补肾法对酒精干预成骨样细胞蛋白质表达影响的实验及相关临床研究
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摘要
1研究目的
股骨头坏死(osteonecrosis of the femoral head，ONFH)是由于多种病因破坏股骨头血供使骨的活性成分死亡的一种病理过程。酒精性股骨头坏死是常见的类型之一，目前病机认识方面比较肤浅，治疗也处于被动，往往出现坏死后才进行治疗，而且办法不多，其发病机理和防治技术尚需不断研究，尤其对其病机和中医防治机理方面进行研究更具有重要意义。
过度摄入酒精是酒精性股骨头的主要原因，目前发病机理主要有酒精的细胞毒作用、骨细胞脂肪变性坏死等学说。股骨坏死的基本病理变化是骨小梁表面成排的成骨细胞消失，骨细胞陷窝空虚。现代医学对股骨头坏死的防治尚缺乏理想手段。“肾主骨”是中医治疗骨病的理论依据，股骨头坏死是中医骨病中越来越受注目的一种，补肾法治疗酒精性股骨头坏死已经在临床上广泛应用，但是中医方剂的治疗作用机理一直未得到现代科学的合理阐述，不利于中医现代化的发展。
蛋白质组学是研究蛋白质组的一个新领域，主要研究蛋白质的特性，可以在蛋白质水平上了解细胞的各项功能、各种生理生化过程以及疾病的病理过程等，通过比较患病细胞与正常细胞中蛋白质的表达变化，或者比较患病细胞与药物处理后细胞中蛋白质的表达变化，可能找到疾病的发病机制，同时也可能发现一些新的药物靶点及新药物。蛋白组学对中医领域的辨证论治研究提供了全新概念的技术平台，从现代技术讲，补肾、补髓是有其基因学或蛋白质学的依据的。
目前在股骨头坏死的机理和中药治疗的机制研究中进行体外模拟成骨细胞受损并进行治疗的实验研究尚未见报道。本课题进行体外细胞实验，首先模拟酒精性股骨头坏死细胞受损的模型，然后采用补肾法代表方剂六味地黄丸含药血清进行治疗，再用蛋白组学的双向电泳技术，找出酒精干预和治疗后的差异蛋白质点，并进行初步分析，为进一步发现差异蛋白质进而研究酒精性股骨头坏死的发病机理和补肾法的治疗机制打下基础。
2研究方法与结果
2．1补肾法对OS-732成骨样细胞增殖作用的研究
2．1．1细胞生长曲线绘制
选择合适密度的OS-732成骨样细胞，给以血清浓度为10％的六味地黄丸高、中、低剂量组和空白对照组(即生理盐水灌胃)血清连续培养7天。显微镜观察并计数，绘制细胞生长曲线，发现药物组的增殖作用均较空白血清组强，高、中剂量组含药血清对细胞有较强的刺激作用，而又以中剂量组的作用最强，3—6d为对数生长期。
2．1．2 MTT法测定OS-732成骨样细胞的增殖试验
选择合适密度的OS-732成骨样细胞，给以血清浓度为10％的六味地黄丸高、中、低剂量组和生理盐水空白对照组血清进行连续培养5天，每天酶标仪测定吸光值，结果：培养24h、48h未见明显差异，但培养第3d、4d、5d后，高、中、低剂量组的增殖作用均较生理盐水空白对照组强，又以中剂量组最为明显，具有统计学意义，高、低剂量组无统计学意义
2．1．3 OS-732成骨样细胞分泌ALP的实验研究
选择合适密度的OS-732成骨样细胞，给以血清浓度为10％的六味地黄丸高、中、低剂量组和生理盐水空白对照组血清进行培养，分别在培养第3d、4d、5d后进行碱性磷酸酶试剂盒检测，酶标仪测定OD值
结果：高、中、低各剂量组均比生理盐水空白组高，但以中剂争组最为明显，具有统计学意义。高、低剂量组无统计学意义。
2．2 OS-732成骨样细胞酒精干预和六味地黄丸含药血清治疗作用的实验研究
2．2．1 OS-732成骨样细胞不同浓度酒精干预下细胞凋亡率检测
选择生长良好合适密度的OS-732成骨样细胞，分别加入不同浓度的酒精进行孵育，空白对照组仅加入同等量RPMl1640。分别于干预后的第24h、48h与72h收集细胞，冷乙醇固定、PI液染、流式细胞仪检测。
结果：酒精干预各组细胞凋亡率明显比空白对照组增高，具有显著意义(P＜0.05)，随着酒精浓度的增加，干预时间的延长，凋亡率逐渐升高，凋亡率与酒精浓度呈正相关，与干预的时间成正相关。
2．2．2 OS-732成骨样细胞酒精干预后电子显微镜观察
分为正常OS-732成骨样细胞组，酒精干预组，酒精干预后加六味地黄丸含药血清治疗组。
透射电镜下的表现：正常成骨样细胞核较大，呈多型性，核仁明显，细胞质中可见较多的核糖体、高尔基体及溶酶体样颗粒。给予酒精干预后部分细胞器发生变化，核糖体、内质网减少，出现较多空泡，可以见到典型的细胞凋亡表现，细胞核固缩碎裂。药物治疗后细胞质内的细胞器从结构上有所恢复，内质网、高尔基体清晰可见。
2．3 OS-732成骨样细胞酒精干预和六味地黄丸含药血清治疗后差异蛋白表达的初步分析
成骨样细胞分为酒精干预组、药物治疗组、空白对照组。每组细胞提取总蛋白后进行双向凝胶电泳技术分离，得出蛋白质点电泳凝胶图谱，然后进行计算机图像分析软件分析。结果，发现酒精干预组与空白正常组相比，表现明显差异的蛋白点有5个(即SSP1002、SSP1401、SSP1602、SSP8501、SSP8504)，均表现为表达降低。这5个蛋白在药物组中其表达差异得到不同程度的逆转。
2．4补肾法治疗酒精性股骨头坏死的临床研究
以六味地黄丸合并生脉成骨片与单一生脉成骨片进行疗效比较。对69例酒精性股骨头坏死患者进行系统的临床观察。结果表明，两组均有较高疗效，合并用药组有效率83.78％，单一用药组81.25％，两组比较无显著差异。从缓解疼痛指标看，合并用药组优于单一用药组，具有显著差异性(P=0.0028)；从治疗前后的体征改善方面观察，合并用药明显优于单纯用药组(P＜0.05)。在影像学方面，两组的死骨吸收，新骨形成，骨膜反应等方面没有明显差异。
3结论
3．1六味地黄丸含药血清对OS-732成骨样细胞具有增殖作用，促进细胞分泌ALP。
3．2一定浓度的酒精可以引起OS-732成骨样细胞凋亡，细胞凋亡与酒精浓度和干预时间成正相关。从倒置显微镜、透射电子显微镜不同角度观察到细胞受损的表现，可以见到细胞死亡和凋亡，存活的细胞可见空泡样改变。
3．3采用蛋白组学双向电泳技术发现酒精干预后受损的0S-732成骨样细胞部分蛋白质表达下调，中药六味地黄丸含药血清可以逆转其部分表达，说明具有治疗作用。
3．4六味地黄丸为补肾法的代表方，在临床观察中发现对酒精性股骨头坏死有较好疗效，结合活血化瘀方法可以提高疗效。
1 Objective of research
Osteonecrosis of the femoral head (ONFH) was a pathologic process caused by multi-pathogenic factors that damaged the blood supply of the femoral head, which led to the death of active constitute. It was a familiar type that caused by alcohol. The pathogenesis and control technique of this type needed continual study. Researches on its pathogenesis and prevention and cure by Chinese medicine were important.
The over intake of alcohol was the first cause of Osteonecrosis of the femoral head caused by alcohol. Its pathogenesis included cell toxic action and fatty degeneration and necrosis of osteocyte.
The basic pathological change of osteonecrosis was the losing of banks of osteoblast on the surface of bone trabecula and the blankness of lacuna in osteocyte. There was no ideal method for the prevention and cure of osteonecrosis of the femoral head by the method of western medicine.
The kidney being in charge of the bone was the basic conception in the treatment on osteopathy by traditional Chinese medicine. There had an extensive use for the treatment on alcoholic osteonecrosis of the femoral head by the method of invigorating the kidney. But its therapeutic mechanism was not well formulized with modern scientific means.
Proteomics was a frontier in studying protein groups. It was mainly investigate the characters of proteins, thus we could know the cellular functions, physiological and biochemical process and pathogenic process at the level of proteins. The pathogenesis, pharmaceutical target point and new drugs could be found by comparing the express of proteins both in normal cells and sicken cells, and in sicken cells and medicated sicken cells. Proteomics provided a conceptual technical platform for investigating the syndrome differentiation in TCM.
From the point of view of modern technology, there was a genic or protein essence in the treatment of this disease by invigorating the kidney and invigorating the marrow. This study processed with cellar experiment in vitro. We simulated the model of alcoholic osteonecrosis of the femoral head. Afterwards, cells were treated with pastille serum of bolus of six drugs including rehmannia that was a reprehensive prescription of the methods of invigorating the kidney. Then we used two-dimensional electrophoresis techniques to find out the differential protein points after the intervention of alcohol and treatment of pastille serum. The preliminary analysis established basement for discovering the structural function of the differential protein and pathogenesis of this disease and therapeutic mechanism of the treatment of invigorating the kidney.
