【作者】 吴淮；

【导师】 何伟；

【作者基本信息】 广州中医药大学 ， 中医骨伤科学， 2010， 博士

【摘要】 1研究目的随着激素在临床广泛应用,激素性股骨头坏死(SONFH)发病率有上升的趋势。该病是骨科临床的疑难病,病因病机复杂,缺乏公认有效的治疗手段。中医药治疗SONFH有一定效果,但其优势在于预防和早期的治疗,对于中晚期SONFH,单纯应用中药治疗效果欠佳。在临床上研究中药预防应用激素的高危人群的SONFH发病存在一些困难。本研究的目的是研究补肾活血祛痰中药组方健骨方在预防家兔SONFH中的效果和作用机制,为临床研究的开展提供实验依据和理论支持。2研究方法将30只健康SPF级新西兰家兔,按体重均衡原则随机分成4组：即正常组、模型组、补肾活血祛痰组方健骨方低剂量组(简称中药低剂量组)、健骨方高剂量组(简称中药高剂量组)。正常组6只,模型组、中药低剂量组、中药高剂量组各8只。正常对照组予灌胃等剂量生理盐水10ml／kg；中药低级量组予每天灌胃健骨方混悬液(含生药3.6g／kg／d)。中药高剂量组予每天灌胃健骨方混悬液(含生药7.19g／kg／d)；连续用药3周后,模型组、中药低剂量组、中药高剂量组均选择在左侧大腿内侧肌肉注射甲基泼尼龙琥珀酸钠20mg／kg／d一次,再连续用药3周。在实验开始3周后抽血查各组血脂三项、血液流变学进行检测,离心取部分血清置于—70℃的低温冰箱备用待检测骨保护素(osteoprotegerin,OPG)和破骨细胞分化因子(receptoractivactivatorofunclear factor kappa B ligand, RANKL);肌肉注射甲基泼尼龙琥珀酸钠造模3周后,再予以查各组血脂三项、血液流变学。连同造模前所取血清标本一起采用酶联免疫吸附试验法(ELISA)检测血清骨保护素(OPG)和破骨细胞分化因子(RANKL);并处死家兔,剖取右后侧股骨头,10%福尔马林溶液固定,进行制片、HE染色及病理组织学检查：把制作好的HE染色切片置于光学显微镜下,从低倍到高倍,参照Matsui病理分级方法观察骨髓、骨小梁有无坏死。采用Image-Pro Plus 6.0软件计算股骨头坏死面积及坏死率。其余数据采用SPSS18.0统计软件包进行统计。各组数据采用均数±标准差(x±S)表示,各组比较用单因素方差分析(one-way ANOVA),组间比较采用S-N-K法进行两两比较。变量间的相关性采用Spearman秩相关分析统计。以P<0.05为显著性差异,P<0.01为非常显著性差异。3研究结果3.1一般情况：中药低剂量组死亡1只,为灌胃操作方法失误,导致药液误入气道急性窒息而死；模型组造模后死亡2只。3.2血脂水平比较：造模前：各组血清高密度脂蛋白(HDL)、胆固醇(TCHO)、甘油三脂水平对比,F=0.733,P=0.542>0.05；S-N-K法组间两两比较亦无显著性差异(P>0.05),具有可比性。造模后：①各组血清HDL水平对比：F=2.534,P=0.082>0.05；S-N-K法组间两两比较提示：模型组血清HDL水平低于正常组,有显著性差异(P=0.042<0.05),低于中药高剂量组,有显著性差异(P=0.018<0.05)；②各组血清TCHO水平对比：F=0.458,P=0.714>0.05；S-N-K法组间两两比较亦无显著性差异；③各组血清TRIG水平对比：F=5.041,P=0.008<0.01；S-N-K法组间两两比较提示：模型组明显高于正常组,有非常显著性差异(P=0.001<0.01)；高于中药低剂量组(P=0.019<0.05),高于中药高剂量组(P=0.013<0.05),有显著性差异。3.2血流变学水平比较：造模前：各组血流变学指标对比：P>0.05,S-N-K法组间两两比较亦无显著性差异(P>0.05),具有可比性。