基于破骨细胞分化调节通路OPG/RANKL探讨补肾健脾方治疗骨质疏松症的作用机理
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摘要
目的
在建立骨质疏松症脾肾两虚型病证结合动物模型的基础上,基于破骨细胞分化调节通路OPG/RANKL,同时采用与脾虚和骨代谢皆密切相关的VIP/VPAC2作为观察指标,探讨在脾虚、肾虚和脾肾两虚状态下,骨吸收与骨形成的变化情况以及OPG/RANKL和VIP/VPAC2的变化特点；另外通过对比健脾方、补肾方及补肾健脾方对脾肾两虚型骨质疏松大鼠OPG/RANKL信号传导通路及VIP/VPAC2的影响,部分阐明补肾健脾方治疗骨质疏松症的作用机制,从而为阐明“脾肾相关”的科学内涵提供进一步的实验依据。
方法
选用健康雌性Wistar大鼠110只,随机分为4组：正常对照组12只,脾虚模型组12只,假手术组12只,卵巢切除组74只。卵巢切除组大鼠行双侧卵巢切除术,术后10周,再将卵巢切除组大鼠随机分为：肾虚模型组、脾肾两虚模型组、己烯雌酚组、健脾组各12只,补肾组及补肾健脾组各13只。
肾虚模型组大鼠以戊巴比妥钠腹腔注射麻醉,摘除双侧卵巢；假手术组只摘取卵巢周围的小块脂肪组织；脾虚模型组采用200%大黄浓煎液灌胃的方法,连续2周后改为每周2次,再持续3个月；脾肾两虚模型组在切除大鼠双侧卵巢10周后,再采用上述脾虚模型制作方法。
己烯雌酚组灌胃给予己烯雌酚,浓度为0.0048mg/ml；健脾组、补肾组及补肾健脾组分别灌胃给予四君子汤、龟鹿二仙汤以及龟鹿二仙汤合四君子汤,每日下午1次,连续6天,休息一天,再连续给药6天,如此给药3个月。正常对照组、假手术组、脾虚模型组、肾虚模型组、脾肾两虚模型组按同法灌服等体积的纯净水。每周称重一次,据此调整给药量。
各组大鼠在处死前16天和前3天分别腹腔注射盐酸四环素30mg/kg体重,以对骨进行荧光标记。给药结束后,采用大鼠麻醉后股动脉取血法,血液离心后用于酶联免疫吸附法(ELISA)检测；取血后将各组大鼠处死,取左侧股骨,用于骨密度检测；取右侧胫骨近端1/3,制作不脱钙骨切片,进行骨组织形态计量学指标的检测；取左侧胫骨近端1/3,制作大鼠胫骨脱钙骨的冰冻切片,用作免疫组化法和原位杂交法对骨髓腔中成骨细胞和骨髓基质细胞中VPAC2、OPG和RANKL蛋白和mRNA表达的检测；取结肠组织,制作石蜡切片,用作免疫组化法和原位杂交法对结肠组织中VIP蛋白和mRNA表达的检测。
实验结果皆以均数±标准差(x±s)表示,正态分布且方差齐性的数据,采用单因素方差分析,组间两两比较,LSD检验；方差不齐的则采用其项下的Dunnett's T3检验。非正态分布数据采用非参数检验。
结果
1补肾健脾方对脾肾两虚型骨质疏松大鼠股骨βMD的影响
表1显示,与假手术组相比,肾虚及脾肾两虚模型组大鼠股骨BMD显著降低,且脾肾两虚模型组BMD降低较之肾虚组更为明显。脾虚模型组大鼠股骨BMD与假手术组无明显差异。
与脾肾两虚模型组相比,健脾组、补肾组、补肾健脾组、己烯雌酚组大鼠股骨BMD均显著增高,但仍显著低于假手术组。与健脾组相比,补肾组及补肾健脾组的BMD均明显增高,且两者之间无差异。
注：与假手术组比较：*P<0.05,**P<0.01；与脾虚模型组比较：◇P<0.05,◇◇P<0.01；与肾虚模型组比较：▲P<0.05,▲▲P<0.01;与脾肾两虚模型组比较：△P<0.05,△AP<0.01；与健脾组比较：☆P<0.05,☆☆P<0.01；与补肾组比较：
P<0.05,
P<0.01。(下同)
2补肾健脾方对脾肾两虚型骨质疏松大鼠骨组织形态计量学指标的影响
2.1补肾健脾方对脾肾两虚型骨质疏松大鼠胫骨TBV%的影响
表2显示,脾虚模型组大鼠胫骨TBV%与假手术组相比无明显差异。肾虚及脾肾两虚模型组TBV%显著低于假手术组及脾虚模型组,且与肾虚模型组相比,脾肾两虚模型组TBV%降低更为明显。
从各治疗组TBV%结果来看,补肾组、健脾组、补肾健脾组、己烯雌酚组大鼠胫骨TBV%均显著高于脾肾两虚模型组,但明显低于假手术组。与健脾组相比,补肾组及补肾健脾组的TBV%明显增高,且补肾健脾组TBV%增高较补肾组更为明显。
2.2补肾健脾方对脾肾两虚型骨质疏松大鼠胫骨TRS%和TFS%的影响
表3显示,与假手术组相比,肾虚及脾肾两虚模型组大鼠胫骨TRS%和TFS%均显著增高,而脾虚模型组无明显差异。与肾虚模型组相比,脾肾两虚模型组大鼠胫骨TRS%和TFS%增高更为明显。
从各治疗组TRS%和TFS%结果来看,补肾组、健脾组、补肾健脾组、己烯雌酚组大鼠胫骨TRS%和TFS%均显著低于脾肾两虚模型组,且补肾健脾组、己烯雌酚组大鼠胫骨TRS%和TFS%相较于假手术组无明显差异,但补肾组、健脾组大鼠胫骨TRS%和TFS%仍明显高于假手术组。与健脾组相比,补肾组及补肾健脾组的TRS%和TFS%明显降低,且补肾健脾组较补肾组降低更为显著。
2.3补肾健脾方对脾肾两虚型骨质疏松大鼠胫骨OSW、MAR和mAR的影响
表4显示,肾虚及脾肾两虚模型组大鼠胫骨OSW、MAR和mAR较之假手术组均显著增高,而脾虚模型组无明显差异。与肾虚模型组相比,脾肾两虚模型组大鼠胫骨MAR和mAR增高更为显著,但OSW无明显差异。
与脾肾两虚模型组相比,补肾组、健脾组、补肾健脾组、己烯雌酚组大鼠胫骨OSW、MAR和mAR均显著降低。与健脾组及补肾组相比,补肾健脾组的MAR和mAR均明显降低,但OSW无明显差异。
3补肾健脾方对脾肾两虚型骨质疏松大鼠外周血血清中OPG、RANKL、VIP、MTL、GAS含量的影响
3.1补肾健脾方对脾肾两虚型骨质疏松大鼠外周血血清中OPG及RANKL含量的影响
表5显示,脾虚模型组大鼠外周血血清中OPG含量与假手术组无明显差异。肾虚、脾肾两虚模型组OPG含量较之假手术组及脾虚模型组均显著降低,且与肾虚模型组相比,脾肾两虚模型组OPG含量降低更为显著。与脾肾两虚模型组相比,健脾组OPG含量无明显差异；而己烯雌酚组、补肾组、补肾健脾组OPG含量均显著增高,但仍显著低于假手术组。与健脾组相比,补肾组和补肾健脾组OPG含量均明显增高,且补肾健脾组明显高于补肾组。
与假手术组相比,脾虚、肾虚、脾肾两虚模型组大鼠外周血血清中RANKL含量均显著增高,且肾虚组RANKL含量明显高于脾虚组,脾肾两虚组又明显高于肾虚组。与脾肾两虚模型组相比,己烯雌酚组、健脾组、补肾组、补肾健脾组RANKL含量均显著降低,但较之假手术组仍显著增高。与健脾组相比,补肾组和补肾健脾组RANKL含量均显著降低,且补肾健脾组明显低于补肾组。
3.2补肾健脾方对脾肾两虚型骨质疏松大鼠外周血血清中VIP含量的影响
表6显示,与假手术组相比,脾虚、肾虚、脾肾两虚模型组大鼠外周血血清中VIP含量均显著增高,且脾虚组VIP含量明显高于肾虚组,脾肾两虚组又显著高于脾虚组。
从各治疗组VIP含量来看,除己烯雌酚组外,健脾组、补肾组、补肾健脾组VIP含量均显著低于脾肾两虚模型组；但健脾组和补肾组仍明显高于假手术组,而补肾健脾组与假手术组无差异。与补肾组相比,健脾组和补肾健脾组VIP含量明显降低,且两者之间无差异。
3.3补肾健脾方对脾肾两虚型骨质疏松大鼠外周血血清中MTL、GAS含量的影响
表7显示,脾虚、肾虚、脾肾两虚模型组大鼠外周血血清中MTL含量较之假手术组均显著降低,脾肾两虚模型组又显著低于脾虚和肾虚组。与脾肾两虚模型组相比,健脾组和补肾健脾组MTL含量均显著增高,且两者之间无差异；但己烯雌酚组和补肾组无明显变化。健脾组和补肾健脾组MTL含量均显著高于补肾组。
与假手术组相比,脾虚、肾虚、脾肾两虚模型组大鼠外周血血清中GAS含量均显著降低,且脾虚组GAS含量明显低于肾虚组,脾肾两虚组又明显低于脾虚组。与脾肾两虚模型组相比,健脾组和补肾健脾组GAS含量均显著增高,但己烯雌酚组和补肾组无明显差异。补肾健脾组GAS含量明显高于健脾组和补肾组。
4补肾健脾方对脾肾两虚型骨质疏松大鼠OB及MSC中OPG>RANKL及VPAC2蛋白和mRNA表达的影响
4.1补肾健脾方对脾肾两虚型骨质疏松大鼠OB及MSC OPG蛋白和mRNA表达的影响
表8显示,与假手术组相比,脾虚模型组大鼠OB及MSC的OPG蛋白和mRNA表达阳性密度与之无差异,而肾虚、脾肾两虚组的OPG阳性密度明显降低,且脾肾两虚组又明显低于肾虚组。
与脾肾两虚模型组相比,己烯雌酚组、健脾组、补肾组、补肾健脾组OPG蛋白和mRNA表达阳性密度均明显增高,但较之假手术组仍明显降低。与健脾组相比,补肾组和补肾健脾组OPG蛋白和mRNA表达阳性密度明显增高,且两者之间无明显差异。
4.2补肾健脾方对脾肾两虚型骨质疏松大鼠OB及MSC RANKL蛋白和mRNA表达的影响
表9显示,与假手术组相比,脾虚、肾虚、脾肾两虚模型组大鼠OB及MSCRANKL蛋白和mRNA表达的阳性密度均明显增高,且肾虚组阳性密度明显高于脾虚组,脾肾两虚组又明显高于肾虚组。
与脾肾两虚模型组相比,己烯雌酚组、健脾组、补肾组、补肾健脾组RANKL蛋白和mRNA表达阳性密度均明显降低,但仍明显高于假手术组。与健脾组相比,补肾组和补肾健脾组RANKL蛋白和mRNA表达阳性密度均明显降低,且两者之间RANKL蛋白表达无差异,但补肾健脾组RANKL mRNA表达阳性密度降低更为明显。
4.3补肾健脾方对脾肾两虚型骨质疏松大鼠OB及MSCVPAC2蛋白和mRNA表达的影响
表10显示,与假手术组相比,脾虚、肾虚、脾肾两虚模型组大鼠OB及MSC中VPAC2蛋白和mRNA表达的阳性密度均明显增高。与肾虚组相比,脾虚、脾肾两虚组VPAC2阳性密度的增高更为明显,且两者之间无差异。
与脾肾两虚模型组相比,己烯雌酚组VPAC2蛋白和mRNA表达阳性密度无差异,健脾组、补肾组、补肾健脾组均明显降低。与补肾组相比,健脾组和补肾健脾组VPAC2蛋白和mRNA表达阳性密度明显降低,且两者之间无差异。
5补肾健脾方对脾肾两虚型骨质疏松大鼠结肠组织中VIP蛋白和mRNA表达的影响
表11显示,与假手术组相比,肾虚模型组大鼠结肠组织中VIP蛋白和mRNA表达阳性密度与之无差异,脾虚、脾肾两虚组的VIP阳性密度显著增高,且脾肾两虚组又明显高于脾虚组。
与脾肾两虚模型组相比,健脾组、补肾组、补肾健脾组VIP蛋白和mRNA表达阳性密度均显著降低,但仍显著高于假手术组。与补肾组相比,补肾健脾组VIP蛋白和mRNA表达阳性密度均明显降低,健脾组与之无差异。
结论
(1)去卵巢能够造成大鼠肾虚型骨质疏松症,而单纯给予大黄浓煎剂可造成大鼠脾虚,但不能导致骨质疏松症。将两种方法相结合,可造成大鼠脾肾两虚型骨质疏松症,且发病程度较单纯去卵巢明显加重。说明脾、肾两脏在骨质疏松症发病过程中均有重要作用,其中肾虚是根本,脾虚是其重要影响因素之一。
(2)健脾方、补肾方和补肾健脾方对脾肾两虚型大鼠骨质疏松症均具有治疗作用,其中,补肾健脾方的疗效明显优于补肾方和健脾方,而补肾方又优于健脾方。
(3)健脾方、补肾方和补肾健脾方共同的作用途径是：一方面,直接调控破骨细胞分化调控因子OPG、RANKL的表达,使OPG表达上调,而使RANKL表达下调,从而明显降低破骨细胞的活性；另一方面,下调VIP和VPAC2表达,导致VIP与VPAC2结合减少,这可能通过成骨细胞的介导作用,上调OPG水平、并下调RANKL水平,最终使破骨细胞的活性降低,骨吸收减少。此外,还能明显提高外周血中MTL及GAS水平。但三者的调节作用存在差异：健脾方对MTL、GAS、VIP及VPAC2的调节作用较强；补肾方对上述指标的调节作用弱于健脾方,但对OPG、RANKL的调节作用较健脾方显著；而补肾健脾方对上述指标的调节作用均明显强于以上两方。这是补肾健脾方疗效优于补肾方和健脾方的机制之一,也进一步说明在骨质疏松症的发生和治疗过程中,中医的脾肾两脏是密切相关的。
Objective
After establishing rat osteoporosis model with spleen and kidney deficiency syndrome, on the basis of OPG/RANKL which is differentiation regulatory factor of osteoclasts and simultaneously selecting VIP/VPAC2which are closely related with Spleen deficiency syndrome and bone metabolism, we would explore the variation characteristics of OPG/RANKL and VIP/VPAC2and the changes of bone resorption and bone formation in the state of spleen deficiency syndrome, kidney deficiency syndrome, and spleen and kidney deficiency syndrome respectively. Moreover, by contrast of the efficacy of recuperating kidney, invigorating spleen, recuperating kidney and invigorating spleen prescription on rats with spleen and kidney deficiency syndrome and the effect on OPG/RANKL signal pathway and VIP/VPAC2, the mechanism of recuperating kidney and invigorating spleen prescription on osteoporosis may be partly clarified, and experimental evidence for the scientific connotation of "spleen and kidney related" will be further provided.