2 Methods and results
2.1 The effect of the method of invigorating the kidney on cellar proliferation of human osteoblast-like cell 0S-732
2.1.1 Drafting of culler curve of growth
We selected human osteoblast-like cell 0S-732 with concentration. 10% of pastille serum of bolus of six drugs including rehmannia, three groups with high dosage, medium dosage and low dosage, was added to culture the human osteoblast-like cell OS-732 for 7 days continuously. The control group was with the blank serum. Then the cell counting and curve of growth drafting was done. Results showed that there was better effect of cell proliferation in the treatment groups than in the control group. There was strong effect in both the high dosage treatment group and the medium dosage treatment group, among them the medium dosage treatment group showed the stronger effect. The logarithmic phase was 3 to 6 days.
2.1.2 The proliferation experiment on the human osteoblast-like cell OS-732 with the method of MTT
We selected human osteoblast-like cell OS-732 with concentration. 10% of pastille serum of bolus of six drugs including rehmannia, three groups with high dosage, medium dosage and low dosage, was added to culture the human osteoblast-like cell OS-732 for 5 days continuously. The control group was with the blank serum. The optical concentration (OD) was tested daily. Results showed that there was no significant difference in the 24 hours groups and 48 hours groups. But in the 3 days groups, 4 days groups and 5 days groups, there was stronger effect of cell proliferation in the 3 dosages treatment groups than that in the control group. Among them, the medium dosage treatment group took the strongest effect of cell proliferation.
2.1.3 Experimental study on the secretion of alkaline phosphatase (ALP) of human osteoblast-like cell OS-732
We selected human osteoblast-like cell OS-732 with concentration. 10% of pastille serum of bolus of six drugs including rehmannia, three groups with high dosage, medium dosage and low dosage, was added to culture the human osteoblast-like cell OS-732 for 3 days, 4 days and 5 days respectively. The control group was with the blank serum. Then alkaline phosphatase kit and Elisa Machine was used to test ALP and optical concentration.
Results showed that there was stronger effect of cell proliferation higher ALP in the 3 dosages treatment groups than that in the control group. Among them, the medium dosage treatment group took the strongest effect.
2.2 Experimental study of alcohol intervention and pastille serum treatment on human osteoblast-like cell OS-732
2.2.1 Detection of apoptosis rate of human osteoblast-like cell OS-732 intervened by alcohol with different concentrations
We selected the human osteoblast-like cell OS-732 in good condition and added different concentration of alcohol. The same amount of RPMII640 medium was added into the control group. Cells were collected after 24 hours, 48 hours and 72 hours intervention. Then we used flow hyetometer (FCM) to test after cool alcohol fixed and PI liquid stained. Results showed that there was higher apoptosis rate in the intervention group than that in the control group. It had significant meaning in statistics (p＜0.05). The apoptosis rate had positive correlation with the alcohol concentration and the acting time of alcohol. 2.2.2 Electron microscopic observation of the human osteoblast-like cell OS-732 intervened by alcohol
It was divided into the normal group, the intervention of alcohol group and the bolus of six drugs including rehmannia group that was the treatment group. Results of electron microscopic observation showed that the normal human osteoblast-like cell OS-732 was with relatively great and polymorphous nucleolus and was with apparent nucleoli. There was quite a lot of ribosome, golgis apparatus and lysosome-like grain. Changes took place in some cell organ after the intervention of alcohol, which was the reducing of ribosome and endoplasmic reticulum and more bubble, The typical apoptosis such as karyopyknosis could be seen. There was rehabilitation, for example, the endoplasmic reticulum and golgis apparatus was visible, in the cell organ after the treatment of pastille serum.
2.3 Preliminary analysis of differential protein after the intervention of human osteoblast-like cell OS-732 by alcohol and the treatment of invigorating the kidney method
The human osteoblast-like cell OS-732 was divided into the blank control group, the intervention of alcohol group and the bolus of six drugs including rehmannia group that was the treatment group. Total protein was extracted and separated with the two dimensional gel electrophoresis techniques. Then the electropherogram was analyzed by image analysis software. Results showed that there was five apparently different points of protein that was SSP1002, SSP 1401, SSP 1602, SSP 8501 and SSP 8504 after comparison between the intervention of alcohol group, the treatment group and the blank control group. It was low expression of protein in the intervention of alcohol group and was high expression of protein in the treatment group. The bolus of six drugs including rehmannia pastille serum could improve the expression of protein.
2.4 Clinical study on the treatment of alcoholic steonecrosis of the femoral head by the invigorating the kidney method
We compared the curative effect of the shengmai ossify group and the combination of the bolus of six drugs including rehmannia and the shengmai ossify group.
69 cases of patients with alcoholic steonecrosis of the femoral head were systematically observed. Results showed that the effective rate in the combination of the bolus of six drugs including rehmannia and the shengmai ossify group was 83.78%, and that in the shengmai ossify group was 81.25 %. There was no significantly difference between two groups. But as to the pain relief, the effect of the bolus of six drugs including rehmannia and the shengmai ossify group was better than that in the shengmai ossify group (P=0.0028). The improvement of physical signs was better in the bolus of six drugs including rehmannia and the shengmai ossify group than that in the shengmai ossify group (p＜0.05).
There was no significant difference in absorption of sequestra, formation of fresh bone and periosteal reaction between two groups.
3 conclusions
The pastille serum of bolus of six drugs including rehmannia had proliferation promotion, and the promotion of ALP secretion of the human osteoblast-like cell OS-732.
Different concentration of alcohol could lead to apoptosis of the human osteoblast-like cell OS-732. There was positive correlation between the apoptosis and alcohol concentration. The inverted microscope and the transmission electron microscope observation showed that cells were damaged, such as cell death, apoptosis and bubble-like change in survival cells.
The two-dimensional electrophoresis technique of proteomics showed that there was descending of partial protein expression in the human osteoblast-like cell OS-732 intervened by alcohol. But there was ascending of partial protein expression in the human osteoblast-like cell OS-732 treated by bolus of six drugs including rehmannia serum.
The bolus of six drugs including rehmannia was the reprehensive prescription of the method of invigorating the kidney. This prescription had better curative effect on the alcoholic steonecrosis of the femoral head in clinic. The combination of activating blood circulation to dissipate blood stasis could improve curative effect in clinic.
股骨头坏死(osteonecrosis of the femoral head，ONFH)是由于多种病因破坏股骨头血供使骨的活性成分死亡的一种病理过程。酒精性股骨头坏死是常见的类型之一，目前病机认识方面比较肤浅，治疗也处于被动，往往出现坏死后才进行治疗，而且办法不多，其发病机理和防治技术尚需不断研究，尤其对其病机和中医防治机理方面进行研究更具有重要意义。
过度摄入酒精是酒精性股骨头的主要原因，目前发病机理主要有酒精的细胞毒作用、骨细胞脂肪变性坏死等学说。股骨坏死的基本病理变化是骨小梁表面成排的成骨细胞消失，骨细胞陷窝空虚。现代医学对股骨头坏死的防治尚缺乏理想手段。“肾主骨”是中医治疗骨病的理论依据，股骨头坏死是中医骨病中越来越受注目的一种，补肾法治疗酒精性股骨头坏死已经在临床上广泛应用，但是中医方剂的治疗作用机理一直未得到现代科学的合理阐述，不利于中医现代化的发展。
蛋白质组学是研究蛋白质组的一个新领域，主要研究蛋白质的特性，可以在蛋白质水平上了解细胞的各项功能、各种生理生化过程以及疾病的病理过程等，通过比较患病细胞与正常细胞中蛋白质的表达变化，或者比较患病细胞与药物处理后细胞中蛋白质的表达变化，可能找到疾病的发病机制，同时也可能发现一些新的药物靶点及新药物。蛋白组学对中医领域的辨证论治研究提供了全新概念的技术平台，从现代技术讲，补肾、补髓是有其基因学或蛋白质学的依据的。
目前在股骨头坏死的机理和中药治疗的机制研究中进行体外模拟成骨细胞受损并进行治疗的实验研究尚未见报道。本课题进行体外细胞实验，首先模拟酒精性股骨头坏死细胞受损的模型，然后采用补肾法代表方剂六味地黄丸含药血清进行治疗，再用蛋白组学的双向电泳技术，找出酒精干预和治疗后的差异蛋白质点，并进行初步分析，为进一步发现差异蛋白质进而研究酒精性股骨头坏死的发病机理和补肾法的治疗机制打下基础。
2研究方法与结果
2．1补肾法对OS-732成骨样细胞增殖作用的研究
2．1．1细胞生长曲线绘制
选择合适密度的OS-732成骨样细胞，给以血清浓度为10％的六味地黄丸高、中、低剂量组和空白对照组(即生理盐水灌胃)血清连续培养7天。显微镜观察并计数，绘制细胞生长曲线，发现药物组的增殖作用均较空白血清组强，高、中剂量组含药血清对细胞有较强的刺激作用，而又以中剂量组的作用最强，3—6d为对数生长期。
