取物上调维生素D受体表达促骨髓间充质干细胞向成骨分化[J].中草药,2010,41(4)：607-609.
[144]Tang DZ, Hou W, Zhou Q, et al. Osthole Stimulates Osteoblast Differentiation and Bone Formation by Activation of β-Catenin-BMP Signaling[J]. J Bone Miner Res,2010, 25(6):1234-1245.
[145]卞琴,刘书芬,黄建华,等.3种补肾中药有效成分对去卵巢骨质疏松大鼠骨髓间充质干细胞的调控作用[J].中华中医药杂志,2011,5：889-893.
[146]Zhao J, Ohba S, Shinkai M, et al. Icariin induces osteogenic differentiation in vitro in a BMP-and Runx2-dependent manner [J]. Biochem Biophys Res Commun,2008,369(2):444-448.
[147]刘海江,王小平,林娟,等.淫羊藿苷和黄芪苷Ⅰ对骨髓基质细胞增殖及成骨能力的影响[J]. 中药材,2006,29(10)：1062-1065.
[148]田李军,苏健,杨慧,等.补肾柔肝法防治卵巢早衰患者骨质疏松症的临床观察[J].河北中医,2006,28(12)：888-890.
[149]张洪,魏之玉,崔德芝.补肾疏肝健脾方调节性腺轴治疗老年骨质疏松症[J].中国中医药信息杂志,1999,6(4)：43-44.
[150]刘忠厚,杨定焯,朱汉民,等.中国人骨质疏松症建议诊断标准(第2稿)[J].中国骨质疏松杂志,2000,6(1)：1-4.
[151]国家技术监督局.中华人民共和国国家标准(GB/16751.2-1997)中医临床诊疗术语证候部分[M].北京：中国标准出版社,1997,第一版：10-41.
[152]Lenore Kurlowicz. The geriatric depression scale[J]. Home Care Provider,2000,5 (2):76-77.
[153]郑筱萸.中药新药临床研究指导原则[M].北京：中国医药科技出版社,2002,第一版：210-296.
[154]Bird SB, Dickson EW. Clinically significant changes in pain along the visual analog scale Original[J]. Annals of Emergency Medicine,2001,38 (6):639-643.
[155]国家中医药管理局.中华人民共和国中医药行业标准·中医病症诊断疗效标准[M].南京：南京大学出版社,1994,第一版：10.
[156]徐国柱,蔡志基.镇痛药临床评价方法研究[J].中国新药杂志,1995,4(4)：20-22.
[157]徐进秀,焦安钦.中医证候规范化研究的思路与设想[J].中国中医基础医学杂志,2005,11(4)：261-265.
[158]盛彤,杨惠民,田金洲,等.老年性骨量减少的证候特征及其与BMD的相关性研究[J].北京中医药大学学报(中医临床版),2006,13(6)：15-18.
[159]孙江波,李智雄,刘绪银,等.骨质疏松症中医证候与骨密度水平的相关性研究[J].中医临床研究,2011,3(5)：12-14.
[160]廖怀章,孙江波,刘绪银.骨质疏松症中医定性证候与骨密度水平的相关性研究[J].湖南中医杂志,2010,26(5)：51-53.
[16l]Copeland JR.Beekman T,Dewey ME, et al. Depression in Europe Geographical Distribution Among Older People[J]. Brit J Psychiatry,1999,174:312-321.
[162]Irwin MR, Miller AH. Depressive disorders and immunity:20 years of progress and discovery[J]. Brain Behav Immun,2007,21:374-383.
[163]蔡焯基.抑郁症临床与基础[M].北京：科学出版社,1997,第一版：1.
[164]Wong SY, Lau EM, Lynn H, et al. Depression and bone mineral density.is there a relationship in elderly Asian men? Results from Mr. Os (Hong Kong)[J]. Osteoporos Int, 2005,16:610-615.
[165]Konstantynow icz J, KadzielaOlech H, Kaczmarski M, et al. Depression in anorexia nervosa:a risk factor for osteoporosis [J]. J Clin Endocrinol Metab,2005,90:5382-5385.
[166]齐璇.绝经后骨质疏松症的抑郁状态及心理干预治疗[J].中国临床康复,2002,6(23)：1950-1951.
[167]彭文芳,赵文穗.绝经后患者焦虑、抑郁状态对骨质疏松和生活质量的影响[J].中国骨质疏松杂志,2007,13(9)：642-644.
[168]Giovanni Cizza. Svetlana Primma, Gyorgy Csako. Depression as a risk factor for osteoporosis Original[J]. Trends in Endocrinology & Metabolism,2009,20 (8):367-373.