Methods
First, one hundred and ten female Wistar rats were selected and divided into four groups randomly:twelve in normal control group, spleen deficiency model group, sham operation group; seventy-four in bilateral ovariectomized group. After the surgery for10weeks, these seventy-four bilateral ovariectomized rats were divided into six sub-groups again:twelve in kidney deficiency model group, spleen and kidney deficiency model group, diethylstilbestrol group and invigorating spleen group; thirteen in recuperating kidney group and recuperating kidney and invigorating spleen group.
Kidney deficiency syndrome model was Established by Ovariectomy after anesthetized by intraperitoneal injection of sodium pentobarbital, while only the fatty tissue near the ovaries of the rats were extirpated in sham operation group;200% rhubarb concentrated decoction was given orally every day for two weeks, then changed to2times a week for three months to establish the spleen deficiency syndrome model; on this basis, the rat osteoporosis model with spleen and kidney deficiency syndrome has been established by the above two methods after Ovariectomy for ten weeks.
Diethylstilbestrol group was administrated diethylstilbestrol orally which was diluted to0.0048mg/ml with normal saline; invigorating spleen group, recuperating kidney group, recuperating kidney and invigorating spleen group were given orally Sijunzitang, Guiluerxiantang, and the two combined respectively, The drug or decoctions were given once a day, six days continuously then1day pause, the whole period lasted3months. Distilled water was administrated orally to other groups, at the same time. The rats of all groups were weighed once a week, and adjusted the dose accordingly.
At the third day and the sixteenth day before the day all the rats were killed, hydrochloric acheomycin was injected into the abdomen of the rats respectively according to30mg/kg to mark the bone with fluorescence. After Medication, The rats were anesthetized to get artery blood, then the blood was centrifuged to detect the level of Various cytokines in serum by enzyme-linked immunosorbent assay (ELISA).After the rats being killed, the left femur were taken for bone mineral density testing; the right tibia of the rats were retained for bone tissue section which weren't decalcified to evaluate the change of bone histomorphometry; at the same time the left tibia of the rats were used for frozen tissue section after being decalcified to observe the protein and mRNA expression of VPAC2,OPG and RANKL by means of immunohistochemistry and in situ hybridization; in addition,colon tissue were taken for paraffin tissue section to observe the protein and mRNA expression of VIP by means of immunohistochemistry and in situ hybridization.
Results were presented as Mean±SE, normal distribution and homogeneity of variance of the data were analyzed by one-way ANOVA, between any two groups, LSD test; heterogeneity of variance of the data were analyzed by Dunnett's T3test. And non-normal data by non-parametric tests.
Results
1Effects of recuperating kidney and invigorating spleen prescription on femur BMD of osteoporosis rats of spleen and kidney deficiency syndrome model
Compared with sham operation group, femur BMD in kidney deficiency model group and spleen and kidney deficiency model group decreased significantly, and the latter was more significantly. But Spleen model group was not different.
Compared with spleen and kidney deficiency syndrome model group, femur BMD in all treatment groups increased obviously. In recuperating kidney group and recuperating kidney and invigorating spleen group, femur BMD increased more significantly compared with invigorating spleen group, but there was no difference between them.
2Effects of recuperating kidney and invigorating spleen prescription on the bone histomorphometry indicators of osteoporosis rats of spleen and kidney deficiency syndrome model
Compared with sham operation group, TBV%in kidney deficiency model group and spleen and kidney deficiency model group decreased significantly, TRS%, TFS%, OSW, MAR and mAR increased significantly, while Spleen model group was not significantly different. Compared with kidney deficiency model group, TBV%in spleen and kidney deficiency model group decreased obviously, TRS%and TFS%increased significantly.
Compared with spleen and kidney deficiency syndrome model group, TBV%in all treatment groups increased obviously, TRS%、 TFS%、OSW、 MAR and mAR decreased obviously. Compared with invigorating spleen group, TBV%increased and TRS%、 TFS%decreased obviously in recuperating kidney group and recuperating kidney and invigorating spleen group, and the latter reversed these indicators more significantly.
3Effects of recuperating kidney and invigorating spleen prescription on serum level of OPQ RANKL, VIP, MTL and GAS
Compared with sham operation group, the serum level of OPG, MTL and GAS decreased significantly in three model groups, except that the serum level of OPG in spleen deficiency model group was not obviously different. In addition, Spleen and kidney deficiency model group was the lowest. While the serum level of RANKL and VIP increased significantly in three model groups, moreover, Spleen and kidney deficiency model group was the highest.
Compared with spleen and kidney deficiency syndrome model group, the serum level of OPG increased significantly in recuperating kidney group and recuperating kidney and invigorating spleen group, and the latter increased more obviously; the serum level of MTL and GAS increased significantly in invigorating spleen group and recuperating kidney and invigorating spleen group. While the serum level of RANKL decreased significantly in all treatment groups, and among which diethylstilbestrol group and recuperating kidney and invigorating spleen group were optimal; the serum level of VIP decreased significantly in all treatment groups except for diethylstilbestrol group, invigorating spleen group and recuperating kidney and invigorating spleen group decreased more significantly than recuperating kidney group.
4Effects of recuperating kidney and invigorating spleen prescription on protein and mRNA expression of OPG, RANKL and VPAC2
Compared with sham operation group, the protein and mRNA expression of OPG was not obviously different in spleen deficiency model group and decreased significantly in kidney deficiency model group and spleen and kidney deficiency model group, in addition, the latter was significantly lower than the former. Different from OPG, the protein and mRNA expression of RANKL increased significantly in all model groups, and kidney deficiency model group was significantly higher than spleen deficiency model group, spleen and kidney deficiency group was higher than kidney deficiency group. In respect of VPAC2, the protein and mRNA expression in three model groups increased significantly compared with sham operation group, compared with kidney deficiency model group, spleen deficiency model group, spleen and kidney deficiency group increased more, and there was no obvious difference between the two groups.
Compared with spleen and kidney deficiency syndrome model group, the protein and mRNA expression of OPG increased obviously in all treatment groups, but still lower than sham operation group. Compared with invigorating spleen group, recuperating kidney group and recuperating kidney and invigorating spleen group increased more and no obvious difference between them. In contrast, the protein and mRNA expression of RANKL decreased obviously in all treatment groups, but still higher than sham operation group. Compared with invigorating spleen group, recuperating kidney group and recuperating kidney and invigorating spleen group decreased obviously, and the latter decreased more significantly in mRNA expression. In respect of VPAC2, the protein and mRNA expression decreased obviously in all treatment groups except for diethylstilbestrol group. Compared with recuperating kidney group, invigorating spleen group and recuperating kidney and invigorating spleen group decreased obviously, and the former decreased more significantly.5Effects of recuperating kidney and invigorating spleen prescription on protein and mRNA expression of VIP in colon tissue
Compared with sham operation group, the protein and mRNA expression of VIP in colon tissue was not obviously different in kidney deficiency model group and increased significantly in spleen deficiency model group and spleen and kidney deficiency model group, in addition, the latter was significantly higher than the former.
Compared with spleen and kidney deficiency syndrome model group, the protein and mRNA expression of VIP in colon tissue decreased obviously in invigorating spleen group, recuperating kidney group and recuperating kidney and invigorating spleen group, but was not different in diethyl stilbestrol group. The protein and mRNA expression of invigorating spleen group and recuperating kidney and invigorating spleen group decreased significantly compared with recuperating kidney group, and there was no difference between them.
Conclusion
(1) Ovariectomy can cause rat osteoporosis with kidney deficiency syndrome, simply giving the rhubarb concentrated decoction can cause rat spleen deficiency syndrome, but can not lead to osteoporosis. If the two methods combined, which can lead to rat osteoporosis with spleen and kidney deficiency syndrome, and the disease is more serious than that which led by Simple ovariectomy. This shows that both spleen and kidney have important role in the process of osteoporosis, and among which, kidney is fundamental, spleen is one of the important factors.
(2) The prescription of recuperating kidney, invigorating spleen, recuperating kidney and invigorating spleen all have therapeutic effects on osteoporosis rats with spleen and kidney deficiency syndrome, and that of recuperating kidney and invigorating spleen works best, followed by recuperating kidney, and followed by invigorating spleen.
(3) The common pathway of three prescriptions is:On the one hand, it can directly regulate the expression of OPG and RANKL, the expression of OPG up-regulated and RANKL down-regulated, so that the osteoclasts activity decreased obviously; on the other hand, it can down-regulate the expression of VIP and VPAC2and thereby reduce the combination of VIP and VPAC2, which can up-regulate OPG levels and down-regulate RANKL levels probably by osteoblast-mediated effect, and ultimately led to reduction of the osteoclasts activity and the bone resorption. In addition, MTL and GAS levels can also be raised significantly in peripheral blood. However, there are some differences among the three ways:invigorating spleen prescription played a stronger role in MTL, GAS, VIP and VPAC2; although the effects of recuperating kidney Prescription on these indicators were weaker than the former, but it can play more significant role in OPG and RANKL; while these indicators were improved significantly by recuperating kidney and invigorating spleen prescription. This may be one of the mechanisms of recuperating kidney and invigorating spleen prescription which was more effective than the other two prescriptions, it further demonstrates that spleen and kidney in Traditional Chinese Medicine are closely related in the occurrence and treatment process of osteoporosis.