2．1．2 MTT法测定OS-732成骨样细胞的增殖试验
选择合适密度的OS-732成骨样细胞，给以血清浓度为10％的六味地黄丸高、中、低剂量组和生理盐水空白对照组血清进行连续培养5天，每天酶标仪测定吸光值，结果：培养24h、48h未见明显差异，但培养第3d、4d、5d后，高、中、低剂量组的增殖作用均较生理盐水空白对照组强，又以中剂量组最为明显，具有统计学意义，高、低剂量组无统计学意义
2．1．3 OS-732成骨样细胞分泌ALP的实验研究
选择合适密度的OS-732成骨样细胞，给以血清浓度为10％的六味地黄丸高、中、低剂量组和生理盐水空白对照组血清进行培养，分别在培养第3d、4d、5d后进行碱性磷酸酶试剂盒检测，酶标仪测定OD值
结果：高、中、低各剂量组均比生理盐水空白组高，但以中剂争组最为明显，具有统计学意义。高、低剂量组无统计学意义。
2．2 OS-732成骨样细胞酒精干预和六味地黄丸含药血清治疗作用的实验研究
2．2．1 OS-732成骨样细胞不同浓度酒精干预下细胞凋亡率检测
选择生长良好合适密度的OS-732成骨样细胞，分别加入不同浓度的酒精进行孵育，空白对照组仅加入同等量RPMl1640。分别于干预后的第24h、48h与72h收集细胞，冷乙醇固定、PI液染、流式细胞仪检测。
结果：酒精干预各组细胞凋亡率明显比空白对照组增高，具有显著意义(P＜0.05)，随着酒精浓度的增加，干预时间的延长，凋亡率逐渐升高，凋亡率与酒精浓度呈正相关，与干预的时间成正相关。
2．2．2 OS-732成骨样细胞酒精干预后电子显微镜观察
分为正常OS-732成骨样细胞组，酒精干预组，酒精干预后加六味地黄丸含药血清治疗组。
透射电镜下的表现：正常成骨样细胞核较大，呈多型性，核仁明显，细胞质中可见较多的核糖体、高尔基体及溶酶体样颗粒。给予酒精干预后部分细胞器发生变化，核糖体、内质网减少，出现较多空泡，可以见到典型的细胞凋亡表现，细胞核固缩碎裂。药物治疗后细胞质内的细胞器从结构上有所恢复，内质网、高尔基体清晰可见。
2．3 OS-732成骨样细胞酒精干预和六味地黄丸含药血清治疗后差异蛋白表达的初步分析
成骨样细胞分为酒精干预组、药物治疗组、空白对照组。每组细胞提取总蛋白后进行双向凝胶电泳技术分离，得出蛋白质点电泳凝胶图谱，然后进行计算机图像分析软件分析。结果，发现酒精干预组与空白正常组相比，表现明显差异的蛋白点有5个(即SSP1002、SSP1401、SSP1602、SSP8501、SSP8504)，均表现为表达降低。这5个蛋白在药物组中其表达差异得到不同程度的逆转。
2．4补肾法治疗酒精性股骨头坏死的临床研究
以六味地黄丸合并生脉成骨片与单一生脉成骨片进行疗效比较。对69例酒精性股骨头坏死患者进行系统的临床观察。结果表明，两组均有较高疗效，合并用药组有效率83.78％，单一用药组81.25％，两组比较无显著差异。从缓解疼痛指标看，合并用药组优于单一用药组，具有显著差异性(P=0.0028)；从治疗前后的体征改善方面观察，合并用药明显优于单纯用药组(P＜0.05)。在影像学方面，两组的死骨吸收，新骨形成，骨膜反应等方面没有明显差异。
3结论
3．1六味地黄丸含药血清对OS-732成骨样细胞具有增殖作用，促进细胞分泌ALP。
3．2一定浓度的酒精可以引起OS-732成骨样细胞凋亡，细胞凋亡与酒精浓度和干预时间成正相关。从倒置显微镜、透射电子显微镜不同角度观察到细胞受损的表现，可以见到细胞死亡和凋亡，存活的细胞可见空泡样改变。
3．3采用蛋白组学双向电泳技术发现酒精干预后受损的0S-732成骨样细胞部分蛋白质表达下调，中药六味地黄丸含药血清可以逆转其部分表达，说明具有治疗作用。
3．4六味地黄丸为补肾法的代表方，在临床观察中发现对酒精性股骨头坏死有较好疗效，结合活血化瘀方法可以提高疗效。
1 Objective of research
Osteonecrosis of the femoral head (ONFH) was a pathologic process caused by multi-pathogenic factors that damaged the blood supply of the femoral head, which led to the death of active constitute. It was a familiar type that caused by alcohol. The pathogenesis and control technique of this type needed continual study. Researches on its pathogenesis and prevention and cure by Chinese medicine were important.
The over intake of alcohol was the first cause of Osteonecrosis of the femoral head caused by alcohol. Its pathogenesis included cell toxic action and fatty degeneration and necrosis of osteocyte.
The basic pathological change of osteonecrosis was the losing of banks of osteoblast on the surface of bone trabecula and the blankness of lacuna in osteocyte. There was no ideal method for the prevention and cure of osteonecrosis of the femoral head by the method of western medicine.
The kidney being in charge of the bone was the basic conception in the treatment on osteopathy by traditional Chinese medicine. There had an extensive use for the treatment on alcoholic osteonecrosis of the femoral head by the method of invigorating the kidney. But its therapeutic mechanism was not well formulized with modern scientific means.
Proteomics was a frontier in studying protein groups. It was mainly investigate the characters of proteins, thus we could know the cellular functions, physiological and biochemical process and pathogenic process at the level of proteins. The pathogenesis, pharmaceutical target point and new drugs could be found by comparing the express of proteins both in normal cells and sicken cells, and in sicken cells and medicated sicken cells. Proteomics provided a conceptual technical platform for investigating the syndrome differentiation in TCM.
From the point of view of modern technology, there was a genic or protein essence in the treatment of this disease by invigorating the kidney and invigorating the marrow. This study processed with cellar experiment in vitro. We simulated the model of alcoholic osteonecrosis of the femoral head. Afterwards, cells were treated with pastille serum of bolus of six drugs including rehmannia that was a reprehensive prescription of the methods of invigorating the kidney. Then we used two-dimensional electrophoresis techniques to find out the differential protein points after the intervention of alcohol and treatment of pastille serum. The preliminary analysis established basement for discovering the structural function of the differential protein and pathogenesis of this disease and therapeutic mechanism of the treatment of invigorating the kidney.
2 Methods and results
2.1 The effect of the method of invigorating the kidney on cellar proliferation of human osteoblast-like cell 0S-732
2.1.1 Drafting of culler curve of growth
We selected human osteoblast-like cell 0S-732 with concentration. 10% of pastille serum of bolus of six drugs including rehmannia, three groups with high dosage, medium dosage and low dosage, was added to culture the human osteoblast-like cell OS-732 for 7 days continuously. The control group was with the blank serum. Then the cell counting and curve of growth drafting was done. Results showed that there was better effect of cell proliferation in the treatment groups than in the control group. There was strong effect in both the high dosage treatment group and the medium dosage treatment group, among them the medium dosage treatment group showed the stronger effect. The logarithmic phase was 3 to 6 days.
2.1.2 The proliferation experiment on the human osteoblast-like cell OS-732 with the method of MTT
We selected human osteoblast-like cell OS-732 with concentration. 10% of pastille serum of bolus of six drugs including rehmannia, three groups with high dosage, medium dosage and low dosage, was added to culture the human osteoblast-like cell OS-732 for 5 days continuously. The control group was with the blank serum. The optical concentration (OD) was tested daily. Results showed that there was no significant difference in the 24 hours groups and 48 hours groups. But in the 3 days groups, 4 days groups and 5 days groups, there was stronger effect of cell proliferation in the 3 dosages treatment groups than that in the control group. Among them, the medium dosage treatment group took the strongest effect of cell proliferation.
2.1.3 Experimental study on the secretion of alkaline phosphatase (ALP) of human osteoblast-like cell OS-732
We selected human osteoblast-like cell OS-732 with concentration. 10% of pastille serum of bolus of six drugs including rehmannia, three groups with high dosage, medium dosage and low dosage, was added to culture the human osteoblast-like cell OS-732 for 3 days, 4 days and 5 days respectively. The control group was with the blank serum. Then alkaline phosphatase kit and Elisa Machine was used to test ALP and optical concentration.
Results showed that there was stronger effect of cell proliferation higher ALP in the 3 dosages treatment groups than that in the control group. Among them, the medium dosage treatment group took the strongest effect.
2.2 Experimental study of alcohol intervention and pastille serum treatment on human osteoblast-like cell OS-732
2.2.1 Detection of apoptosis rate of human osteoblast-like cell OS-732 intervened by alcohol with different concentrations
We selected the human osteoblast-like cell OS-732 in good condition and added different concentration of alcohol. The same amount of RPMII640 medium was added into the control group. Cells were collected after 24 hours, 48 hours and 72 hours intervention. Then we used flow hyetometer (FCM) to test after cool alcohol fixed and PI liquid stained. Results showed that there was higher apoptosis rate in the intervention group than that in the control group. It had significant meaning in statistics (p＜0.05). The apoptosis rate had positive correlation with the alcohol concentration and the acting time of alcohol. 2.2.2 Electron microscopic observation of the human osteoblast-like cell OS-732 intervened by alcohol
It was divided into the normal group, the intervention of alcohol group and the bolus of six drugs including rehmannia group that was the treatment group. Results of electron microscopic observation showed that the normal human osteoblast-like cell OS-732 was with relatively great and polymorphous nucleolus and was with apparent nucleoli. There was quite a lot of ribosome, golgis apparatus and lysosome-like grain. Changes took place in some cell organ after the intervention of alcohol, which was the reducing of ribosome and endoplasmic reticulum and more bubble, The typical apoptosis such as karyopyknosis could be seen. There was rehabilitation, for example, the endoplasmic reticulum and golgis apparatus was visible, in the cell organ after the treatment of pastille serum.
