【作者】 刘武；

【导师】 樊粤光；

【作者基本信息】 广州中医药大学 ， 中医骨伤科学， 2007， 博士

【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。更多还原

【Abstract】 1 Objective of researchOsteonecrosis 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 results2.1 The effect of the method of invigorating the kidney on cellar proliferation of human osteoblast-like cell 0S-7322.1.1 Drafting of culler curve of growthWe 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-732We 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-7322.2.1 Detection of apoptosis rate of human osteoblast-like cell OS-732 intervened by alcohol with different concentrationsWe 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 alcoholIt 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 methodThe 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 methodWe 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 conclusionsThe 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.更多还原