在建立骨质疏松症脾肾两虚型病证结合动物模型的基础上,基于破骨细胞分化调节通路OPG/RANKL,同时采用与脾虚和骨代谢皆密切相关的VIP/VPAC2作为观察指标,探讨在脾虚、肾虚和脾肾两虚状态下,骨吸收与骨形成的变化情况以及OPG/RANKL和VIP/VPAC2的变化特点；另外通过对比健脾方、补肾方及补肾健脾方对脾肾两虚型骨质疏松大鼠OPG/RANKL信号传导通路及VIP/VPAC2的影响,部分阐明补肾健脾方治疗骨质疏松症的作用机制,从而为阐明“脾肾相关”的科学内涵提供进一步的实验依据。
方法
选用健康雌性Wistar大鼠110只,随机分为4组：正常对照组12只,脾虚模型组12只,假手术组12只,卵巢切除组74只。卵巢切除组大鼠行双侧卵巢切除术,术后10周,再将卵巢切除组大鼠随机分为：肾虚模型组、脾肾两虚模型组、己烯雌酚组、健脾组各12只,补肾组及补肾健脾组各13只。
肾虚模型组大鼠以戊巴比妥钠腹腔注射麻醉,摘除双侧卵巢；假手术组只摘取卵巢周围的小块脂肪组织；脾虚模型组采用200%大黄浓煎液灌胃的方法,连续2周后改为每周2次,再持续3个月；脾肾两虚模型组在切除大鼠双侧卵巢10周后,再采用上述脾虚模型制作方法。
己烯雌酚组灌胃给予己烯雌酚,浓度为0.0048mg/ml；健脾组、补肾组及补肾健脾组分别灌胃给予四君子汤、龟鹿二仙汤以及龟鹿二仙汤合四君子汤,每日下午1次,连续6天,休息一天,再连续给药6天,如此给药3个月。正常对照组、假手术组、脾虚模型组、肾虚模型组、脾肾两虚模型组按同法灌服等体积的纯净水。每周称重一次,据此调整给药量。
各组大鼠在处死前16天和前3天分别腹腔注射盐酸四环素30mg/kg体重,以对骨进行荧光标记。给药结束后,采用大鼠麻醉后股动脉取血法,血液离心后用于酶联免疫吸附法(ELISA)检测；取血后将各组大鼠处死,取左侧股骨,用于骨密度检测；取右侧胫骨近端1/3,制作不脱钙骨切片,进行骨组织形态计量学指标的检测；取左侧胫骨近端1/3,制作大鼠胫骨脱钙骨的冰冻切片,用作免疫组化法和原位杂交法对骨髓腔中成骨细胞和骨髓基质细胞中VPAC2、OPG和RANKL蛋白和mRNA表达的检测；取结肠组织,制作石蜡切片,用作免疫组化法和原位杂交法对结肠组织中VIP蛋白和mRNA表达的检测。
实验结果皆以均数±标准差(x±s)表示,正态分布且方差齐性的数据,采用单因素方差分析,组间两两比较,LSD检验；方差不齐的则采用其项下的Dunnett's T3检验。非正态分布数据采用非参数检验。
结果
1补肾健脾方对脾肾两虚型骨质疏松大鼠股骨βMD的影响
表1显示,与假手术组相比,肾虚及脾肾两虚模型组大鼠股骨BMD显著降低,且脾肾两虚模型组BMD降低较之肾虚组更为明显。脾虚模型组大鼠股骨BMD与假手术组无明显差异。
与脾肾两虚模型组相比,健脾组、补肾组、补肾健脾组、己烯雌酚组大鼠股骨BMD均显著增高,但仍显著低于假手术组。与健脾组相比,补肾组及补肾健脾组的BMD均明显增高,且两者之间无差异。
注：与假手术组比较：*P<0.05,**P<0.01；与脾虚模型组比较：◇P<0.05,◇◇P<0.01；与肾虚模型组比较：▲P<0.05,▲▲P<0.01;与脾肾两虚模型组比较：△P<0.05,△AP<0.01；与健脾组比较：☆P<0.05,☆☆P<0.01；与补肾组比较：
P<0.05,
P<0.01。(下同)
2补肾健脾方对脾肾两虚型骨质疏松大鼠骨组织形态计量学指标的影响
2.1补肾健脾方对脾肾两虚型骨质疏松大鼠胫骨TBV%的影响
表2显示,脾虚模型组大鼠胫骨TBV%与假手术组相比无明显差异。肾虚及脾肾两虚模型组TBV%显著低于假手术组及脾虚模型组,且与肾虚模型组相比,脾肾两虚模型组TBV%降低更为明显。
从各治疗组TBV%结果来看,补肾组、健脾组、补肾健脾组、己烯雌酚组大鼠胫骨TBV%均显著高于脾肾两虚模型组,但明显低于假手术组。与健脾组相比,补肾组及补肾健脾组的TBV%明显增高,且补肾健脾组TBV%增高较补肾组更为明显。
2.2补肾健脾方对脾肾两虚型骨质疏松大鼠胫骨TRS%和TFS%的影响
表3显示,与假手术组相比,肾虚及脾肾两虚模型组大鼠胫骨TRS%和TFS%均显著增高,而脾虚模型组无明显差异。与肾虚模型组相比,脾肾两虚模型组大鼠胫骨TRS%和TFS%增高更为明显。
从各治疗组TRS%和TFS%结果来看,补肾组、健脾组、补肾健脾组、己烯雌酚组大鼠胫骨TRS%和TFS%均显著低于脾肾两虚模型组,且补肾健脾组、己烯雌酚组大鼠胫骨TRS%和TFS%相较于假手术组无明显差异,但补肾组、健脾组大鼠胫骨TRS%和TFS%仍明显高于假手术组。与健脾组相比,补肾组及补肾健脾组的TRS%和TFS%明显降低,且补肾健脾组较补肾组降低更为显著。
2.3补肾健脾方对脾肾两虚型骨质疏松大鼠胫骨OSW、MAR和mAR的影响
表4显示,肾虚及脾肾两虚模型组大鼠胫骨OSW、MAR和mAR较之假手术组均显著增高,而脾虚模型组无明显差异。与肾虚模型组相比,脾肾两虚模型组大鼠胫骨MAR和mAR增高更为显著,但OSW无明显差异。
与脾肾两虚模型组相比,补肾组、健脾组、补肾健脾组、己烯雌酚组大鼠胫骨OSW、MAR和mAR均显著降低。与健脾组及补肾组相比,补肾健脾组的MAR和mAR均明显降低,但OSW无明显差异。
3补肾健脾方对脾肾两虚型骨质疏松大鼠外周血血清中OPG、RANKL、VIP、MTL、GAS含量的影响
3.1补肾健脾方对脾肾两虚型骨质疏松大鼠外周血血清中OPG及RANKL含量的影响
表5显示,脾虚模型组大鼠外周血血清中OPG含量与假手术组无明显差异。肾虚、脾肾两虚模型组OPG含量较之假手术组及脾虚模型组均显著降低,且与肾虚模型组相比,脾肾两虚模型组OPG含量降低更为显著。与脾肾两虚模型组相比,健脾组OPG含量无明显差异；而己烯雌酚组、补肾组、补肾健脾组OPG含量均显著增高,但仍显著低于假手术组。与健脾组相比,补肾组和补肾健脾组OPG含量均明显增高,且补肾健脾组明显高于补肾组。
与假手术组相比,脾虚、肾虚、脾肾两虚模型组大鼠外周血血清中RANKL含量均显著增高,且肾虚组RANKL含量明显高于脾虚组,脾肾两虚组又明显高于肾虚组。与脾肾两虚模型组相比,己烯雌酚组、健脾组、补肾组、补肾健脾组RANKL含量均显著降低,但较之假手术组仍显著增高。与健脾组相比,补肾组和补肾健脾组RANKL含量均显著降低,且补肾健脾组明显低于补肾组。
3.2补肾健脾方对脾肾两虚型骨质疏松大鼠外周血血清中VIP含量的影响
表6显示,与假手术组相比,脾虚、肾虚、脾肾两虚模型组大鼠外周血血清中VIP含量均显著增高,且脾虚组VIP含量明显高于肾虚组,脾肾两虚组又显著高于脾虚组。
从各治疗组VIP含量来看,除己烯雌酚组外,健脾组、补肾组、补肾健脾组VIP含量均显著低于脾肾两虚模型组；但健脾组和补肾组仍明显高于假手术组,而补肾健脾组与假手术组无差异。与补肾组相比,健脾组和补肾健脾组VIP含量明显降低,且两者之间无差异。
3.3补肾健脾方对脾肾两虚型骨质疏松大鼠外周血血清中MTL、GAS含量的影响
表7显示,脾虚、肾虚、脾肾两虚模型组大鼠外周血血清中MTL含量较之假手术组均显著降低,脾肾两虚模型组又显著低于脾虚和肾虚组。与脾肾两虚模型组相比,健脾组和补肾健脾组MTL含量均显著增高,且两者之间无差异；但己烯雌酚组和补肾组无明显变化。健脾组和补肾健脾组MTL含量均显著高于补肾组。
与假手术组相比,脾虚、肾虚、脾肾两虚模型组大鼠外周血血清中GAS含量均显著降低,且脾虚组GAS含量明显低于肾虚组,脾肾两虚组又明显低于脾虚组。与脾肾两虚模型组相比,健脾组和补肾健脾组GAS含量均显著增高,但己烯雌酚组和补肾组无明显差异。补肾健脾组GAS含量明显高于健脾组和补肾组。
4补肾健脾方对脾肾两虚型骨质疏松大鼠OB及MSC中OPG>RANKL及VPAC2蛋白和mRNA表达的影响
4.1补肾健脾方对脾肾两虚型骨质疏松大鼠OB及MSC OPG蛋白和mRNA表达的影响
表8显示,与假手术组相比,脾虚模型组大鼠OB及MSC的OPG蛋白和mRNA表达阳性密度与之无差异,而肾虚、脾肾两虚组的OPG阳性密度明显降低,且脾肾两虚组又明显低于肾虚组。
与脾肾两虚模型组相比,己烯雌酚组、健脾组、补肾组、补肾健脾组OPG蛋白和mRNA表达阳性密度均明显增高,但较之假手术组仍明显降低。与健脾组相比,补肾组和补肾健脾组OPG蛋白和mRNA表达阳性密度明显增高,且两者之间无明显差异。
4.2补肾健脾方对脾肾两虚型骨质疏松大鼠OB及MSC RANKL蛋白和mRNA表达的影响
表9显示,与假手术组相比,脾虚、肾虚、脾肾两虚模型组大鼠OB及MSCRANKL蛋白和mRNA表达的阳性密度均明显增高,且肾虚组阳性密度明显高于脾虚组,脾肾两虚组又明显高于肾虚组。
与脾肾两虚模型组相比,己烯雌酚组、健脾组、补肾组、补肾健脾组RANKL蛋白和mRNA表达阳性密度均明显降低,但仍明显高于假手术组。与健脾组相比,补肾组和补肾健脾组RANKL蛋白和mRNA表达阳性密度均明显降低,且两者之间RANKL蛋白表达无差异,但补肾健脾组RANKL mRNA表达阳性密度降低更为明显。
4.3补肾健脾方对脾肾两虚型骨质疏松大鼠OB及MSCVPAC2蛋白和mRNA表达的影响
表10显示,与假手术组相比,脾虚、肾虚、脾肾两虚模型组大鼠OB及MSC中VPAC2蛋白和mRNA表达的阳性密度均明显增高。与肾虚组相比,脾虚、脾肾两虚组VPAC2阳性密度的增高更为明显,且两者之间无差异。
与脾肾两虚模型组相比,己烯雌酚组VPAC2蛋白和mRNA表达阳性密度无差异,健脾组、补肾组、补肾健脾组均明显降低。与补肾组相比,健脾组和补肾健脾组VPAC2蛋白和mRNA表达阳性密度明显降低,且两者之间无差异。
5补肾健脾方对脾肾两虚型骨质疏松大鼠结肠组织中VIP蛋白和mRNA表达的影响
表11显示,与假手术组相比,肾虚模型组大鼠结肠组织中VIP蛋白和mRNA表达阳性密度与之无差异,脾虚、脾肾两虚组的VIP阳性密度显著增高,且脾肾两虚组又明显高于脾虚组。
与脾肾两虚模型组相比,健脾组、补肾组、补肾健脾组VIP蛋白和mRNA表达阳性密度均显著降低,但仍显著高于假手术组。与补肾组相比,补肾健脾组VIP蛋白和mRNA表达阳性密度均明显降低,健脾组与之无差异。
结论
(1)去卵巢能够造成大鼠肾虚型骨质疏松症,而单纯给予大黄浓煎剂可造成大鼠脾虚,但不能导致骨质疏松症。将两种方法相结合,可造成大鼠脾肾两虚型骨质疏松症,且发病程度较单纯去卵巢明显加重。说明脾、肾两脏在骨质疏松症发病过程中均有重要作用,其中肾虚是根本,脾虚是其重要影响因素之一。
(2)健脾方、补肾方和补肾健脾方对脾肾两虚型大鼠骨质疏松症均具有治疗作用,其中,补肾健脾方的疗效明显优于补肾方和健脾方,而补肾方又优于健脾方。
(3)健脾方、补肾方和补肾健脾方共同的作用途径是：一方面,直接调控破骨细胞分化调控因子OPG、RANKL的表达,使OPG表达上调,而使RANKL表达下调,从而明显降低破骨细胞的活性；另一方面,下调VIP和VPAC2表达,导致VIP与VPAC2结合减少,这可能通过成骨细胞的介导作用,上调OPG水平、并下调RANKL水平,最终使破骨细胞的活性降低,骨吸收减少。此外,还能明显提高外周血中MTL及GAS水平。但三者的调节作用存在差异：健脾方对MTL、GAS、VIP及VPAC2的调节作用较强；补肾方对上述指标的调节作用弱于健脾方,但对OPG、RANKL的调节作用较健脾方显著；而补肾健脾方对上述指标的调节作用均明显强于以上两方。这是补肾健脾方疗效优于补肾方和健脾方的机制之一,也进一步说明在骨质疏松症的发生和治疗过程中,中医的脾肾两脏是密切相关的。
Objective
After establishing rat osteoporosis model with spleen and kidney deficiency syndrome, on the basis of OPG/RANKL which is differentiation regulatory factor of osteoclasts and simultaneously selecting VIP/VPAC2which are closely related with Spleen deficiency syndrome and bone metabolism, we would explore the variation characteristics of OPG/RANKL and VIP/VPAC2and the changes of bone resorption and bone formation in the state of spleen deficiency syndrome, kidney deficiency syndrome, and spleen and kidney deficiency syndrome respectively. Moreover, by contrast of the efficacy of recuperating kidney, invigorating spleen, recuperating kidney and invigorating spleen prescription on rats with spleen and kidney deficiency syndrome and the effect on OPG/RANKL signal pathway and VIP/VPAC2, the mechanism of recuperating kidney and invigorating spleen prescription on osteoporosis may be partly clarified, and experimental evidence for the scientific connotation of "spleen and kidney related" will be further provided.
Methods
First, one hundred and ten female Wistar rats were selected and divided into four groups randomly:twelve in normal control group, spleen deficiency model group, sham operation group; seventy-four in bilateral ovariectomized group. After the surgery for10weeks, these seventy-four bilateral ovariectomized rats were divided into six sub-groups again:twelve in kidney deficiency model group, spleen and kidney deficiency model group, diethylstilbestrol group and invigorating spleen group; thirteen in recuperating kidney group and recuperating kidney and invigorating spleen group.