2.3 Preliminary analysis of differential protein after the intervention of human osteoblast-like cell OS-732 by alcohol and the treatment of invigorating the kidney method
The human osteoblast-like cell OS-732 was divided into the blank control group, the intervention of alcohol group and the bolus of six drugs including rehmannia group that was the treatment group. Total protein was extracted and separated with the two dimensional gel electrophoresis techniques. Then the electropherogram was analyzed by image analysis software. Results showed that there was five apparently different points of protein that was SSP1002, SSP 1401, SSP 1602, SSP 8501 and SSP 8504 after comparison between the intervention of alcohol group, the treatment group and the blank control group. It was low expression of protein in the intervention of alcohol group and was high expression of protein in the treatment group. The bolus of six drugs including rehmannia pastille serum could improve the expression of protein.
2.4 Clinical study on the treatment of alcoholic steonecrosis of the femoral head by the invigorating the kidney method
We compared the curative effect of the shengmai ossify group and the combination of the bolus of six drugs including rehmannia and the shengmai ossify group.
69 cases of patients with alcoholic steonecrosis of the femoral head were systematically observed. Results showed that the effective rate in the combination of the bolus of six drugs including rehmannia and the shengmai ossify group was 83.78%, and that in the shengmai ossify group was 81.25 %. There was no significantly difference between two groups. But as to the pain relief, the effect of the bolus of six drugs including rehmannia and the shengmai ossify group was better than that in the shengmai ossify group (P=0.0028). The improvement of physical signs was better in the bolus of six drugs including rehmannia and the shengmai ossify group than that in the shengmai ossify group (p＜0.05).
There was no significant difference in absorption of sequestra, formation of fresh bone and periosteal reaction between two groups.
3 conclusions
The pastille serum of bolus of six drugs including rehmannia had proliferation promotion, and the promotion of ALP secretion of the human osteoblast-like cell OS-732.
Different concentration of alcohol could lead to apoptosis of the human osteoblast-like cell OS-732. There was positive correlation between the apoptosis and alcohol concentration. The inverted microscope and the transmission electron microscope observation showed that cells were damaged, such as cell death, apoptosis and bubble-like change in survival cells.
The two-dimensional electrophoresis technique of proteomics showed that there was descending of partial protein expression in the human osteoblast-like cell OS-732 intervened by alcohol. But there was ascending of partial protein expression in the human osteoblast-like cell OS-732 treated by bolus of six drugs including rehmannia serum.
The bolus of six drugs including rehmannia was the reprehensive prescription of the method of invigorating the kidney. This prescription had better curative effect on the alcoholic steonecrosis of the femoral head in clinic. The combination of activating blood circulation to dissipate blood stasis could improve curative effect in clinic.
引文
[1] 张佩玉，王红霞，蒋海燕，等．人成骨肉瘤细胞系OS-732的建立及其特征的观察．中华骨科杂志，1985：5(2)：100～103．
[2] 骆鹏，吕慧贤，王立兰．MTT法测金葡液对成骨样细胞OS-732生长的影响．中国生物制品学杂志，2000；13(3)：172．
[3] 扬艳兰，柳洁，薛延．1，25—二羟维生素D_3对OS—732人骨样细胞增殖与分化的影响．首都医药，2004；24：18～19．
[4] 扬艳兰，柳洁，薛延．2β-(3羟丙氧)-骨化三醇对OS-732人成骨样细胞增殖与分化的影响．中华老年医学杂志，2005；24(7)：508～510．
[5] 薛延，袁润英，相东等．降钙素对人成骨样细胞增殖与细胞膜流动性的影响．卫生研究，2000；29(3)：168～169．
[6] Park SR, Oreffo RO, Triffitt JT. Bone, 1999; 24(6): 549～554.
[7] Nuttall ME, Patton AJ, Olivera DL, et al. J Bone Miner Res. 1998; 13(3): 371～382.
[8] Thompson DL, Lum KD, Nygaard SC. J Bone Miner Res, 1998; 13(2): 195～204.
[9] Satomura K, Krebsbach P, Bianco P, et al. J Cell Biochem, 2000; 78(3): 391～403.
[10] Matsuo K, Hirohata T, Sugioka Y, et al. Influence of alcohol intake, cigarette smoking, and occupational status on idiopathic osteonecrosis of the femoral head. Clin Orthopm, 1988; 234: 115～123.
[11] Hirota Y, Hirohata T, Fukuda K, et al. Association of alcohol intake, cigarette smoking and occupational status with the risk of idiopathic osteonecrosis of the femoral head. Am J Epidemiol. 1993; 137(5): 530～538.
[12] 樊粤光，袁浩，何伟，等．692例股骨头缺血坏死病因调查分析．广州中医学院学报，1999；11(1)：29．
[13] 王义生，张春霖，王利民，等．双支撑骨柱术治疗成人晚期股骨头缺血性坏死．中华骨科杂志，1995，15；584．
[14] 孙洪江，张洪．非创伤性股骨头缺血性坏死的临床病因分析．创伤骨科论坛，2002；31(3)：179．
[15] Castro FP Jr. Harris MB inferences in age, laterality and steinberg stage at initial presentation in patients with steroid-induced, alcohol-induced and idiopathic femoral head osteonecrosis. J Arthroplasty, 1999; 14(6): 672～676.
[16] 刘忠堂，王坤正，池永龙，等．酒精和激素性股骨头缺血性坏死发病机制的实 验研究．温州医学院学报，1999；29(3)：184．
[17] Manggold J, Sergi C, Becker K, et al. A new animal model of femoral head necrosis induced by intraosseous injection of ethanol. Lab Anim, 2002; 36(2): 173～180.
[18] 徐晓良，同志勤，王坤正，等．bBMP／胶原／珊瑚复合人工骨修复股骨头骨缺损实验研究，骨与关节损伤杂志，2000；15(3)：209～211．
[19] Solomon L. Idiopathic necrosis of the femoral head: Pathogenesis and treatment Can J Surg, 1981; 24(6): 573～578.
[20] Boskey AL, Raggio CL, Bullough PG, et al. Changes in the bone tissue lipids inpersons with steroid and alcohol-induced osteonecrosis. ClinOrthopm 1983; (172): 289～295.
[21] 王义生，毛克亚，李月白．酒精性股骨头缺血性坏死发病机理的实验研究．中华骨科杂志，1998；18(4)：231～233．
[22] Jones JP. Pat embolism and osteonecrosis. Ortbop Clin NorthAm,1985; 16: 595.
[23] JonesJP. Intravaseular coagulation and osteonecrosis. Clin Ortbop, 1992; 277: 41.
[24] Arlet J, LarocheM, SolerR, et al. Histopathology ofthe vessels ofthe femoral heads in specimens of osteonecrosis, osteoarthritis and algodystrophy. Clin Rheumatol, 1993; 12(2): 162.
[25] Rico H, Gomez Castresana F, Cabranes JA, et al. Increased blood cortisol in alcoholic patients with aseptic necrosis of the femoral head. Calcif Tissue Int, 1985; 37(6): 585～587.
[26] 黄永镇，骆旭东，郭坤亮，等．内源性糖皮质激素对酒精性股骨头缺血坏死骨细胞凋亡相关蛋白表达的影响．贵州医药，2004；28(12)：1059～1062．
[27] AnttiPoika I, Karahar Ju E, Vankka E, et al. Alcohol-associated femoral head necrosis. Ann chir Gynaecol, 1987; 76(6): 318～322.
[28]] 刘忠堂，王坤正，池永龙，等．PGE2和TXB2在酒精性股骨头骨坏死中的意义．临床骨科杂志，2002；5(2)：90．
[29] 何伟，徐传毅，李雄，等．酒精性股骨头坏死血液流变学改变及意义．中医正骨，2001；3(10)：8～9．
[30] Glueck CJ, Crawfard A, Dennis R, et al. Association of antithrombotic factor deficiencies and hypofibrinolysis with Legg-Perthes diseases. J Bone and Joint Surg, 1996; 78 (1): 3.
[31] 刘慧松，董天华．藻酸双酯钠、二磷酸盐联合应用对酒精性股骨头坏死预防作用的实验研究．苏州大学学报(医学版)，2003；23(5)：52．
[32] Wang Y, Li Y, Mao K, et al. alcohol-induced adipogenesis in bone and marrow: a possible mechanism for osteonecrosis. Clin Orthop, 2003, (410): 213～224.
[33] 李骁，于学忠，陈晓亮，等．酒精导致股骨头缺血性坏死早期变化实验研究．中国误诊学杂志，2003；3(9)：1304．
[34] Cheung RC, GrayC, Boyde A, et al. Effects of ethanol on bone cells in vitro resulting in increased resorption. Bone, 1995; 16(1): 143.
[35] Klein RF. Alcohol induced bone disease: Impact of ethanol on osteoblast proliferation. Alcohol clin Exp Res, 1997; 21(3): 392.
[36] Gold EW, Cangemi PI. Incidence and pathogensis of alcohol-indnced osteonecrosis of femoral head. CIin Orthop, 1979; 143: 222.
[37] 郭哲，赵德伟．酒精性股骨头缺血坏死的诊断与治疗．骨与关节损伤杂志，2000；15(4)：258．
[38] simank HG, Brocai DR, Strauck Km, et al. Core decompression in osteonecrosis of the femroal head: risk-factor-dependent outcome evaluation using survivorship analysis. Int Orthop, 1999; 23(3): 154～159.