[169]Reginster JY, Deroisy R, Paul I, et al. Depressive vulnerabil ity is not an independent risk factor for osteoporosis in postmenopausal women[J]. Maturitas,1999,33(2):133-137.
[170]Williams LJ, Pasco JA, Jacka FN, et al. Depression and bone metabolism. Psychother Psychosom,2009,78(1):16-25.
[171]Yirmiya R, Inbal Goshen, Alon Bajayo, et al. Depression induces bone loss through stimulation of the sympathetic nervous system[J]. PNAS,2006,103 (45):16876-16881.
[172]Yirmiya R, Bab I. Major depression is a risk factor for low bone mineral density: A meta-analysis[J]. Biological Psychiatry,2009,66:423-32.
[173]Massimo Cocchi, Lucio Tonello, Fabio Gabrielli, et al. Depression, osteoporosis, serotonin and cell membrane viscosity between biology and philosophical anthropology [J]. Ann Gen Psychiatry,2011,10 (9):1-7.
[174]邵大清.绝经后骨质疏松症患者应重视抑郁症状的评估.浙江省医学会-浙江省医学会麻醉学术年会,2007,274-275.
[175]Coelho R, Silva C, Maia A, et al. Bone mineral density and depression:a community study in women[J]. Psychosom Res,1999,46(1):29-35.
[176]刘存斌.骨质疏松症中医研究进展[J].中医临床研究,2011,3(7)：16-17.
[177]黄平,王会仍.骨质疏松症骨密度测定与中医辨证分型[J].浙江中西医杂志,1999,9(1)：21-22.
[178]魏之玉,张洪,朱振铎,等.196例原发性骨质疏松症辨证分析[J].山东中医学院学报,1996,1：30-31.
[179]张学娅,许东云,张颖,等.从“肝肾同源”论探讨骨质疏松症病因病机及其治疗原则[J].辽宁中医杂志,2011,38(12)：2362-2363.
[180]张荣华,丘和明.中医防治退行性骨质疏松症用药分析[J].中医药学报,1997,25(4)：30-31.
[181]杨帆,徐妙容,杨妙馥,等.中药治疗骨质疏松症的用药规律[J].中国临床康复,2005,9(31)：203-205.
[182]黄连芳,吴铁,谢华,等.何首鸟煎剂对去卵巢大鼠骨质丢失的防治作用[J].中国老年学杂志,2005,25(6)：709-710.
[183]陈象青,王钦茂,方华武,等.白芍总苷和当归提取物对小鼠免疫性肝损伤的保护作用[J]. 承德医学院学报,2004,21(1)：8-10.
[184]郝习,赵明耀.枸杞多糖对树突状细胞的成熟及免疫学功能的影响[J].中医研究,2012,23(17)：24-27.
[185]高成芬.培补肝肾为主治疗老年骨质疏松症临床观察[J].现代康复,2001,5(6)：91.
[186]章薇,金华,袁静.养血.调肝方防治绝经后骨质疏松症的研究思路[J].中国中医骨伤科杂志,2005,13(3)：52-53.
[187]Rugang Zhang, Maxim V, Xiaofen Ye, et al. Formation of MacroH2A-Containing Senescence-Associated Heterochromatin Foci and Senescence Driven by ASFla and HIRA[J]. Development Cell,2005,8:19-30.
[188]肖建德.实用骨质疏松学[M].北京：科学出版社,2004,第一版：3-5.
[189]江军亮.骨质疏松症的中医药治疗进展[J].陕西中医,2012,33(1)：119-121.
[190]代平,詹瑞森,王卫国.骨质疏松患者体外培养BM SCs的生物学特性[J].医学临床研究,2007,12(24)：2019-2023.
[191]Bei Chen, Yi Zhong, Wei Peng, et al. Age-related changes in the central auditory system:Comparison of d-galactose-induced aging rats and naturally aging rats Original [J]. Brain Research,2010,1344 (16):43-53.
[192]裴凌鹏,董福慧,惠伯棣.D-半乳糖致衰老作用对大鼠骨质的影响[J].中医正骨,2007,19(3)：1-3.
[193]邢玉芝,杨玲麟,林洪,等.D-半乳糖对大鼠骨髓间充质干细胞拟衰老作用的实验研究[J].中国修复重建外科杂志,2006,20(4)：475-479.
[194]张进,徐志伟,丁富平.“肾藏精”的现代实质新理论[J].世界科学技术-中医药现代化,2010,12(4)：550-552.
[195]邓元江,易受乡,严洁.细胞信号转导理论在中医药研究中的应用[J].中国中医药信息杂志,2004；11(9)：836-839.
[196]陈希,梁祖建,邵敏,等.补肾健脾活血方对骨质疏松症信号转导基因表达的调控作用[J].新中医,2008,40(3)：60-62.