Kidney deficiency syndrome model was Established by Ovariectomy after anesthetized by intraperitoneal injection of sodium pentobarbital, while only the fatty tissue near the ovaries of the rats were extirpated in sham operation group;200% rhubarb concentrated decoction was given orally every day for two weeks, then changed to2times a week for three months to establish the spleen deficiency syndrome model; on this basis, the rat osteoporosis model with spleen and kidney deficiency syndrome has been established by the above two methods after Ovariectomy for ten weeks.
Diethylstilbestrol group was administrated diethylstilbestrol orally which was diluted to0.0048mg/ml with normal saline; invigorating spleen group, recuperating kidney group, recuperating kidney and invigorating spleen group were given orally Sijunzitang, Guiluerxiantang, and the two combined respectively, The drug or decoctions were given once a day, six days continuously then1day pause, the whole period lasted3months. Distilled water was administrated orally to other groups, at the same time. The rats of all groups were weighed once a week, and adjusted the dose accordingly.
At the third day and the sixteenth day before the day all the rats were killed, hydrochloric acheomycin was injected into the abdomen of the rats respectively according to30mg/kg to mark the bone with fluorescence. After Medication, The rats were anesthetized to get artery blood, then the blood was centrifuged to detect the level of Various cytokines in serum by enzyme-linked immunosorbent assay (ELISA).After the rats being killed, the left femur were taken for bone mineral density testing; the right tibia of the rats were retained for bone tissue section which weren't decalcified to evaluate the change of bone histomorphometry; at the same time the left tibia of the rats were used for frozen tissue section after being decalcified to observe the protein and mRNA expression of VPAC2,OPG and RANKL by means of immunohistochemistry and in situ hybridization; in addition,colon tissue were taken for paraffin tissue section to observe the protein and mRNA expression of VIP by means of immunohistochemistry and in situ hybridization.
Results were presented as Mean±SE, normal distribution and homogeneity of variance of the data were analyzed by one-way ANOVA, between any two groups, LSD test; heterogeneity of variance of the data were analyzed by Dunnett's T3test. And non-normal data by non-parametric tests.
Results
1Effects of recuperating kidney and invigorating spleen prescription on femur BMD of osteoporosis rats of spleen and kidney deficiency syndrome model
Compared with sham operation group, femur BMD in kidney deficiency model group and spleen and kidney deficiency model group decreased significantly, and the latter was more significantly. But Spleen model group was not different.
Compared with spleen and kidney deficiency syndrome model group, femur BMD in all treatment groups increased obviously. In recuperating kidney group and recuperating kidney and invigorating spleen group, femur BMD increased more significantly compared with invigorating spleen group, but there was no difference between them.
2Effects of recuperating kidney and invigorating spleen prescription on the bone histomorphometry indicators of osteoporosis rats of spleen and kidney deficiency syndrome model
Compared with sham operation group, TBV%in kidney deficiency model group and spleen and kidney deficiency model group decreased significantly, TRS%, TFS%, OSW, MAR and mAR increased significantly, while Spleen model group was not significantly different. Compared with kidney deficiency model group, TBV%in spleen and kidney deficiency model group decreased obviously, TRS%and TFS%increased significantly.
Compared with spleen and kidney deficiency syndrome model group, TBV%in all treatment groups increased obviously, TRS%、 TFS%、OSW、 MAR and mAR decreased obviously. Compared with invigorating spleen group, TBV%increased and TRS%、 TFS%decreased obviously in recuperating kidney group and recuperating kidney and invigorating spleen group, and the latter reversed these indicators more significantly.
3Effects of recuperating kidney and invigorating spleen prescription on serum level of OPQ RANKL, VIP, MTL and GAS
Compared with sham operation group, the serum level of OPG, MTL and GAS decreased significantly in three model groups, except that the serum level of OPG in spleen deficiency model group was not obviously different. In addition, Spleen and kidney deficiency model group was the lowest. While the serum level of RANKL and VIP increased significantly in three model groups, moreover, Spleen and kidney deficiency model group was the highest.
Compared with spleen and kidney deficiency syndrome model group, the serum level of OPG increased significantly in recuperating kidney group and recuperating kidney and invigorating spleen group, and the latter increased more obviously; the serum level of MTL and GAS increased significantly in invigorating spleen group and recuperating kidney and invigorating spleen group. While the serum level of RANKL decreased significantly in all treatment groups, and among which diethylstilbestrol group and recuperating kidney and invigorating spleen group were optimal; the serum level of VIP decreased significantly in all treatment groups except for diethylstilbestrol group, invigorating spleen group and recuperating kidney and invigorating spleen group decreased more significantly than recuperating kidney group.
4Effects of recuperating kidney and invigorating spleen prescription on protein and mRNA expression of OPG, RANKL and VPAC2
Compared with sham operation group, the protein and mRNA expression of OPG was not obviously different in spleen deficiency model group and decreased significantly in kidney deficiency model group and spleen and kidney deficiency model group, in addition, the latter was significantly lower than the former. Different from OPG, the protein and mRNA expression of RANKL increased significantly in all model groups, and kidney deficiency model group was significantly higher than spleen deficiency model group, spleen and kidney deficiency group was higher than kidney deficiency group. In respect of VPAC2, the protein and mRNA expression in three model groups increased significantly compared with sham operation group, compared with kidney deficiency model group, spleen deficiency model group, spleen and kidney deficiency group increased more, and there was no obvious difference between the two groups.
Compared with spleen and kidney deficiency syndrome model group, the protein and mRNA expression of OPG increased obviously in all treatment groups, but still lower than sham operation group. Compared with invigorating spleen group, recuperating kidney group and recuperating kidney and invigorating spleen group increased more and no obvious difference between them. In contrast, the protein and mRNA expression of RANKL decreased obviously in all treatment groups, but still higher than sham operation group. Compared with invigorating spleen group, recuperating kidney group and recuperating kidney and invigorating spleen group decreased obviously, and the latter decreased more significantly in mRNA expression. In respect of VPAC2, the protein and mRNA expression decreased obviously in all treatment groups except for diethylstilbestrol group. Compared with recuperating kidney group, invigorating spleen group and recuperating kidney and invigorating spleen group decreased obviously, and the former decreased more significantly.5Effects of recuperating kidney and invigorating spleen prescription on protein and mRNA expression of VIP in colon tissue
Compared with sham operation group, the protein and mRNA expression of VIP in colon tissue was not obviously different in kidney deficiency model group and increased significantly in spleen deficiency model group and spleen and kidney deficiency model group, in addition, the latter was significantly higher than the former.
Compared with spleen and kidney deficiency syndrome model group, the protein and mRNA expression of VIP in colon tissue decreased obviously in invigorating spleen group, recuperating kidney group and recuperating kidney and invigorating spleen group, but was not different in diethyl stilbestrol group. The protein and mRNA expression of invigorating spleen group and recuperating kidney and invigorating spleen group decreased significantly compared with recuperating kidney group, and there was no difference between them.
Conclusion
(1) Ovariectomy can cause rat osteoporosis with kidney deficiency syndrome, simply giving the rhubarb concentrated decoction can cause rat spleen deficiency syndrome, but can not lead to osteoporosis. If the two methods combined, which can lead to rat osteoporosis with spleen and kidney deficiency syndrome, and the disease is more serious than that which led by Simple ovariectomy. This shows that both spleen and kidney have important role in the process of osteoporosis, and among which, kidney is fundamental, spleen is one of the important factors.
(2) The prescription of recuperating kidney, invigorating spleen, recuperating kidney and invigorating spleen all have therapeutic effects on osteoporosis rats with spleen and kidney deficiency syndrome, and that of recuperating kidney and invigorating spleen works best, followed by recuperating kidney, and followed by invigorating spleen.