[39] 曾意荣，袁浩，何伟，等．中西医结合治疗激素性或酒精性股骨头坏死．广州中医药大学学报，1999；16(3)：179～182．
[40] 杨康，马在山．马在山治疗酒精中毒性股骨头坏死100例疗效观察．北京中医，2001；20(2)：17～18．
[41] 杨艳萍，沈霖，周丕琪，等．补肾活血方对酒精性股骨头坏死患者血液流变学的影响．中国中医骨伤科杂志，2002；10(1)：28～30．
[42] Wang Y, Li Y, Man K, et al. Alcohol-induced adipogenesis in bone and marrow: a possible mechanism for osteonecrosis. Clin Orthop, 2003; (410): 213.
[43] Wang Y, Cui Q, Wang WJ, et al. Alcohol-induce regulation of genes involved in bone marrow cell osteogenesis and adipogenesis. Proceeding of the 48th Orthopaedic Research Society Meeting Dallas, 2002.
[44] 郭坤亮，赵德伟，等．早期酒精性股骨头缺血坏死骨细胞凋亡的实验研究．重庆医科大学学报，2003；28(3)：313～315．
[45] 骆旭东，郭坤亮，安洪．酒精性股骨细胞凋亡及其p53等基因的表达．中国公共卫生，2004；20(10)：1209～1210．
[46] Weinstein RS, Jilka RL, Parfitt AM, et al. Inhibition ofosteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocortoroids. Clin Invest, 1998; 102: 274～282.
[47] Parfitt AM, Villanueva AR, Foldes J, et al. Relations between histologic indices of bone formation: implications for the pathogenesis of spinal osteoporosis. Bone Miner Res, 1995; 10: 466～473.
[48] Sharma G, Sandhir R, et al. Effect of ethanol on cadmium uptake and metabolism of zinc and copper in rats exposed to cadmium. Nutr, 1991; 121: 87.
[49] John M, Rogers J. Zinc deficiency causes apoptosis but not cell cycle alterations in organogenesis stage rat embryos. Teratology, 1995; 52: 149.
[50] 焦健．酒精性营养不良．中国社区医师，2001；10：24～25．
[51] 高采平．肿瘤坏死因子—a与酒精性肝病．国外医学·消化系疾病分，2005，25(6)：379～381
[52] 黄永镇，骆旭东，郭坤亮．内源性糖皮质激素对酒精性股骨头缺血坏死骨细胞凋亡相关蛋白表达的影响．贵州医药，2004；28(12)：1059～1062．
[53] Prockop DJ. Marrow stromal cells as stem cells for nonhematopietic tissues. Science, 1997; 276(5309): 71～74.
[54] Bemsford JN, Bennett JH, Devlin C, et al. Evidence for an inverse relationship between the diferentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci, 1992; 102: 341～351.
[55] 王义生，李杰，殷力，等．酒精性骨坏死发病机制的分子生物学实验研究．河南医学研究，2004；13(1)：1～5．
[56] 肖鲁伟，王伟东，童培建，等．补肾复方含药血清对大鼠成骨细胞OPG、ODF表达的影响．中医正骨，2006；18(9)：1～3．
[57] Yasuda H, Shima N, kagawa N, et al. Osteoclast diferentiation factor is ligand for osteoprotegerin/ostecclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA, 1998; 95: 3597.
[58] Roodman GD. Cell biology Of the Osteoclast. Exprimental Hematology, 1999; 27: 1229.
[59] 胡冰，傅炳国，沈霖，等．复方中药含药血清对大鼠成骨细胞增殖及矿化功能的影响．中国中医骨伤科杂志，2003；11(4)：8～10．
[60] Bellows CG, Aubin JEM, Antosa ME. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria populations. Calcif Tissue Int, 1986; 38: 143.
[61] 马涛，崔燎，吴铁，等．老年大鼠含淫羊藿血清对成骨细胞的增殖与分化的响．中国骨质疏松杂志，2002；8(1)：55-57．
[62] 魏义勇，石印玉，冯伟．补肾中药对增龄成骨细胞VDR蛋白表达的影响．中国骨质疏松杂志，2006；12(2)：177～180．
[63] 秧荣昆，郭磊磊．骨碎补提取液对成骨细胞增殖的影响．贵阳中医学院学报， 2006；28(4)：61～62．
[64] 俞索静，肖鲁伟，吴承亮，等．右归饮对体外成骨细胞增殖和分化影响的实验研究．浙江临床医学，2004；6(12)：1027～1028．
[65] Wilkins MR, Sanchez JC, Gooley AA, et al. Progress with proteome project: why all proteins expressed by a genome should be identified and how to do it?. Biotechnol Genet Eng Rev, 1995; 13: 19.
[66] Wasbager V C, Humphery SmithI. Williams K L, et al. Progress with gene product mapping of the molicutes: Mycoplasmagenitalium. Plectrophoresis, 1995, 16, 1090—1094.
[67] JennyL. Proteomics: Capacity versus utility. Electro-phoresis, 2000, 21: 1071～1081.
[68] Hanghui L. Development of mutichannel devices with an array of electrosray tips for high-through put mass spectrometry. Anal Chem, 2000, 72: 3303～3310.
[69] Chambers G, Lawrie L, Cash P et al. A new approach to the study of disease. J Path, 2000, 192: 280.
[70] Blackstock WP and Weri WP. Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol, 1999, 17(3): 121.
[71] Jurgens M, Schrader M. Peptidomic approaches in proteomic research. Curr Opin Mol Ther, 2002, 4(3): 236～241.
[72] Dutt MJ and Lee KH. Proteomic analysis. Curr Opin Biotechnol, 2000; 11: 176.
[73] Gevaert K and Vandekerckhove J. Protein identification methods in proteomics. Electrophoresis, 2000; 21: 1145.
[74] Powell K. Proteomics delivers on promise of cancer biomarkers. Nat Med, 2003; 9: 980.
[75] Speicher DW. Proteomics: an infinite problem with infinite potential. The Scientist, 2002, 16: 12～14.
[76] Weinberger SR, Morris TS, Pawlak M. Recent trends in protein biochip technology. armacogenomics, 2000; 1(4): 395～416.
[77] Issaq HJ, Veenstra TD, Conrads TP et al. The SFLDI-TOF MS approach to proteomics: protein profiling and biomarker identification. Biochem Bioophys Commun, 2002; 292(3): 587～592.
[78] Vlahou A, Schellhammer PF, Mendrinos S, et al. Development of a novel proteomic approach for the detection of transitional cell carcinoma of the bladder in urine. Am J Pathol, 2001; 158 (4): 1491～1502.
[79] Petricoin EF, Ardekani AM, Hitt BA, et al. Use of proteomic patterms in serum to identify ovarian cancer. Lancet, 2002; 359 (9306): 572～577.
[80] Jr GW, Cazares LH, Leung SM, et al. Proteinchip (R) surface enhanced laser desorption/ionization (SFLDI)spectrometry: a novel protein biochip technology for detection of prostate cancer biomarkers in complex protein??mixtures. Prostate Cancer Prostatic Dis, 1999; 2 (5/6): 214～276.[81] Macbeath G, Schreiber SL. Printing proteins as microarrays for high-throughput function determination. Science, 2000; 278: 1760～1763.[82] 王斌．蛋白质芯片SFLDI-TOF-MS技与泌尿系肿瘤研究．国外医学泌尿系统分册，2005；25(3)：297～3000．[83] IssaqHJ, ConradsTP, PrietoDA, et al. SELDI-TOF MS for diagnost proteomics. Anal Chem, 2003; 75: 148～155.[84] Hutchens TW, Yip TT. New desorption strategies for the masss pectromeric analysis of macromolecules. Rapid Commun Mass Spectrom, 1993; 7: 576～580.[85] 沈自尹．有关证与神经内分泌免疫网络的研究．中医药学刊，2003；21(1)：10～11．[86] 申维玺．论中医“肾本质”的科学内涵．中国中医基础医学志，2001，7(6)：10～13．[87] 张杰，利用方证相应学说探寻中医证的物质基础．中国中医药信息杂志．2005；12(11)：3～5．[88] 马增春，高月，谭洪玲，等．用分子中药组学技术研究四物汤补血的作用机理．世界科学技术．中医药现代化
基础研究，2005；7(3)：23～27．[89] 马增春，高月，谭洪玲，等．四物汤对辐射致血虚证小鼠血清蛋白质表达的影响．中国中药杂志，2003；28(11)：1050～1054．[90] Iwama H, Amagaya S, Ogihara Y, et al. Effect of shosaikoto a Japanese and Chinese traditional herbal medical mixture on the mitogentic activity of lipopolysaccharid: a new pharmacological testing model. JEthonpharmacolm 1987; 21(1): 45.[91] 曹勇，张丹，郑广娟，等．补肾化瘀解毒方对肺癌耐药细胞内Ca~(2+)浓度的影响的研究．四川中医，2003；21(11)：24～26．[92] 张金梁，崔志清．中药血清药理学实验方法的研究探微．中医药学刊，2004；22(12)：2271～2272．[93] 孟李，王宁生．含药血清的制备方法研究．中药新药与临床药理，1999；10(5)：290．[94] 冯伟，石印玉．MTT法分析中药含药血清对体外软骨细胞增殖影响的研究．上海中医药大学学报，2000；14(1)：43．[95] 胡冰，傅炳国，沈霖，等．复方中药含药血清对大鼠成骨细胞增殖及矿化功能的影响．中国中医骨伤科杂志，2003；11(4)：8～10．[96] 冯伟，傅文彧，张玥，等．接骨中药含药血清对成骨细胞生物功能的影响．上海 第二医科大学学报，2004；24(7)：542～544．[97] 郑志永，杨久山，毕荣修，等．人成骨细胞的离体培养和鉴定．中国中医骨伤科杂志，2006；14(4)：8～9．
[98] 王元善，孙道升，杨荣，等．转化生长因子β调节体外培养人成骨细胞结缔组织生长因子mRNA的表达．中国临床康复，2006；10(45)：94～96．
[99] 詹红生，赵咏芳，冯伟，等．含药血清方法在中药调节骨与软骨代谢基础研究中的应用．中国骨伤，2000；13(11)：661～662．
[100] 李娟，吴伟康，余克强，等．不同实验浓度补肾中药血清对人成骨细胞增殖及分化的促进作用．中国临床康复，2005；9(19)：82～84．
[101] 肖鲁伟，王伟东，童培建，等．补肾复方含药血清对大鼠成骨细胞OPG、ODF表达的影响．中医正骨，2○○6；18(9)：1～3．
[102] 魏义勇，石印玉，冯伟．补肾中药对增龄成骨细胞VDR蛋白表达的影响．中国骨质疏松杂志，2006；12(2)：177～180．
[103] 李芳芳，李恩，宋士军，等．补肾方剂及不同分离组分对成骨细胞增殖分化的影响．中国骨质疏松杂志，1998；4(3)：71～75．
[104] suda T, Takahashi N. Osteoblasts are esential for osteoclast Formation. Calcif Tissue Int, 1989; 44(1): 45～47.
[105] 橱小东，陈安玉．氟对大鼠成骨样细胞增殖的影响．华西口腔医学杂志，1993；11(3)：230～232．
[106] 邢国胜，谈志龙，王淑云，等．补肾健骨汤对成骨细胞增殖及碱性磷酸酶、骨钙素合成的影响．中草药，2001；32(11)：1020～1022．
[107] Matsuo K, Hirohata T, Sugioka Y, et al. Influence of alcoholintake, cigarette smoking, and occupational status on idiopathie osteonecrosis of the femoral head. Clin Orthopm, 1988; 234: 115～123.
[108] Solonmon, J Clinical and therapeutic concepts in ischemic femur head necrosis. Orthopade, 1990; 19: 200.
[109] Wang GJ, Sweet DE Reger Sl, et al. Fat-cell changes as a mechanism of avascular necrosis of the femoral head in cortisone treated rabbit. Bone josurg(Am), 1977; 59: 729.
[110] 王义生，毛克亚，李月白．酒精性股骨头缺血坏死发病机的实验研究．中华骨科杂志，1998；18：231．
[111] Shih CH, Yang WE, Lee Zl et al. Effect of long-term alcohol on the femoral head of rabbit. Formos Med Assoc, 1991; 90: 443.
[112] Solomon L, Schntzler H, Seftel H, et al. Alcoholic osteonecrosis: an attempted experimental model in the adult rat. in: Arlet J, Ficat RP, Hungerford DS ed. Bone Circulation Baltimore. Williams & Wilkins. 1984, 238.
[113] Weinstein RS, Jilka RL, Parfitt AM, et al. Inhibition of osteoblastogenesis and promotion of of apoptosis of osteoblasts and osleocytes by glucocortoroidsClin Invest, 1998; 102: 274～282.
[114] Parfitt AM, Villanueva AR, Foldes J et al. Relations between histologic indices of bone formation: implications for the pathogenesis of spinal osteoporosis. Bone Miner Res, 1995; 10: 466～473.
[115] Sharma G, Sandhir R, et al. Effect of ethanol on cadmium uptake and metabolism of zinc and copper in rats exposed to cadmium. Nutr, 1991; 121: 87.
[116] John M, Rogers J. Zinc deficiency causes apoptosis but not cell cycle alterations in organogenesis stage rat embryos. Teratology, 1995; 52～149.
[117] Thuluvath PJ, Triger DR. Evaluation of nutritional status by using anthropometry in adults with alcoholic and nonalcoholic liver disease. AM Clin Nutr, 1994; 60: 269.
[118] Schlorff EC, Husain K, Somani SM. Dose and time dependent effects of ethanol on antioxdant system in rat testes. Alcohol, 1999; 18(2-3): 203～214.
[119] 马萍，李红．苍耳子的研究进展．中草药，1999；30(8)：634～636．
[120] AndsonL, SerhamerJ. A comparison of select mRNA and Protein aboundances in humanliver. Electrophoresis, 1997; 18: 533.
[121] LuYQ, GaoY. The development of pharmacology research in Chinese medicine herb compounds modernization. New Med Clin Pharm Res Chin Med Herb(中药新药与临床研究)，2002；13(1)：59～61．
[122] 中华人民共和国卫生部 中药新药临床研究指导原则(第3辑)．北京，1997：136．
[123] 李子荣．股骨头坏死的ARCO分期．中华外科杂志，1996；34(3)：186～187．
[124] 李宇明．袁浩教授治疗股骨头缺血性坏死的学术思想和经验．全国股骨头无菌性坏死学术研讨会论文汇编(北京)，1999，11．
[125] 邓沂，张晓刚，任远，等．中医对股骨头坏死的认识．甘肃中医学院报，1988；15(4)：54～56．
[126] 刘少军，袁浩．股骨头坏死的中医临床思路与方法探讨．中国医药学报，2002；17(1)：44～47．
[127] 龚梅芳，徐传毅，邹季，等．活血健骨汤对兔缺血性股骨头坏死TXB2和TBX2-6-Ket0-PGFI的作用．中国微循环，2001；5(2)：123～124．
[128] 肖正权．健骨颗粒治疗无菌性股骨头坏死120例临床观察．中医药学报，2002；30(4)：33．
[129] 左萍萍，吴业华，李学坤，等．新骨生胶囊治疗股骨头坏死的药理研究．中国康复理论与实验，2002；8(1)：11～12．
[130] 洪加源，许书亮，阮景绰，等．复元散对激素性股骨头环死脂代谢的影响．中医正骨，2001；13(4)：6～8．
[131] 茆军，郭玉成．补肾通络汤治疗早期非刨伤性股骨头坏死的临床研究．河北医学，2002；8(7)：672～673．
[2] 骆鹏，吕慧贤，王立兰．MTT法测金葡液对成骨样细胞OS-732生长的影响．中国生物制品学杂志，2000；13(3)：172．
[3] 扬艳兰，柳洁，薛延．1，25—二羟维生素D_3对OS—732人骨样细胞增殖与分化的影响．首都医药，2004；24：18～19．
[4] 扬艳兰，柳洁，薛延．2β-(3羟丙氧)-骨化三醇对OS-732人成骨样细胞增殖与分化的影响．中华老年医学杂志，2005；24(7)：508～510．
[5] 薛延，袁润英，相东等．降钙素对人成骨样细胞增殖与细胞膜流动性的影响．卫生研究，2000；29(3)：168～169．
[6] Park SR, Oreffo RO, Triffitt JT. Bone, 1999; 24(6): 549～554.
[7] Nuttall ME, Patton AJ, Olivera DL, et al. J Bone Miner Res. 1998; 13(3): 371～382.
[8] Thompson DL, Lum KD, Nygaard SC. J Bone Miner Res, 1998; 13(2): 195～204.
[9] Satomura K, Krebsbach P, Bianco P, et al. J Cell Biochem, 2000; 78(3): 391～403.
[10] Matsuo K, Hirohata T, Sugioka Y, et al. Influence of alcohol intake, cigarette smoking, and occupational status on idiopathic osteonecrosis of the femoral head. Clin Orthopm, 1988; 234: 115～123.
[11] Hirota Y, Hirohata T, Fukuda K, et al. Association of alcohol intake, cigarette smoking and occupational status with the risk of idiopathic osteonecrosis of the femoral head. Am J Epidemiol. 1993; 137(5): 530～538.
[12] 樊粤光，袁浩，何伟，等．692例股骨头缺血坏死病因调查分析．广州中医学院学报，1999；11(1)：29．
[13] 王义生，张春霖，王利民，等．双支撑骨柱术治疗成人晚期股骨头缺血性坏死．中华骨科杂志，1995，15；584．
[14] 孙洪江，张洪．非创伤性股骨头缺血性坏死的临床病因分析．创伤骨科论坛，2002；31(3)：179．
[15] Castro FP Jr. Harris MB inferences in age, laterality and steinberg stage at initial presentation in patients with steroid-induced, alcohol-induced and idiopathic femoral head osteonecrosis. J Arthroplasty, 1999; 14(6): 672～676.
[16] 刘忠堂，王坤正，池永龙，等．酒精和激素性股骨头缺血性坏死发病机制的实 验研究．温州医学院学报，1999；29(3)：184．
[17] Manggold J, Sergi C, Becker K, et al. A new animal model of femoral head necrosis induced by intraosseous injection of ethanol. Lab Anim, 2002; 36(2): 173～180.
[18] 徐晓良，同志勤，王坤正，等．bBMP／胶原／珊瑚复合人工骨修复股骨头骨缺损实验研究，骨与关节损伤杂志，2000；15(3)：209～211．
[19] Solomon L. Idiopathic necrosis of the femoral head: Pathogenesis and treatment Can J Surg, 1981; 24(6): 573～578.
[20] Boskey AL, Raggio CL, Bullough PG, et al. Changes in the bone tissue lipids inpersons with steroid and alcohol-induced osteonecrosis. ClinOrthopm 1983; (172): 289～295.