(3) The common pathway of three prescriptions is:On the one hand, it can directly regulate the expression of OPG and RANKL, the expression of OPG up-regulated and RANKL down-regulated, so that the osteoclasts activity decreased obviously; on the other hand, it can down-regulate the expression of VIP and VPAC2and thereby reduce the combination of VIP and VPAC2, which can up-regulate OPG levels and down-regulate RANKL levels probably by osteoblast-mediated effect, and ultimately led to reduction of the osteoclasts activity and the bone resorption. In addition, MTL and GAS levels can also be raised significantly in peripheral blood. However, there are some differences among the three ways:invigorating spleen prescription played a stronger role in MTL, GAS, VIP and VPAC2; although the effects of recuperating kidney Prescription on these indicators were weaker than the former, but it can play more significant role in OPG and RANKL; while these indicators were improved significantly by recuperating kidney and invigorating spleen prescription. This may be one of the mechanisms of recuperating kidney and invigorating spleen prescription which was more effective than the other two prescriptions, it further demonstrates that spleen and kidney in Traditional Chinese Medicine are closely related in the occurrence and treatment process of osteoporosis.
引文
[1]Cooper CEP idemiology of osteoporosis[J].osteoporos Intl999;9(SupplZ):52-8
[2]中华医学会编著.临床诊疗指南·骨质疏松症和骨矿盐疾病分册[M].北京：人民卫生出版社,2010
[3]Nguye TV.Association between brest cancel and bone mineral density:the Dubbo Osteporosis Epidemiology Study[J].Maturitas,2007,36(1):27-34
[4]李宁华,区品中,朱汉民,等.中国部分地区中老年人群原发性骨质疏松症患病率研究[J].中华骨科杂志,2001,21(5)：275-278
[5]张亚军,刘忠厚,张鹏.中国原发性骨质疏松症流行病学[J].中国骨质疏松杂志2010,16(3)：229-234
[6]薛延.骨质疏松症防治指南[M].北京：人民卫生出版社,2008
[7]郭世绂.骨质疏松基础与临床[M].天津：天津科技出版社,2001：535
[8]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,2005,17(2)：3-5
[9]张进,徐志伟.“肾藏精,主骨,生髓”理论内涵辨析[J].中国中医基础医学杂志,2009,15(11)：805-809
[10]张颖,鞠大宏,张博,等.从肝脾肾三脏探讨绝经后骨质疏松症的发病机理[J].中国中医基础医学杂志,2012,18(1)：36-37
[11]杨芳.骨质疏松症与中医脾虚证的关系探讨[J].山东中医药大学学报,2009,33(5)：368-369
[12]冯尔宥,宋敏.绝经后骨质疏松症病机的探讨[J].甘肃中医学院学报.2002,19(1)： 8
[13]刘忠厚.骨质疏松学[M].北京：科学出版社,1998：319-320
[14]杨帆,邓兆智,韩云,等.中药治疗骨质疏松症的用药规律探析[J].广东药学院学报,2003,19(1)：70
[15]张洪,魏之玉,王景存.补肾疏肝健脾方治疗老年原发性骨质疏松症38例[J].山东中医药大学学报,1997,21(5)：375
[16]郭郡浩,蔡辉.从瘀论治原发性骨质疏松症研究进展[J].河北中医,2008,30(11)：1225-1227
[17]Meumie HE.Predictions on future diagnosis and treatment of osteoporosis [J].Calcif-Tissue-Int.1995,57(2):83
[18]马中兴,高文杰,魏小堂,等.中医学对骨质疏松症病因病机的认识[J].中医研究,2012,25(1)：14-15
[19]葛瑞.从络病学说论治骨质疏松症[J].辽宁中医药大学学报,2010,12(1)：65-66
[20]眭承志,刘志坤,陈少玫,等.绝经后骨质疏松症与血瘀[J].中国中西医结合杂志,2005,25(5)：456-458
[21]周亚娜.补肾化痰法影响骨髓间充质干细胞成脂和成骨分化的实验研究[D].武汉：湖北中医学院,2008
[22]鞠大宏,张春英,王安民,等.左归丸对去卵巢所致大鼠骨质疏松症的治疗作用[J].中国中医基础医学杂志,2001,7(3)：17-21
[23]鞠大宏,吴萍,贾红伟,等.左归丸对卵巢切除所致骨质疏松大鼠骨钙素和降钙素含量的影响[J].中国中医药信息杂志.2003,10(1)：16-20
[24]鞠大宏,张春英,徐世杰,等.温补肾阳方对去卵巢大鼠骨质疏松症的治疗作用[J].中国中医基础医学杂志,2000,6(4)：29-33
[25]鞠大宏,张丽坤,吴皓,等.温补肾阳方对卵巢切除所致骨质疏松大鼠外周血浆中促肾上腺皮质激素和p-内啡肽含量的影响[J].中国中医基础医学杂志,2003,9(5)：30-33
[26]黄连芳,吴铁,谢华,等.何首乌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
[27]罗小玲,梁晓萍,文锦丽.蛇床子总香豆素对骨质疏松大鼠骨密度、骨形态计量学影响[J].中国中医急症,2008,17(3)：368-369
[28]李春雯,史晓林,刘康,等.强骨饮与福善美对去势大鼠腰椎骨形态计量学的对比实验研究[J].中国中医骨伤科杂志,2007,15(9)：41-43
[29]阮祥燕,齐久梅,刘玉兰,等.中药对绝经妇女股骨颈骨密度及股骨颈抗骨折能力的影响[J].中国骨质疏松杂志,2006,12(2)：181-184
[30]玄明实,哈孝贤,于顺禄,等.中药强骨丹对去卵巢骨质疏松大鼠骨密度和骨生物力学的影响[J].天津中医药,2007,24(2)：147-148
[31]刘剑梅,葛宝丰,白孟海,等.红景天壮骨丸治疗骨质疏松症的实验研究[J].中 国临床康复,2004,8(27)：5911
[32]廖进民,李青南.延藿骨宝对实验性大鼠骨质疏松的骨密度和生物力学影响[J].中药材,2002,25(11)：809
[33]Teitelbaum SL. Osteoclasts:what do they do and how do they do it? [J].Am J Pathol,2007,170:427-435
[34]刘庆思,庄洪,黄宏兴,等.骨质疏松症中西医结合治疗[M].北京：人民卫生出版社,2006：146-151
[35]张宁,刘世巍,韩凤岳,等.补肾活血中药对体外培养成骨细胞活性的影响[J].中国中医药信息杂志,2005：12(1)：40-42
[36]马金,郑洪新.补肾中药防治骨质疏松症的细胞分子生物学机理探要[J].中医药学刊,2002,20(2)：161-163
[37]林煜,吴银生,卢天祥,等.健骨颗粒对成骨细胞分化的影响[J].中华中医药杂志,2012,27(1)：162-165
[38]高晓燕,杜晓鹃,赵春颖.补肾中药对成骨样细胞UMR106增殖的影响[J].承德医学院学报,2001,18(4)：283
[39]陈顺,关延彬.骨碎补药理作用的研究进展[J].医药导报,2006,25(7)：685
[40]刘剑刚,谢雁鸣,赵晋宁,等.骨碎补总黄酮胶囊对实验性骨质疏松症和镇痛作用的影响[J].中国实验方剂学杂志,2004,10(5)：31-34
[41]王威,史红,何永志,等.中药续断含药血清对成骨细胞增殖和骨基质蛋白产生的影响[J].中国骨质疏松杂志,2009,15(2)：103-106
[42]Kim HK, Woo ER, L ee HW, et al. The correlation of Salvia miltiorrhiza extract induced regulation of osteoclas togenesis with the amount of components tan-shinone I, tanshinone IIA, cryptotanshinone,and dihydrot anshin one[J].Immu nopharmacol Im munot oxicol,2008,30(2):347-364
[43]吴小霞,周则卫.雌激素的研究进展[J].医药导报,2008,27(10)：1234-1236
[44]张颖.左归丸对去卵巢所致骨质疏松症大股骨骨髓中相关蛋白质的影响[D].北京：中国中医科学院,2012
[45]Khosla S, Melton LJ 3rd, Atkinson EJ, et al. Relationship of serum sex steroid levels and bone turnover markers with bone mineral density in men and women: a key role for bioavailable estrogen[J]. J Clin Endocrinol Metab,1998,83:2266-2268
[46]梅敏.骨质疏松症治疗研究进展[J].吉林医学,2011,32(4)：749-751
[47]Arijmandi BH, Salih MA, Herbert DC,et al.Evidence for estrogen recaptor-link-ed calcium transport in the intestine. J Bone Miner 1993,21:63
[48]支英杰,谢雁鸣.中医药对绝经后骨质疏松症的现代研究进展[J].中医杂志,2010：51(1)：241-243
[49]邓伟民,黄海,曾东,等.补肾壮骨冲剂对更年期综合征患者性激素及血脂的影响[J].中医杂志,2011,52(3)：208-213
[50]郑建靖,徐美娟.益肾活血汤对骨质疏松模型大鼠作用的研究[J].药物研究,2001,13(8)：7-8
[51]单丽娟,李凤森,张伟.骨健颗粒剂对去势大鼠激素水平影响的实验研究[J].中医药学刊,2004,22(1)：66
[52]Scheidt-Nave C, Bismar H, Leidig-Bruckner G, et al.Serum interleukin-6 is a major predictor of bone loss in women specific to the first decade past menopause[J]. Clin Endocrinol Metab,2001,86(5):2032-2042
[53]谭清武,陈俊文.补肾健脾活血方药对老年骨质疏松大鼠血清L-6的影响[J].中国中医药科技,2002,9(6)：331-339
[54]鞠大宏,赵宏艳,刘梅洁,等.左归丸含药血清对成骨细胞IL-1、IL-6和COX-2表达的影响[J].中国实验动物学报,2006,14(2)：27-30
[55]吴昊,查振刚,姚平,等.淫羊藿苷对骨髓间充质干细胞骨向诱导的实验研究[J].中国中西医结合杂志,2010,30(4)：410-414
[56]张荣华,杨丽,朱晓峰,等.益骨胶囊对去卵巢骨质疏松大鼠骨组织TGF-β1 mRNA表达的影响[J].中药材,2005,28(4)：312-315
[57]Myers M GJ, Sun XJ, Chertham B, et al.IRS-1 is a common element ininsu-lin and insulin-like growth factor-I sign al ingt otheprosphatidy linositol3-kinase [J].Endocrinology,1993,132(4):1421
[58]王勇刚,昝强,徐武清,等.补肾活血法对去势大鼠骨质疏松模型血清IGF-I的影响[J].江苏中医药,2009,41(1)：70-71
[59]周立飞,高肖波,刘振东.补肾健脾汤对绝经后骨质疏松症患者细胞因子、 骨密度及雌激素水平的影响[J].中国中医药科技,2008,15(3)：170-171
[60]何志鹏.葛根对去卵巢大鼠骨质疏松作用的研究[J].牡丹江医学院学报,2008,29(5)：9-11
[61]付萍,李伟,杨铭,等.蛇床子总香豆素对去卵巢大鼠骨质疏松症大鼠骨矿和骨微量元素的影响[J].世界元素医学,2008,15(3)：41-43
[62]刘梅洁,吕国红,邹军,等.左归丸含药血清对成骨细胞分泌骨钙素的影响[J].中国中医基础医学杂志,2007,15(8)：581-582
[63]吕海波.左归丸对去卵巢大鼠骨组织中TGF-β、 Smad信号转导通路的研究[D].沈阳：辽宁中医药大学,2011
[64]龚建文.从P38信号通路探讨中药骨康方对去势大鼠MSCs成骨分化的机制研究[D].广州：广州中医药大学,2011
[65]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
[66]Gori F, Hofbauer LC, Dunstan CR, et al. The expression of osteoprotegerin and RANK ligand and the support of osteoclast by stromalosteoblast lineage cells is developmentaly regulated[J]. Endocrinology,2000,141(12):4768-4776
[67]齐尚峰,张永波.中医药防治骨质疏松症的研究概况[J].中国骨质疏松杂志,2009,15(3)：230-235
[68]江湧,杨海韵.骨质疏松症从痿痹瘀痉辨治[J].新中医,2007,39(7)：3
[69]谢雁鸣,朱云茵,葛继荣,等.基于临床流行病学调查的原发性骨质疏松症中医基本证候研究[J].世界科学技术-中医药现代化,2007,9(2)：38-44
[70]方朝晖,耿家金,张有志.1000例老年性骨质疏松症证候分布与组合规律研究[J].中国中医药信息杂志,2007,14(5)：15-17
[71]史晓,蔡奇文,杜修东,等.原发性骨质疏松症患者中医量表的信度及效度研究[J].中医杂志,2009,15(2)：124-126
[72]徐祖健,等.绝经后妇女原发性骨质疏松症中医辨证分型与骨密度的相关性研究[J].泸州医学院学报,2008,31(4)：390
[73]孙江波,刘绪银,廖怀章.骨质疏松症中医定位辨证与骨密度水平的相关性研究[J].世界中西医结合杂志,2011,6(1)：33-34
[74]穆刚,王平,张君涛.对原发性骨质疏松症证型实质的探讨[J].中国中医骨 伤科杂志,2006,14(1)：66-67
[75]Nelson HD,Humphrey LL,Nygren P,et al,Postmenopausal hormonereplacement theraphy:Scientific review [J] JAMA,2002,288(7):8722881
[76]Pak CY,Zerwekh JE,Antich P.Anabolic effects of fluoride on bone7[J].Trends Endocrinol Metab,1995,6(7):2229-2234
[77]谢雁鸣,朱芸茵,吴泰相.中医药治疗绝经后骨质疏松的疗效及安全性系统评价[J].中国循证医学杂志,2005,(01)：29-41
[78]阳波,杨静.黄芪对绝经后骨质疏松症患者影响的临床研究[J].四川中医,2007,28(3)：291
[79]邹丽宜,吴铁,崔燎.黄芪多糖对肝纤维化小鼠骨丢失的防治作用[J].中西医结合肝病杂志,2002,12(2)：95-98
[80]刘海全,秦佳佳.骨康颗粒治疗肾阳虚型绝经后骨质疏松症的疗效观[J].山东中医药大学学报,2010,34(1)：48-49
[81]王照康.补肾健脾密骨方对老年骨质疏松患者疼痛及其相关生化指标的改善作用[J].中国临床康复,2004,8(27)：5930-5949
[82]王惠礼,杨晓玉.骨康宁治疗原发性骨质疏松症的疗效观察[J].中国医学创新,2012,9(1)：29-30
[83]刘树胜,解增友,耿读海.中医学对骨质疏松症病因病机的认识[J].河北中医,2012,34(1)：32-33
[84]史传道,袁普卫,杨晓航.抗疏健骨颗粒治疗绝经后骨质疏松症40例[J].山西中医,2012,28(1)：16-17
[85]白洁,白孟海,刘茗露,等.中药骨疏康治疗绝经后骨质疏松症临床观察[J].西部中医药,2012,25(1)：22-24
[86]梁冬波,黄承军,娄宇明,等.补阳还五汤治疗绝经后骨质疏松症疗效观察[J].现代中西医结合杂志,2012,21(8)：829-830
[87]王建伟,马勇,尹恒,等.金匮肾气丸联用葡萄糖酸钙对原发性骨质疏松症疼痛临床疗效观察[J].辽宁中医药大学学报,2012,14(2)：5-6
[88]何成奇,熊恩富,饶凡,等.骨痛宁擦剂治疗骨质疏松疼痛的临床疗效观察[J].中医研究,2001,14(2)：27-29
[89]赵利华,农泽宁,钟旋,等.温针灸对绝经后骨质疏松症患者骨密度及生化 指标的影响[J].中国针灸,2005,25(12)：597-900
[90]吴明霞,吴炳煌,刘献祥,等.灸对绝经后骨质疏松症影响的实验和临床研究[J].中国针灸,2002,22(5)：334-336
[91]徐娅莉,金建军,徐登玉,等.针刺加穴位贴敷对原发性骨质疏松症患者骨密度及疼痛的影响[J].中国针灸,2006,26(2)：87-90
[92]林莺,许金榜,李沛.针药结合对绝经后妇女骨代谢调节作用的临床研究[J].福建医药杂志,2009,31(6)：131-133
[93]林志苇,黎健,高丽萍等.肾俞穴位埋线对原发性骨质疏松症骨密度影响的研究[J].中国骨质疏松杂志,2006,12(4)：381-383
[94]熊芳丽,肖亚平.耳针治疗老年妇女骨质疏松症60例临床观察[J].贵阳中医学院学报,2000,22(2)：33-34
[95]李小平.运用低频脉冲电磁场治疗原发性骨质疏松症[J].湖北中医杂志,2011,33(3)：60-61
[96]Kanis JA, Burlet N, Cooper C, et al. European guidance for the diagnosis and management of osteoporosis in postmenopausal women[J]. Osteoporos Int, 2008,19:399-428
[97]National Osteoporosis Foundation. Physician's guide to prevention and treat-ment of osteoporosis[J].National osteoporosis foundation,2008
[98]杨玺.骨质疏松症防治[M].上海：科学技术文献出版社,2003：13
[99]杨芬分.骨质疏松症的预防[J].柳州医学,2007,20(2)：71-73
[100]李新民,宋艳.合理营养与骨质疏松症的预防[J].中国现代药物应用,2007,1(1)：52-53
[101]Porthouse J,Cockekayne S,King C,et al.Randomised controlled trial of calcium and supplementation with cholecalciferol(vitamin D3)for prevention of fractures in primary Care[J]. BMJ,2005,30(330),1005
[102]Grant AM,Avenell A,Campbell,MK,et al.Oral vitamin D3 and calcium for secondary prevention of low-trauma fractures in elderly people (Randomised Evaluation for Calcium Or vitamin D RECORD);a randomized placebo controlled trial[J].Lancet,2005,365 (9471):1621-1628
[103]李燕燕,韩继红.中医食疗对老年骨质疏松症患者临床症状和骨密度的影 响研究[J].护士进修杂志,2010,25(6)：490-492
[104]程晓明.骨质疏松症预防保健新进展[J].现代预防医学,2008,35(24)：64-65
[105]郭晓东,赵民生,曹秀虹.骨质疏松症的预防与治疗[J].社区医学杂志,2010,8(12)：16-17
[106]Devine A,Hodgson JM,Dick IM,et al.Tea drinking is associated with benefits on bone density in older women[J].AM J Clin Nutr2007,86(4):1243-1247
[107]戴闽,昌军.骨科运动康复[M].北京：人民卫生出版社,2008：243.
[108]张林,杨锡让,薛延.运动与人体骨密度变化研究进展[J].北京体育大学学报,2000,23(1)：64
[109]阮彩莲,白亚军,刘运磊.不同有氧运动时间对衰老大鼠骨质成分的影响[J].中国组织工程研究与临床康复,2009,28(13)：5500-5504
[110]张磊,包可,王勇.骨质疏松症中西医结合预防及治疗的研究进展[J].中西医结合研究,2011,3(3)：153-155
[111]Sagiv M,Vogelaere PP,Soudry M,et al.Role of physical activity training I attenuation of height loss through aging[J]. Gerontology,2000,46(5):266-270
[112]郁嫣嫣,祁奇,余波,等.太极拳锻炼对绝经后女性骨密度的影响[J].中国康复理论与实践,2012,18(2)：155-157
[113]邹军,林菲,张丽,等.长期太极拳运动对绝经后妇女骨密度和平衡能力的影响[J].中国康复理论与实践,2011,17(1)：80-82
[114]中华中医药学会.中医体质分类与判定[M].北京：中国中医药出版社,2009.