[21] 王义生，毛克亚，李月白．酒精性股骨头缺血性坏死发病机理的实验研究．中华骨科杂志，1998；18(4)：231～233．
[22] Jones JP. Pat embolism and osteonecrosis. Ortbop Clin NorthAm,1985; 16: 595.
[23] JonesJP. Intravaseular coagulation and osteonecrosis. Clin Ortbop, 1992; 277: 41.
[24] Arlet J, LarocheM, SolerR, et al. Histopathology ofthe vessels ofthe femoral heads in specimens of osteonecrosis, osteoarthritis and algodystrophy. Clin Rheumatol, 1993; 12(2): 162.
[25] Rico H, Gomez Castresana F, Cabranes JA, et al. Increased blood cortisol in alcoholic patients with aseptic necrosis of the femoral head. Calcif Tissue Int, 1985; 37(6): 585～587.
[26] 黄永镇，骆旭东，郭坤亮，等．内源性糖皮质激素对酒精性股骨头缺血坏死骨细胞凋亡相关蛋白表达的影响．贵州医药，2004；28(12)：1059～1062．
[27] AnttiPoika I, Karahar Ju E, Vankka E, et al. Alcohol-associated femoral head necrosis. Ann chir Gynaecol, 1987; 76(6): 318～322.
[28]] 刘忠堂，王坤正，池永龙，等．PGE2和TXB2在酒精性股骨头骨坏死中的意义．临床骨科杂志，2002；5(2)：90．
[29] 何伟，徐传毅，李雄，等．酒精性股骨头坏死血液流变学改变及意义．中医正骨，2001；3(10)：8～9．
[30] Glueck CJ, Crawfard A, Dennis R, et al. Association of antithrombotic factor deficiencies and hypofibrinolysis with Legg-Perthes diseases. J Bone and Joint Surg, 1996; 78 (1): 3.
[31] 刘慧松，董天华．藻酸双酯钠、二磷酸盐联合应用对酒精性股骨头坏死预防作用的实验研究．苏州大学学报(医学版)，2003；23(5)：52．
[32] Wang Y, Li Y, Mao K, et al. alcohol-induced adipogenesis in bone and marrow: a possible mechanism for osteonecrosis. Clin Orthop, 2003, (410): 213～224.
[33] 李骁，于学忠，陈晓亮，等．酒精导致股骨头缺血性坏死早期变化实验研究．中国误诊学杂志，2003；3(9)：1304．
[34] Cheung RC, GrayC, Boyde A, et al. Effects of ethanol on bone cells in vitro resulting in increased resorption. Bone, 1995; 16(1): 143.
[35] Klein RF. Alcohol induced bone disease: Impact of ethanol on osteoblast proliferation. Alcohol clin Exp Res, 1997; 21(3): 392.
[36] Gold EW, Cangemi PI. Incidence and pathogensis of alcohol-indnced osteonecrosis of femoral head. CIin Orthop, 1979; 143: 222.
[37] 郭哲，赵德伟．酒精性股骨头缺血坏死的诊断与治疗．骨与关节损伤杂志，2000；15(4)：258．
[38] simank HG, Brocai DR, Strauck Km, et al. Core decompression in osteonecrosis of the femroal head: risk-factor-dependent outcome evaluation using survivorship analysis. Int Orthop, 1999; 23(3): 154～159.
[39] 曾意荣，袁浩，何伟，等．中西医结合治疗激素性或酒精性股骨头坏死．广州中医药大学学报，1999；16(3)：179～182．
[40] 杨康，马在山．马在山治疗酒精中毒性股骨头坏死100例疗效观察．北京中医，2001；20(2)：17～18．
[41] 杨艳萍，沈霖，周丕琪，等．补肾活血方对酒精性股骨头坏死患者血液流变学的影响．中国中医骨伤科杂志，2002；10(1)：28～30．
[42] Wang Y, Li Y, Man K, et al. Alcohol-induced adipogenesis in bone and marrow: a possible mechanism for osteonecrosis. Clin Orthop, 2003; (410): 213.
[43] Wang Y, Cui Q, Wang WJ, et al. Alcohol-induce regulation of genes involved in bone marrow cell osteogenesis and adipogenesis. Proceeding of the 48th Orthopaedic Research Society Meeting Dallas, 2002.
[44] 郭坤亮，赵德伟，等．早期酒精性股骨头缺血坏死骨细胞凋亡的实验研究．重庆医科大学学报，2003；28(3)：313～315．
[45] 骆旭东，郭坤亮，安洪．酒精性股骨细胞凋亡及其p53等基因的表达．中国公共卫生，2004；20(10)：1209～1210．
[46] Weinstein RS, Jilka RL, Parfitt AM, et al. Inhibition ofosteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocortoroids. Clin Invest, 1998; 102: 274～282.
[47] Parfitt AM, Villanueva AR, Foldes J, et al. Relations between histologic indices of bone formation: implications for the pathogenesis of spinal osteoporosis. Bone Miner Res, 1995; 10: 466～473.
[48] Sharma G, Sandhir R, et al. Effect of ethanol on cadmium uptake and metabolism of zinc and copper in rats exposed to cadmium. Nutr, 1991; 121: 87.
[49] John M, Rogers J. Zinc deficiency causes apoptosis but not cell cycle alterations in organogenesis stage rat embryos. Teratology, 1995; 52: 149.
[50] 焦健．酒精性营养不良．中国社区医师，2001；10：24～25．
[51] 高采平．肿瘤坏死因子—a与酒精性肝病．国外医学·消化系疾病分，2005，25(6)：379～381
[52] 黄永镇，骆旭东，郭坤亮．内源性糖皮质激素对酒精性股骨头缺血坏死骨细胞凋亡相关蛋白表达的影响．贵州医药，2004；28(12)：1059～1062．
[53] Prockop DJ. Marrow stromal cells as stem cells for nonhematopietic tissues. Science, 1997; 276(5309): 71～74.
[54] Bemsford JN, Bennett JH, Devlin C, et al. Evidence for an inverse relationship between the diferentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci, 1992; 102: 341～351.
[55] 王义生，李杰，殷力，等．酒精性骨坏死发病机制的分子生物学实验研究．河南医学研究，2004；13(1)：1～5．
[56] 肖鲁伟，王伟东，童培建，等．补肾复方含药血清对大鼠成骨细胞OPG、ODF表达的影响．中医正骨，2006；18(9)：1～3．
[57] Yasuda H, Shima N, kagawa N, et al. Osteoclast diferentiation factor is ligand for osteoprotegerin/ostecclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA, 1998; 95: 3597.
[58] Roodman GD. Cell biology Of the Osteoclast. Exprimental Hematology, 1999; 27: 1229.
[59] 胡冰，傅炳国，沈霖，等．复方中药含药血清对大鼠成骨细胞增殖及矿化功能的影响．中国中医骨伤科杂志，2003；11(4)：8～10．
[60] Bellows CG, Aubin JEM, Antosa ME. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria populations. Calcif Tissue Int, 1986; 38: 143.
[61] 马涛，崔燎，吴铁，等．老年大鼠含淫羊藿血清对成骨细胞的增殖与分化的响．中国骨质疏松杂志，2002；8(1)：55-57．
[62] 魏义勇，石印玉，冯伟．补肾中药对增龄成骨细胞VDR蛋白表达的影响．中国骨质疏松杂志，2006；12(2)：177～180．
[63] 秧荣昆，郭磊磊．骨碎补提取液对成骨细胞增殖的影响．贵阳中医学院学报， 2006；28(4)：61～62．
[64] 俞索静，肖鲁伟，吴承亮，等．右归饮对体外成骨细胞增殖和分化影响的实验研究．浙江临床医学，2004；6(12)：1027～1028．
[65] Wilkins MR, Sanchez JC, Gooley AA, et al. Progress with proteome project: why all proteins expressed by a genome should be identified and how to do it?. Biotechnol Genet Eng Rev, 1995; 13: 19.
[66] Wasbager V C, Humphery SmithI. Williams K L, et al. Progress with gene product mapping of the molicutes: Mycoplasmagenitalium. Plectrophoresis, 1995, 16, 1090—1094.
[67] JennyL. Proteomics: Capacity versus utility. Electro-phoresis, 2000, 21: 1071～1081.
[68] Hanghui L. Development of mutichannel devices with an array of electrosray tips for high-through put mass spectrometry. Anal Chem, 2000, 72: 3303～3310.
[69] Chambers G, Lawrie L, Cash P et al. A new approach to the study of disease. J Path, 2000, 192: 280.
[70] Blackstock WP and Weri WP. Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol, 1999, 17(3): 121.
[71] Jurgens M, Schrader M. Peptidomic approaches in proteomic research. Curr Opin Mol Ther, 2002, 4(3): 236～241.
[72] Dutt MJ and Lee KH. Proteomic analysis. Curr Opin Biotechnol, 2000; 11: 176.
[73] Gevaert K and Vandekerckhove J. Protein identification methods in proteomics. Electrophoresis, 2000; 21: 1145.
[74] Powell K. Proteomics delivers on promise of cancer biomarkers. Nat Med, 2003; 9: 980.
[75] Speicher DW. Proteomics: an infinite problem with infinite potential. The Scientist, 2002, 16: 12～14.
[76] Weinberger SR, Morris TS, Pawlak M. Recent trends in protein biochip technology. armacogenomics, 2000; 1(4): 395～416.
[77] Issaq HJ, Veenstra TD, Conrads TP et al. The SFLDI-TOF MS approach to proteomics: protein profiling and biomarker identification. Biochem Bioophys Commun, 2002; 292(3): 587～592.
[78] Vlahou A, Schellhammer PF, Mendrinos S, et al. Development of a novel proteomic approach for the detection of transitional cell carcinoma of the bladder in urine. Am J Pathol, 2001; 158 (4): 1491～1502.
[79] Petricoin EF, Ardekani AM, Hitt BA, et al. Use of proteomic patterms in serum to identify ovarian cancer. Lancet, 2002; 359 (9306): 572～577.
[80] Jr GW, Cazares LH, Leung SM, et al. Proteinchip (R) surface enhanced laser desorption/ionization (SFLDI)spectrometry: a novel protein biochip technology for detection of prostate cancer biomarkers in complex protein??mixtures. Prostate Cancer Prostatic Dis, 1999; 2 (5/6): 214～276.[81] Macbeath G, Schreiber SL. Printing proteins as microarrays for high-throughput function determination. Science, 2000; 278: 1760～1763.[82] 王斌．蛋白质芯片SFLDI-TOF-MS技与泌尿系肿瘤研究．国外医学泌尿系统分册，2005；25(3)：297～3000．[83] IssaqHJ, ConradsTP, PrietoDA, et al. SELDI-TOF MS for diagnost proteomics. Anal Chem, 2003; 75: 148～155.[84] Hutchens TW, Yip TT. New desorption strategies for the masss pectromeric analysis of macromolecules. Rapid Commun Mass Spectrom, 1993; 7: 576～580.[85] 沈自尹．有关证与神经内分泌免疫网络的研究．中医药学刊，2003；21(1)：10～11．[86] 申维玺．论中医“肾本质”的科学内涵．中国中医基础医学志，2001，7(6)：10～13．[87] 张杰，利用方证相应学说探寻中医证的物质基础．中国中医药信息杂志．2005；12(11)：3～5．[88] 马增春，高月，谭洪玲，等．用分子中药组学技术研究四物汤补血的作用机理．世界科学技术．中医药现代化
基础研究，2005；7(3)：23～27．[89] 马增春，高月，谭洪玲，等．四物汤对辐射致血虚证小鼠血清蛋白质表达的影响．中国中药杂志，2003；28(11)：1050～1054．[90] Iwama H, Amagaya S, Ogihara Y, et al. Effect of shosaikoto a Japanese and Chinese traditional herbal medical mixture on the mitogentic activity of lipopolysaccharid: a new pharmacological testing model. JEthonpharmacolm 1987; 21(1): 45.[91] 曹勇，张丹，郑广娟，等．补肾化瘀解毒方对肺癌耐药细胞内Ca~(2+)浓度的影响的研究．四川中医，2003；21(11)：24～26．[92] 张金梁，崔志清．中药血清药理学实验方法的研究探微．中医药学刊，2004；22(12)：2271～2272．[93] 孟李，王宁生．含药血清的制备方法研究．中药新药与临床药理，1999；10(5)：290．[94] 冯伟，石印玉．MTT法分析中药含药血清对体外软骨细胞增殖影响的研究．上海中医药大学学报，2000；14(1)：43．[95] 胡冰，傅炳国，沈霖，等．复方中药含药血清对大鼠成骨细胞增殖及矿化功能的影响．中国中医骨伤科杂志，2003；11(4)：8～10．[96] 冯伟，傅文彧，张玥，等．接骨中药含药血清对成骨细胞生物功能的影响．上海 第二医科大学学报，2004；24(7)：542～544．[97] 郑志永，杨久山，毕荣修，等．人成骨细胞的离体培养和鉴定．中国中医骨伤科杂志，2006；14(4)：8～9．
[98] 王元善，孙道升，杨荣，等．转化生长因子β调节体外培养人成骨细胞结缔组织生长因子mRNA的表达．中国临床康复，2006；10(45)：94～96．
[99] 詹红生，赵咏芳，冯伟，等．含药血清方法在中药调节骨与软骨代谢基础研究中的应用．中国骨伤，2000；13(11)：661～662．
[100] 李娟，吴伟康，余克强，等．不同实验浓度补肾中药血清对人成骨细胞增殖及分化的促进作用．中国临床康复，2005；9(19)：82～84．
[101] 肖鲁伟，王伟东，童培建，等．补肾复方含药血清对大鼠成骨细胞OPG、ODF表达的影响．中医正骨，2○○6；18(9)：1～3．
[102] 魏义勇，石印玉，冯伟．补肾中药对增龄成骨细胞VDR蛋白表达的影响．中国骨质疏松杂志，2006；12(2)：177～180．
[103] 李芳芳，李恩，宋士军，等．补肾方剂及不同分离组分对成骨细胞增殖分化的影响．中国骨质疏松杂志，1998；4(3)：71～75．
[104] suda T, Takahashi N. Osteoblasts are esential for osteoclast Formation. Calcif Tissue Int, 1989; 44(1): 45～47.
[105] 橱小东，陈安玉．氟对大鼠成骨样细胞增殖的影响．华西口腔医学杂志，1993；11(3)：230～232．
[106] 邢国胜，谈志龙，王淑云，等．补肾健骨汤对成骨细胞增殖及碱性磷酸酶、骨钙素合成的影响．中草药，2001；32(11)：1020～1022．
[107] Matsuo K, Hirohata T, Sugioka Y, et al. Influence of alcoholintake, cigarette smoking, and occupational status on idiopathie osteonecrosis of the femoral head. Clin Orthopm, 1988; 234: 115～123.
[108] Solonmon, J Clinical and therapeutic concepts in ischemic femur head necrosis. Orthopade, 1990; 19: 200.
[109] Wang GJ, Sweet DE Reger Sl, et al. Fat-cell changes as a mechanism of avascular necrosis of the femoral head in cortisone treated rabbit. Bone josurg(Am), 1977; 59: 729.
[110] 王义生，毛克亚，李月白．酒精性股骨头缺血坏死发病机的实验研究．中华骨科杂志，1998；18：231．
[111] Shih CH, Yang WE, Lee Zl et al. Effect of long-term alcohol on the femoral head of rabbit. Formos Med Assoc, 1991; 90: 443.
[112] Solomon L, Schntzler H, Seftel H, et al. Alcoholic osteonecrosis: an attempted experimental model in the adult rat. in: Arlet J, Ficat RP, Hungerford DS ed. Bone Circulation Baltimore. Williams & Wilkins. 1984, 238.
[113] Weinstein RS, Jilka RL, Parfitt AM, et al. Inhibition of osteoblastogenesis and promotion of of apoptosis of osteoblasts and osleocytes by glucocortoroidsClin Invest, 1998; 102: 274～282.
[114] Parfitt AM, Villanueva AR, Foldes J et al. Relations between histologic indices of bone formation: implications for the pathogenesis of spinal osteoporosis. Bone Miner Res, 1995; 10: 466～473.
[115] Sharma G, Sandhir R, et al. Effect of ethanol on cadmium uptake and metabolism of zinc and copper in rats exposed to cadmium. Nutr, 1991; 121: 87.
[116] John M, Rogers J. Zinc deficiency causes apoptosis but not cell cycle alterations in organogenesis stage rat embryos. Teratology, 1995; 52～149.
[117] Thuluvath PJ, Triger DR. Evaluation of nutritional status by using anthropometry in adults with alcoholic and nonalcoholic liver disease. AM Clin Nutr, 1994; 60: 269.
[118] Schlorff EC, Husain K, Somani SM. Dose and time dependent effects of ethanol on antioxdant system in rat testes. Alcohol, 1999; 18(2-3): 203～214.
[119] 马萍，李红．苍耳子的研究进展．中草药，1999；30(8)：634～636．
[120] AndsonL, SerhamerJ. A comparison of select mRNA and Protein aboundances in humanliver. Electrophoresis, 1997; 18: 533.
[121] LuYQ, GaoY. The development of pharmacology research in Chinese medicine herb compounds modernization. New Med Clin Pharm Res Chin Med Herb(中药新药与临床研究)，2002；13(1)：59～61．
[122] 中华人民共和国卫生部 中药新药临床研究指导原则(第3辑)．北京，1997：136．
[123] 李子荣．股骨头坏死的ARCO分期．中华外科杂志，1996；34(3)：186～187．
[124] 李宇明．袁浩教授治疗股骨头缺血性坏死的学术思想和经验．全国股骨头无菌性坏死学术研讨会论文汇编(北京)，1999，11．
[125] 邓沂，张晓刚，任远，等．中医对股骨头坏死的认识．甘肃中医学院报，1988；15(4)：54～56．
[126] 刘少军，袁浩．股骨头坏死的中医临床思路与方法探讨．中国医药学报，2002；17(1)：44～47．
[127] 龚梅芳，徐传毅，邹季，等．活血健骨汤对兔缺血性股骨头坏死TXB2和TBX2-6-Ket0-PGFI的作用．中国微循环，2001；5(2)：123～124．
[128] 肖正权．健骨颗粒治疗无菌性股骨头坏死120例临床观察．中医药学报，2002；30(4)：33．
[129] 左萍萍，吴业华，李学坤，等．新骨生胶囊治疗股骨头坏死的药理研究．中国康复理论与实验，2002；8(1)：11～12．
[130] 洪加源，许书亮，阮景绰，等．复元散对激素性股骨头环死脂代谢的影响．中医正骨，2001；13(4)：6～8．
[131] 茆军，郭玉成．补肾通络汤治疗早期非刨伤性股骨头坏死的临床研究．河北医学，2002；8(7)：672～673．