[115]蔡亮,江静华,杨健松.骨量减少及骨质疏松症中医体质分类与辨证分型相关性研究[J].新中医,2011,43(7)：51-52
[1]支英杰,谢雁鸣.中医药对绝经后骨质疏松症的现代研究进展[J].中医杂志,2010,51(1)：241-243
[2]鞠大宏,刘梅洁,赵宏艳,等.左归丸含药血清对成骨细胞OPG、RANKL mRNA表达的影响[J].北京中医药大学学报,2008,31(5)：312-315
[3]刘梅洁,鞠大宏,赵宏艳,等.“肾主骨”的机理研究-左归丸含药血清对破骨细胞分化调控因子OPG、RANKL蛋白表达的影响[J].中国中医基础医学杂志,2009,15(3)：184-187,196
[4]邓铁涛.略论五脏相关取代五行学说.广州中医学院学报,1988,(2)：4
[5]李浩钢.骨密Ⅰ、Ⅱ号方脾肾同补治疗骨质疏松症32例[J].上海中医药杂志,1997(2)：28
[6]王照康.补肾健脾密骨方对老年骨质疏松患者疼痛及其相关生化指标的改善作用[J].中国临床康复,2004,8(27)：5930-5949
[7]王惠礼,杨晓玉.骨康宁治疗原发性骨质疏松症的疗效观察[J].中国医学创新,2012,9(1)：29-30
[9]欧阳钢,王玲玲,卓铁军,等.不同刺激方法对原发性骨质疏松症骨密度的影响[J].中国针灸,2004,24(1)：25-26
[9]王东岩,欧阳钢.针药结合原发性骨质疏松症患者骨密度的影响[J].中国中医骨伤科杂志,2005,14(12)：911-913
[10]徐敏,刘保新,黄承军,等.原绝经后骨质疏松症的穴位治疗进展[J].浙江中医药大学学报,2011,35(2)：301-302
[11]孙爱华.肾与脾关系物质基础的实验观察[J].中医杂志,1998,39(1)：47-9
[12]高守泉.脾肾相关性的实验研究[J].湖南中医药导报,1998,4(12)：26-27
[13]吕爱平,李德新,崔家鹏.脾肾相关的分子生物学基础—脾肾阳虚证模型大鼠自由基损伤的比较研究[J].辽宁中医杂志,2001,28(3)：189-91
[14]吕爱平,李德新,崔家鹏.脾肾阳虚模型大鼠肝细胞线粒体磷脂组分变化的比较研究[J].中医药学刊,2003,21(1)：78
[15]罗卫芳,郭树仁,程士德,等.中医学肾本质现代研究概述[J].中国中医基础医学杂志,2001,7(4)：75-77
[16]任艳玲,郑洪新,杜松.补肾益气生精中药对去卵巢大鼠骨质疏松发生机制的调节作用[J].北京中医药大学学报,2006,29(2)：94-97
[17]郑洪新,刘伶,林庶茹,等.参茸补血丸对去卵巢大鼠骨质疏松防治作用的实验研究[J].中国老年学杂志,2006,26(3)：385-388
[18]张倩,胡小琪,郭宏霞,等.我国藏族和汉族儿童少年骨转换研究[J].中国骨质疏松杂志,2008,14(6)：454-457
[19]李燕舞,王汝俊,王建华.大黄脾虚模型大鼠胃壁细胞的超微结构观察[J].中国中西医结合消化杂志,2005,13(3)：155-157
[20]南京医学院中西医结合研究组.脾虚泄泻动物模型的研制[JI].浙江中医杂志,1982,17(8)：355
[21]刘汶.番泻叶致脾虚证动物模型的造型方法[J].中国中西医结合脾胃杂志,1998,6(4)：231
[22]曲长江.番泻叶泻下与劳倦过度单、复因素脾虚模型的免疫学研究[J].中国中西医结合脾胃杂志,1999,7(4)：212
[23]任平.四君子汤对脾虚模型大鼠胃动素和前列腺素E2含量的影响[J].中药药理与临床,1994,10(6)：7
[24]周奇志.实验性脾虚证大鼠脑内单胺递质的变化及其择时治疗效应[J].成都中医药大学学报,1998,21(3)：36
[25]易杰,李德新.脾虚证动物模型研究思路和方法探析[J].上海中医药杂志,2001,5(5)：40-42
[26]陈奇.中药药理研究方法学[M].北京：人民卫生出版社.1993,1023
[27]鞠大宏,张丽坤,吴皓,等.温补肾阳方对卵巢切除所致骨质疏松大鼠外周血浆中促肾上腺皮质激素和p-内啡肽含量的影响[J].中国中医基础医学杂志,2003,9(5)：30-33
[28]鞠大宏,吕爱平,张春英,等.左归丸对卵巢切除所致骨质疏松大鼠IL-1和IL-6活性的影响[J-].中医杂志,2002,43(10)：777-779
[29]鞠大宏,张春英,王安民,等.温补肾阳方对去卵巢所致骨质疏松大鼠IL-1和IL-6活性的影响[J].中国中医基础医学杂志,2000,6(10)：5-9
[30]陈津岩,王立峰,赵慧,等.右归丸对脾肾两虚证大鼠甲状腺激素及环核苷酸水平的影响[J].广州中医药大学学报,2009,26(4)：377-380
[31]王立峰,陈芝喜,赵慧,等.强肌健力方对脾肾两虚证大鼠血浆性激素水平的影响[J].放射免疫学杂志,2008,21(2)：147-149
[32]刘梅洁,李鸿泓,鞠大宏,等.骨质疏松症脾肾两虚型病证结合动物模型的建立[J].中国中医基础医学杂志,2012,18(8)：836-838,844
[33]周修通.“龟鹿二仙汤”为主治疗骨质疏松症临床观察[J].上海中医药杂志,2001,47(11)：24
[34]周修通.“龟鹿二仙汤”治疗骨质疏松症45例临床观察[J].江苏中医,2001,22(6)： 28
[35]杨伟岸.龟鹿二仙汤加味治疗老年脊椎骨质疏松性腰背部疼痛63例[J].福建中医药,2005,50(5)：19
[36]尹斯利,金勋杰,吴铁,等.中药成方四君子汤对去卵巢大鼠血清骨钙素水平影响[J].中国骨质疏松杂志,2000,6(3)：69-70
[37]鞠大宏,李鸿泓,刘梅洁,等.补肾健脾方对大鼠脾肾两虚型骨质疏松症的治疗作用[J].中华中医药杂志,2012,12(27)：3207-3210
[38]Simonetws, Lacey D L, Dunstan C R. Osteoprotegerin:A novel secreted protein involved in the regulation ofbone density[J].Cell,1997,89 (2):309-319
[39]Gorif, Hofbauer L C, Dunstan C R. The expressionof osteoprotegerin and RANK ligand and the support ofosteoclast formation by stromal osteoblast lineagecells isdevelopmentally regulated[J].Endocrinology,2009,141 (12):4768-4776
[40]Yasuda H. Bone and bone related biochemical examinations. Bone and collagen related metabolites. Receptor activator of NF-kappaBligand(RANKL) [J].Clin Calcium,2006,16(6):964-970
[41]Shimizu-Ishiura M,Kawana F, Sasaki T.Osteoprotegerin administration reduces femural bone loss in ovariectomizedmice via impairment of osteoclast structure and function[J].Electron Microsc(Tokyo),2002,51(5):315-325
[42]Bekker PJ, Holloway D, Nakanishi A, et al.The effect of a single dose of osteopotegerin in postmenopausal women[J].Bone Mine Res,2001,16:3348-60
[43]Clohisy D.Cellular mechanisms of osteolysis[J].Bone Joint Surg Am,2003,85 Suppl 1:S4-6
[44]Gori F, Hofbauer LC, Dunstan CR, et al. The expression of osteoprotegerin and RANK ligand and the support of osteoclast by stromalosteoblast lineage cells is developmentaly regulated[J]. Endocrinology,2000,141(12):4768-4776
[45]Yano K,Tsuda E,Washida N,et al.Immunological characterization of eirculating osteoprotegerin/osteoclastogenesis inhibitory factor:increased serum concen-trations in postmenopausal women with osteoporosis [J].J Bone Miner Res,1999, 14(4):518-527
[46]Eghbali-Fatourechi QKhosla S,Sanyal A,et al.Role of RANK ligand in media-ting increased bone resorption in early postmenopausal women [J].J Clin Invest, 2003,111(8):1221-1230
[47]Hofbauer LC, Khosla S, Dunstan CR, et al. Estrogen stimulates gene expression and protein production of osteoproteigern in human osteoblastic cells[J].Endori-nology,1999,140(9):4367-4370
[48]郭梁.成骨细胞破骨细胞及OPG/RANKL/RANK轴与骨质疏松[J].中医正骨,2010,22(7)：41-44
[49]喻明丽,沈志强.OPG/RANK/RANKL系统在骨质疏松中的研究进展[J].昆明医学院学报,2012,33(1B)：194-197
[50]张忠兵,张学庸,陈希陶,等.脾虚泄泻患者肠粘膜中SP和VIP初步探讨[J].中西医结合杂志,1991,11(3)：144
[51]贾波,邓中甲,黄秀深,等.白术茯苓汤及其配伍对脾虚大鼠胃肠激素的影响[J].成都中医药大学学报,2002,25(2)：13-15,64
[52]黄学宽,任凌燕,李进,等.六味和中散对胃肠动力及胃肠激素的影响[J].中药新药与临床药理,2007,18(3)：205-207
[53]Ransjo M, Lie A, MukohyamaH, Lundberg P, et al. Microisolate mouse osteoclasts express VIP-1 and PACAP receptor[J].Biochem Biophys Res Commun,2000,274(2),400-40
[54]Lundberg P, Lie A, Bjurholm A, et al. Vasoactive intestinapeptide regulates osteoclast activity via specific binding sites oboth osteoclasts and osteoblasts[J].Bone,2000,27(6):80-10
[55]Perssona E, Voznesenskyc OS, Huang YF, et al. Increased expression of interleukin-6 by vasoactive intestinal peptide is associated with regulation of CREB, AP-1 and C/EBP, but not NF-κB, in mouse calvarial osteoblasts[J].Bone, 2005,37(4):513-529
[56]Perssona E, Lerner UH. The neuropeptide VIP potentiates IL-6 production induced by proinflammatory osteotropic cytokines in calvarial osteoblasts and the osteoblastic cell lineMC3T3-E1[J].Biochem Biophys Res Commun,2005, 335 (3):705-711
[57]Nagata A, Tanaka T, Minezawa A, et al. cAMP activation by PACAP/VIP stimulates IL-6 release and inhibits osteoblastic differentiation through VPAC2 receptor in osteoblastic MC3T3 cells. J Cell Physiol[J].2009,221(1):75-83
[2]中华医学会编著.临床诊疗指南·骨质疏松症和骨矿盐疾病分册[M].北京：人民卫生出版社,2010
[3]Nguye TV.Association between brest cancel and bone mineral density:the Dubbo Osteporosis Epidemiology Study[J].Maturitas,2007,36(1):27-34
[4]李宁华,区品中,朱汉民,等.中国部分地区中老年人群原发性骨质疏松症患病率研究[J].中华骨科杂志,2001,21(5)：275-278
[5]张亚军,刘忠厚,张鹏.中国原发性骨质疏松症流行病学[J].中国骨质疏松杂志2010,16(3)：229-234
[6]薛延.骨质疏松症防治指南[M].北京：人民卫生出版社,2008
[7]郭世绂.骨质疏松基础与临床[M].天津：天津科技出版社,2001：535
[8]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,2005,17(2)：3-5
[9]张进,徐志伟.“肾藏精,主骨,生髓”理论内涵辨析[J].中国中医基础医学杂志,2009,15(11)：805-809
[10]张颖,鞠大宏,张博,等.从肝脾肾三脏探讨绝经后骨质疏松症的发病机理[J].中国中医基础医学杂志,2012,18(1)：36-37
[11]杨芳.骨质疏松症与中医脾虚证的关系探讨[J].山东中医药大学学报,2009,33(5)：368-369
[12]冯尔宥,宋敏.绝经后骨质疏松症病机的探讨[J].甘肃中医学院学报.2002,19(1)： 8
[13]刘忠厚.骨质疏松学[M].北京：科学出版社,1998：319-320
[14]杨帆,邓兆智,韩云,等.中药治疗骨质疏松症的用药规律探析[J].广东药学院学报,2003,19(1)：70
[15]张洪,魏之玉,王景存.补肾疏肝健脾方治疗老年原发性骨质疏松症38例[J].山东中医药大学学报,1997,21(5)：375
[16]郭郡浩,蔡辉.从瘀论治原发性骨质疏松症研究进展[J].河北中医,2008,30(11)：1225-1227
[17]Meumie HE.Predictions on future diagnosis and treatment of osteoporosis [J].Calcif-Tissue-Int.1995,57(2):83
[18]马中兴,高文杰,魏小堂,等.中医学对骨质疏松症病因病机的认识[J].中医研究,2012,25(1)：14-15
[19]葛瑞.从络病学说论治骨质疏松症[J].辽宁中医药大学学报,2010,12(1)：65-66
[20]眭承志,刘志坤,陈少玫,等.绝经后骨质疏松症与血瘀[J].中国中西医结合杂志,2005,25(5)：456-458
[21]周亚娜.补肾化痰法影响骨髓间充质干细胞成脂和成骨分化的实验研究[D].武汉：湖北中医学院,2008
[22]鞠大宏,张春英,王安民,等.左归丸对去卵巢所致大鼠骨质疏松症的治疗作用[J].中国中医基础医学杂志,2001,7(3)：17-21
[23]鞠大宏,吴萍,贾红伟,等.左归丸对卵巢切除所致骨质疏松大鼠骨钙素和降钙素含量的影响[J].中国中医药信息杂志.2003,10(1)：16-20
[24]鞠大宏,张春英,徐世杰,等.温补肾阳方对去卵巢大鼠骨质疏松症的治疗作用[J].中国中医基础医学杂志,2000,6(4)：29-33
[25]鞠大宏,张丽坤,吴皓,等.温补肾阳方对卵巢切除所致骨质疏松大鼠外周血浆中促肾上腺皮质激素和p-内啡肽含量的影响[J].中国中医基础医学杂志,2003,9(5)：30-33
[26]黄连芳,吴铁,谢华,等.何首乌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
[27]罗小玲,梁晓萍,文锦丽.蛇床子总香豆素对骨质疏松大鼠骨密度、骨形态计量学影响[J].中国中医急症,2008,17(3)：368-369
[28]李春雯,史晓林,刘康,等.强骨饮与福善美对去势大鼠腰椎骨形态计量学的对比实验研究[J].中国中医骨伤科杂志,2007,15(9)：41-43
[29]阮祥燕,齐久梅,刘玉兰,等.中药对绝经妇女股骨颈骨密度及股骨颈抗骨折能力的影响[J].中国骨质疏松杂志,2006,12(2)：181-184
[30]玄明实,哈孝贤,于顺禄,等.中药强骨丹对去卵巢骨质疏松大鼠骨密度和骨生物力学的影响[J].天津中医药,2007,24(2)：147-148
[31]刘剑梅,葛宝丰,白孟海,等.红景天壮骨丸治疗骨质疏松症的实验研究[J].中 国临床康复,2004,8(27)：5911
[32]廖进民,李青南.延藿骨宝对实验性大鼠骨质疏松的骨密度和生物力学影响[J].中药材,2002,25(11)：809
[33]Teitelbaum SL. Osteoclasts:what do they do and how do they do it? [J].Am J Pathol,2007,170:427-435
[34]刘庆思,庄洪,黄宏兴,等.骨质疏松症中西医结合治疗[M].北京：人民卫生出版社,2006：146-151
[35]张宁,刘世巍,韩凤岳,等.补肾活血中药对体外培养成骨细胞活性的影响[J].中国中医药信息杂志,2005：12(1)：40-42
[36]马金,郑洪新.补肾中药防治骨质疏松症的细胞分子生物学机理探要[J].中医药学刊,2002,20(2)：161-163
[37]林煜,吴银生,卢天祥,等.健骨颗粒对成骨细胞分化的影响[J].中华中医药杂志,2012,27(1)：162-165
[38]高晓燕,杜晓鹃,赵春颖.补肾中药对成骨样细胞UMR106增殖的影响[J].承德医学院学报,2001,18(4)：283
[39]陈顺,关延彬.骨碎补药理作用的研究进展[J].医药导报,2006,25(7)：685
[40]刘剑刚,谢雁鸣,赵晋宁,等.骨碎补总黄酮胶囊对实验性骨质疏松症和镇痛作用的影响[J].中国实验方剂学杂志,2004,10(5)：31-34
[41]王威,史红,何永志,等.中药续断含药血清对成骨细胞增殖和骨基质蛋白产生的影响[J].中国骨质疏松杂志,2009,15(2)：103-106
[42]Kim HK, Woo ER, L ee HW, et al. The correlation of Salvia miltiorrhiza extract induced regulation of osteoclas togenesis with the amount of components tan-shinone I, tanshinone IIA, cryptotanshinone,and dihydrot anshin one[J].Immu nopharmacol Im munot oxicol,2008,30(2):347-364
[43]吴小霞,周则卫.雌激素的研究进展[J].医药导报,2008,27(10)：1234-1236
[44]张颖.左归丸对去卵巢所致骨质疏松症大股骨骨髓中相关蛋白质的影响[D].北京：中国中医科学院,2012
[45]Khosla S, Melton LJ 3rd, Atkinson EJ, et al. Relationship of serum sex steroid levels and bone turnover markers with bone mineral density in men and women: a key role for bioavailable estrogen[J]. J Clin Endocrinol Metab,1998,83:2266-2268
[46]梅敏.骨质疏松症治疗研究进展[J].吉林医学,2011,32(4)：749-751
[47]Arijmandi BH, Salih MA, Herbert DC,et al.Evidence for estrogen recaptor-link-ed calcium transport in the intestine. J Bone Miner 1993,21:63
[48]支英杰,谢雁鸣.中医药对绝经后骨质疏松症的现代研究进展[J].中医杂志,2010：51(1)：241-243
[49]邓伟民,黄海,曾东,等.补肾壮骨冲剂对更年期综合征患者性激素及血脂的影响[J].中医杂志,2011,52(3)：208-213
[50]郑建靖,徐美娟.益肾活血汤对骨质疏松模型大鼠作用的研究[J].药物研究,2001,13(8)：7-8
[51]单丽娟,李凤森,张伟.骨健颗粒剂对去势大鼠激素水平影响的实验研究[J].中医药学刊,2004,22(1)：66
[52]Scheidt-Nave C, Bismar H, Leidig-Bruckner G, et al.Serum interleukin-6 is a major predictor of bone loss in women specific to the first decade past menopause[J]. Clin Endocrinol Metab,2001,86(5):2032-2042
[53]谭清武,陈俊文.补肾健脾活血方药对老年骨质疏松大鼠血清L-6的影响[J].中国中医药科技,2002,9(6)：331-339
[54]鞠大宏,赵宏艳,刘梅洁,等.左归丸含药血清对成骨细胞IL-1、IL-6和COX-2表达的影响[J].中国实验动物学报,2006,14(2)：27-30
[55]吴昊,查振刚,姚平,等.淫羊藿苷对骨髓间充质干细胞骨向诱导的实验研究[J].中国中西医结合杂志,2010,30(4)：410-414
[56]张荣华,杨丽,朱晓峰,等.益骨胶囊对去卵巢骨质疏松大鼠骨组织TGF-β1 mRNA表达的影响[J].中药材,2005,28(4)：312-315
[57]Myers M GJ, Sun XJ, Chertham B, et al.IRS-1 is a common element ininsu-lin and insulin-like growth factor-I sign al ingt otheprosphatidy linositol3-kinase [J].Endocrinology,1993,132(4):1421
[58]王勇刚,昝强,徐武清,等.补肾活血法对去势大鼠骨质疏松模型血清IGF-I的影响[J].江苏中医药,2009,41(1)：70-71
[59]周立飞,高肖波,刘振东.补肾健脾汤对绝经后骨质疏松症患者细胞因子、 骨密度及雌激素水平的影响[J].中国中医药科技,2008,15(3)：170-171
[60]何志鹏.葛根对去卵巢大鼠骨质疏松作用的研究[J].牡丹江医学院学报,2008,29(5)：9-11
[61]付萍,李伟,杨铭,等.蛇床子总香豆素对去卵巢大鼠骨质疏松症大鼠骨矿和骨微量元素的影响[J].世界元素医学,2008,15(3)：41-43
[62]刘梅洁,吕国红,邹军,等.左归丸含药血清对成骨细胞分泌骨钙素的影响[J].中国中医基础医学杂志,2007,15(8)：581-582
[63]吕海波.左归丸对去卵巢大鼠骨组织中TGF-β、 Smad信号转导通路的研究[D].沈阳：辽宁中医药大学,2011
[64]龚建文.从P38信号通路探讨中药骨康方对去势大鼠MSCs成骨分化的机制研究[D].广州：广州中医药大学,2011
[65]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
[66]Gori F, Hofbauer LC, Dunstan CR, et al. The expression of osteoprotegerin and RANK ligand and the support of osteoclast by stromalosteoblast lineage cells is developmentaly regulated[J]. Endocrinology,2000,141(12):4768-4776
[67]齐尚峰,张永波.中医药防治骨质疏松症的研究概况[J].中国骨质疏松杂志,2009,15(3)：230-235
[68]江湧,杨海韵.骨质疏松症从痿痹瘀痉辨治[J].新中医,2007,39(7)：3
[69]谢雁鸣,朱云茵,葛继荣,等.基于临床流行病学调查的原发性骨质疏松症中医基本证候研究[J].世界科学技术-中医药现代化,2007,9(2)：38-44
[70]方朝晖,耿家金,张有志.1000例老年性骨质疏松症证候分布与组合规律研究[J].中国中医药信息杂志,2007,14(5)：15-17
[71]史晓,蔡奇文,杜修东,等.原发性骨质疏松症患者中医量表的信度及效度研究[J].中医杂志,2009,15(2)：124-126
[72]徐祖健,等.绝经后妇女原发性骨质疏松症中医辨证分型与骨密度的相关性研究[J].泸州医学院学报,2008,31(4)：390
[73]孙江波,刘绪银,廖怀章.骨质疏松症中医定位辨证与骨密度水平的相关性研究[J].世界中西医结合杂志,2011,6(1)：33-34
[74]穆刚,王平,张君涛.对原发性骨质疏松症证型实质的探讨[J].中国中医骨 伤科杂志,2006,14(1)：66-67
[75]Nelson HD,Humphrey LL,Nygren P,et al,Postmenopausal hormonereplacement theraphy:Scientific review [J] JAMA,2002,288(7):8722881
[76]Pak CY,Zerwekh JE,Antich P.Anabolic effects of fluoride on bone7[J].Trends Endocrinol Metab,1995,6(7):2229-2234
[77]谢雁鸣,朱芸茵,吴泰相.中医药治疗绝经后骨质疏松的疗效及安全性系统评价[J].中国循证医学杂志,2005,(01)：29-41
[78]阳波,杨静.黄芪对绝经后骨质疏松症患者影响的临床研究[J].四川中医,2007,28(3)：291
[79]邹丽宜,吴铁,崔燎.黄芪多糖对肝纤维化小鼠骨丢失的防治作用[J].中西医结合肝病杂志,2002,12(2)：95-98
[80]刘海全,秦佳佳.骨康颗粒治疗肾阳虚型绝经后骨质疏松症的疗效观[J].山东中医药大学学报,2010,34(1)：48-49
[81]王照康.补肾健脾密骨方对老年骨质疏松患者疼痛及其相关生化指标的改善作用[J].中国临床康复,2004,8(27)：5930-5949
[82]王惠礼,杨晓玉.骨康宁治疗原发性骨质疏松症的疗效观察[J].中国医学创新,2012,9(1)：29-30
[83]刘树胜,解增友,耿读海.中医学对骨质疏松症病因病机的认识[J].河北中医,2012,34(1)：32-33
[84]史传道,袁普卫,杨晓航.抗疏健骨颗粒治疗绝经后骨质疏松症40例[J].山西中医,2012,28(1)：16-17
[85]白洁,白孟海,刘茗露,等.中药骨疏康治疗绝经后骨质疏松症临床观察[J].西部中医药,2012,25(1)：22-24
[86]梁冬波,黄承军,娄宇明,等.补阳还五汤治疗绝经后骨质疏松症疗效观察[J].现代中西医结合杂志,2012,21(8)：829-830
[87]王建伟,马勇,尹恒,等.金匮肾气丸联用葡萄糖酸钙对原发性骨质疏松症疼痛临床疗效观察[J].辽宁中医药大学学报,2012,14(2)：5-6
[88]何成奇,熊恩富,饶凡,等.骨痛宁擦剂治疗骨质疏松疼痛的临床疗效观察[J].中医研究,2001,14(2)：27-29
[89]赵利华,农泽宁,钟旋,等.温针灸对绝经后骨质疏松症患者骨密度及生化 指标的影响[J].中国针灸,2005,25(12)：597-900
[90]吴明霞,吴炳煌,刘献祥,等.灸对绝经后骨质疏松症影响的实验和临床研究[J].中国针灸,2002,22(5)：334-336
[91]徐娅莉,金建军,徐登玉,等.针刺加穴位贴敷对原发性骨质疏松症患者骨密度及疼痛的影响[J].中国针灸,2006,26(2)：87-90
[92]林莺,许金榜,李沛.针药结合对绝经后妇女骨代谢调节作用的临床研究[J].福建医药杂志,2009,31(6)：131-133
[93]林志苇,黎健,高丽萍等.肾俞穴位埋线对原发性骨质疏松症骨密度影响的研究[J].中国骨质疏松杂志,2006,12(4)：381-383
[94]熊芳丽,肖亚平.耳针治疗老年妇女骨质疏松症60例临床观察[J].贵阳中医学院学报,2000,22(2)：33-34
[95]李小平.运用低频脉冲电磁场治疗原发性骨质疏松症[J].湖北中医杂志,2011,33(3)：60-61
[96]Kanis JA, Burlet N, Cooper C, et al. European guidance for the diagnosis and management of osteoporosis in postmenopausal women[J]. Osteoporos Int, 2008,19:399-428
[97]National Osteoporosis Foundation. Physician's guide to prevention and treat-ment of osteoporosis[J].National osteoporosis foundation,2008
[98]杨玺.骨质疏松症防治[M].上海：科学技术文献出版社,2003：13
[99]杨芬分.骨质疏松症的预防[J].柳州医学,2007,20(2)：71-73
[100]李新民,宋艳.合理营养与骨质疏松症的预防[J].中国现代药物应用,2007,1(1)：52-53
[101]Porthouse J,Cockekayne S,King C,et al.Randomised controlled trial of calcium and supplementation with cholecalciferol(vitamin D3)for prevention of fractures in primary Care[J]. BMJ,2005,30(330),1005
[102]Grant AM,Avenell A,Campbell,MK,et al.Oral vitamin D3 and calcium for secondary prevention of low-trauma fractures in elderly people (Randomised Evaluation for Calcium Or vitamin D RECORD);a randomized placebo controlled trial[J].Lancet,2005,365 (9471):1621-1628
[103]李燕燕,韩继红.中医食疗对老年骨质疏松症患者临床症状和骨密度的影 响研究[J].护士进修杂志,2010,25(6)：490-492
[104]程晓明.骨质疏松症预防保健新进展[J].现代预防医学,2008,35(24)：64-65
[105]郭晓东,赵民生,曹秀虹.骨质疏松症的预防与治疗[J].社区医学杂志,2010,8(12)：16-17
[106]Devine A,Hodgson JM,Dick IM,et al.Tea drinking is associated with benefits on bone density in older women[J].AM J Clin Nutr2007,86(4):1243-1247
[107]戴闽,昌军.骨科运动康复[M].北京：人民卫生出版社,2008：243.
[108]张林,杨锡让,薛延.运动与人体骨密度变化研究进展[J].北京体育大学学报,2000,23(1)：64
[109]阮彩莲,白亚军,刘运磊.不同有氧运动时间对衰老大鼠骨质成分的影响[J].中国组织工程研究与临床康复,2009,28(13)：5500-5504
[110]张磊,包可,王勇.骨质疏松症中西医结合预防及治疗的研究进展[J].中西医结合研究,2011,3(3)：153-155
[111]Sagiv M,Vogelaere PP,Soudry M,et al.Role of physical activity training I attenuation of height loss through aging[J]. Gerontology,2000,46(5):266-270
[112]郁嫣嫣,祁奇,余波,等.太极拳锻炼对绝经后女性骨密度的影响[J].中国康复理论与实践,2012,18(2)：155-157
[113]邹军,林菲,张丽,等.长期太极拳运动对绝经后妇女骨密度和平衡能力的影响[J].中国康复理论与实践,2011,17(1)：80-82
[114]中华中医药学会.中医体质分类与判定[M].北京：中国中医药出版社,2009.
[115]蔡亮,江静华,杨健松.骨量减少及骨质疏松症中医体质分类与辨证分型相关性研究[J].新中医,2011,43(7)：51-52
[1]支英杰,谢雁鸣.中医药对绝经后骨质疏松症的现代研究进展[J].中医杂志,2010,51(1)：241-243
[2]鞠大宏,刘梅洁,赵宏艳,等.左归丸含药血清对成骨细胞OPG、RANKL mRNA表达的影响[J].北京中医药大学学报,2008,31(5)：312-315
[3]刘梅洁,鞠大宏,赵宏艳,等.“肾主骨”的机理研究-左归丸含药血清对破骨细胞分化调控因子OPG、RANKL蛋白表达的影响[J].中国中医基础医学杂志,2009,15(3)：184-187,196
[4]邓铁涛.略论五脏相关取代五行学说.广州中医学院学报,1988,(2)：4
[5]李浩钢.骨密Ⅰ、Ⅱ号方脾肾同补治疗骨质疏松症32例[J].上海中医药杂志,1997(2)：28
[6]王照康.补肾健脾密骨方对老年骨质疏松患者疼痛及其相关生化指标的改善作用[J].中国临床康复,2004,8(27)：5930-5949
[7]王惠礼,杨晓玉.骨康宁治疗原发性骨质疏松症的疗效观察[J].中国医学创新,2012,9(1)：29-30
[9]欧阳钢,王玲玲,卓铁军,等.不同刺激方法对原发性骨质疏松症骨密度的影响[J].中国针灸,2004,24(1)：25-26
[9]王东岩,欧阳钢.针药结合原发性骨质疏松症患者骨密度的影响[J].中国中医骨伤科杂志,2005,14(12)：911-913
[10]徐敏,刘保新,黄承军,等.原绝经后骨质疏松症的穴位治疗进展[J].浙江中医药大学学报,2011,35(2)：301-302
[11]孙爱华.肾与脾关系物质基础的实验观察[J].中医杂志,1998,39(1)：47-9
[12]高守泉.脾肾相关性的实验研究[J].湖南中医药导报,1998,4(12)：26-27
[13]吕爱平,李德新,崔家鹏.脾肾相关的分子生物学基础—脾肾阳虚证模型大鼠自由基损伤的比较研究[J].辽宁中医杂志,2001,28(3)：189-91
[14]吕爱平,李德新,崔家鹏.脾肾阳虚模型大鼠肝细胞线粒体磷脂组分变化的比较研究[J].中医药学刊,2003,21(1)：78
[15]罗卫芳,郭树仁,程士德,等.中医学肾本质现代研究概述[J].中国中医基础医学杂志,2001,7(4)：75-77
[16]任艳玲,郑洪新,杜松.补肾益气生精中药对去卵巢大鼠骨质疏松发生机制的调节作用[J].北京中医药大学学报,2006,29(2)：94-97
[17]郑洪新,刘伶,林庶茹,等.参茸补血丸对去卵巢大鼠骨质疏松防治作用的实验研究[J].中国老年学杂志,2006,26(3)：385-388
[18]张倩,胡小琪,郭宏霞,等.我国藏族和汉族儿童少年骨转换研究[J].中国骨质疏松杂志,2008,14(6)：454-457
[19]李燕舞,王汝俊,王建华.大黄脾虚模型大鼠胃壁细胞的超微结构观察[J].中国中西医结合消化杂志,2005,13(3)：155-157
[20]南京医学院中西医结合研究组.脾虚泄泻动物模型的研制[JI].浙江中医杂志,1982,17(8)：355
[21]刘汶.番泻叶致脾虚证动物模型的造型方法[J].中国中西医结合脾胃杂志,1998,6(4)：231
[22]曲长江.番泻叶泻下与劳倦过度单、复因素脾虚模型的免疫学研究[J].中国中西医结合脾胃杂志,1999,7(4)：212
[23]任平.四君子汤对脾虚模型大鼠胃动素和前列腺素E2含量的影响[J].中药药理与临床,1994,10(6)：7
[24]周奇志.实验性脾虚证大鼠脑内单胺递质的变化及其择时治疗效应[J].成都中医药大学学报,1998,21(3)：36
[25]易杰,李德新.脾虚证动物模型研究思路和方法探析[J].上海中医药杂志,2001,5(5)：40-42
[26]陈奇.中药药理研究方法学[M].北京：人民卫生出版社.1993,1023
[27]鞠大宏,张丽坤,吴皓,等.温补肾阳方对卵巢切除所致骨质疏松大鼠外周血浆中促肾上腺皮质激素和p-内啡肽含量的影响[J].中国中医基础医学杂志,2003,9(5)：30-33
[28]鞠大宏,吕爱平,张春英,等.左归丸对卵巢切除所致骨质疏松大鼠IL-1和IL-6活性的影响[J-].中医杂志,2002,43(10)：777-779
[29]鞠大宏,张春英,王安民,等.温补肾阳方对去卵巢所致骨质疏松大鼠IL-1和IL-6活性的影响[J].中国中医基础医学杂志,2000,6(10)：5-9
[30]陈津岩,王立峰,赵慧,等.右归丸对脾肾两虚证大鼠甲状腺激素及环核苷酸水平的影响[J].广州中医药大学学报,2009,26(4)：377-380
[31]王立峰,陈芝喜,赵慧,等.强肌健力方对脾肾两虚证大鼠血浆性激素水平的影响[J].放射免疫学杂志,2008,21(2)：147-149
[32]刘梅洁,李鸿泓,鞠大宏,等.骨质疏松症脾肾两虚型病证结合动物模型的建立[J].中国中医基础医学杂志,2012,18(8)：836-838,844
[33]周修通.“龟鹿二仙汤”为主治疗骨质疏松症临床观察[J].上海中医药杂志,2001,47(11)：24
[34]周修通.“龟鹿二仙汤”治疗骨质疏松症45例临床观察[J].江苏中医,2001,22(6)： 28
[35]杨伟岸.龟鹿二仙汤加味治疗老年脊椎骨质疏松性腰背部疼痛63例[J].福建中医药,2005,50(5)：19
[36]尹斯利,金勋杰,吴铁,等.中药成方四君子汤对去卵巢大鼠血清骨钙素水平影响[J].中国骨质疏松杂志,2000,6(3)：69-70
[37]鞠大宏,李鸿泓,刘梅洁,等.补肾健脾方对大鼠脾肾两虚型骨质疏松症的治疗作用[J].中华中医药杂志,2012,12(27)：3207-3210
[38]Simonetws, Lacey D L, Dunstan C R. Osteoprotegerin:A novel secreted protein involved in the regulation ofbone density[J].Cell,1997,89 (2):309-319
[39]Gorif, Hofbauer L C, Dunstan C R. The expressionof osteoprotegerin and RANK ligand and the support ofosteoclast formation by stromal osteoblast lineagecells isdevelopmentally regulated[J].Endocrinology,2009,141 (12):4768-4776
[40]Yasuda H. Bone and bone related biochemical examinations. Bone and collagen related metabolites. Receptor activator of NF-kappaBligand(RANKL) [J].Clin Calcium,2006,16(6):964-970
[41]Shimizu-Ishiura M,Kawana F, Sasaki T.Osteoprotegerin administration reduces femural bone loss in ovariectomizedmice via impairment of osteoclast structure and function[J].Electron Microsc(Tokyo),2002,51(5):315-325
[42]Bekker PJ, Holloway D, Nakanishi A, et al.The effect of a single dose of osteopotegerin in postmenopausal women[J].Bone Mine Res,2001,16:3348-60
[43]Clohisy D.Cellular mechanisms of osteolysis[J].Bone Joint Surg Am,2003,85 Suppl 1:S4-6
[44]Gori F, Hofbauer LC, Dunstan CR, et al. The expression of osteoprotegerin and RANK ligand and the support of osteoclast by stromalosteoblast lineage cells is developmentaly regulated[J]. Endocrinology,2000,141(12):4768-4776
[45]Yano K,Tsuda E,Washida N,et al.Immunological characterization of eirculating osteoprotegerin/osteoclastogenesis inhibitory factor:increased serum concen-trations in postmenopausal women with osteoporosis [J].J Bone Miner Res,1999, 14(4):518-527
[46]Eghbali-Fatourechi QKhosla S,Sanyal A,et al.Role of RANK ligand in media-ting increased bone resorption in early postmenopausal women [J].J Clin Invest, 2003,111(8):1221-1230
[47]Hofbauer LC, Khosla S, Dunstan CR, et al. Estrogen stimulates gene expression and protein production of osteoproteigern in human osteoblastic cells[J].Endori-nology,1999,140(9):4367-4370
[48]郭梁.成骨细胞破骨细胞及OPG/RANKL/RANK轴与骨质疏松[J].中医正骨,2010,22(7)：41-44
[49]喻明丽,沈志强.OPG/RANK/RANKL系统在骨质疏松中的研究进展[J].昆明医学院学报,2012,33(1B)：194-197
[50]张忠兵,张学庸,陈希陶,等.脾虚泄泻患者肠粘膜中SP和VIP初步探讨[J].中西医结合杂志,1991,11(3)：144
[51]贾波,邓中甲,黄秀深,等.白术茯苓汤及其配伍对脾虚大鼠胃肠激素的影响[J].成都中医药大学学报,2002,25(2)：13-15,64
[52]黄学宽,任凌燕,李进,等.六味和中散对胃肠动力及胃肠激素的影响[J].中药新药与临床药理,2007,18(3)：205-207
[53]Ransjo M, Lie A, MukohyamaH, Lundberg P, et al. Microisolate mouse osteoclasts express VIP-1 and PACAP receptor[J].Biochem Biophys Res Commun,2000,274(2),400-40
[54]Lundberg P, Lie A, Bjurholm A, et al. Vasoactive intestinapeptide regulates osteoclast activity via specific binding sites oboth osteoclasts and osteoblasts[J].Bone,2000,27(6):80-10
[55]Perssona E, Voznesenskyc OS, Huang YF, et al. Increased expression of interleukin-6 by vasoactive intestinal peptide is associated with regulation of CREB, AP-1 and C/EBP, but not NF-κB, in mouse calvarial osteoblasts[J].Bone, 2005,37(4):513-529
[56]Perssona E, Lerner UH. The neuropeptide VIP potentiates IL-6 production induced by proinflammatory osteotropic cytokines in calvarial osteoblasts and the osteoblastic cell lineMC3T3-E1[J].Biochem Biophys Res Commun,2005, 335 (3):705-711
[57]Nagata A, Tanaka T, Minezawa A, et al. cAMP activation by PACAP/VIP stimulates IL-6 release and inhibits osteoblastic differentiation through VPAC2 receptor in osteoblastic MC3T3 cells. J Cell Physiol[J].2009,221(1):75-83