造模后：①全血粘度值切变率：中药低剂量组与模型组全血粘度值切变率(200、30、5、1)对比无显著性差异(P>0.05)；模型组全血粘度值切变率(200、30、5、1)明显高于正常组(P<0.05),有非常显著性差异；模型组全血粘度值切变率200高于正常组(P<0.05),有显著性差异,全血粘度值切变率(30、5、1)明显高于正常组(P<0.01),有非常显著性差异；中药低剂量组全血粘度值切变率200高于正常组(P<0.05),有显著性差异,全血粘度值切变率(30、5、1)明显高于正常组(P<0.01),有非常显著性差异；②红细胞压积：模型组明显高于正常组,有非常显著性差异(P=0.000<0.01),中药低剂量组明显高于正常组,有非常显著性差异(P=0.000<0.01),中药高剂量组高于正常组,有显著性差异(P=0.017<0.05)；与模型组对比：中药低剂量组无显著性差异(P=0.306>0.05),中药高剂量组明显低于模型组,有非常显著性差异(P=0.007<0.01)；③血浆粘度值切变率200：模型组明显高于正常组,有非常显著性差异(P=0.000<0.01),中药低剂量组高于正常组,有显著性差异(P=0.012<0.05),中药高剂量组无显著性差异(P=0.070>0.05)；与模型组对比：中药低剂量组无显著性差异(P=0.142>0.05),中药高剂量组(P=0.020<0.05),有显著性差异；④各组全血低切相对粘度对比：模型组高于正常组、中药低剂量组和中药高剂量组,有显著性差异(P<0.05)；⑤全血高切相对粘度对比：模型组明显高于正常组、中药低剂量组和模型组,有非常显著性差异(P<0.01)；⑥其余各组红细胞刚性指数、红细胞聚集指数、红细胞变形指数、全血高切还原粘度、全血低切还原粘度对比(P>0.05),无统计学意义。3.3血清OPG、RANKL、OPG/RANKL水平比较：造模前：各组血清OPG水平对比：F=0.046,P>0.05；各组血清RANKL浓度对比：F=0.844,P=0.483>0.05；各组OPG/RANKL对比：F=0.89,P=0.459>0.05。S-N-K法组间两两比较亦无显著性差异。说明造模前各组动物OPG、RANKL、OPG/RANKL水平对比无显著性差异,具有可比性。造模后：①血清OPG水平对比：F=6.667,P=0.002<0.01；模型组明显高于正常组,有非常显著性差异(P=0.000<0.01),中药低剂量组和中药高剂量组低于正常组,有显著性差异(P<0.05)；中药低剂量组高于模型组,有显著性差异(P=0.026<0.05)；中药高剂量组高于模型组,有显著性差异(P=0.034<0.05)。②各组血清RANKL浓度对比：F=0.616,P=0.612>0.05；S-N-K法组间两两比较亦无显著性差异。③各组OPG/RANKL对比：模型组明显低于正常组,有非常显著性差异(P=0.000<0.01),中药低剂量组低于正常组,有显著性差异(P=0.011<0.05)；中药高剂量组明显高于正常组,有非常显著性差异(P=0.002<0.01)；中药低剂量组明显高于模型组,有非常显著性差异(P=0.001<0.01)；中药高剂量组明显高于模型组,有非常显著性差异(P=0.002<0.01)。3.4股骨头病理表现结果：①正常组：6例病理正常,符合0级病变；模型组：造模后死亡2例,6例病理学改变符合2级病变；中药低剂组：灌胃死亡1例,1例病理学改变符合2级病变,6例病理学改变符合1级病变；中药高剂组：1例病理学改变符合2级病变,7例病理学改变符合1级病变。②造模后模型组股骨头坏死面积和坏死率明显高于正常组、中药低剂量组、中药高剂量组(P<0.01),有非常显著性差异。3.5血清OPG、RANKL、OPG/RANKL与股骨头病理表现相关性分析：血清OPG水平与股骨头坏死面积的相关系数是-0.638,P=0.00<0.01,二者有负相关关系；血清RANKL水平与股骨头坏死面积的相关系数是0.131,P=0.516>0.05,无显著性差异；OPG/RANKL与股骨头坏死面积的相关系数是-0.636,P=0.00<0.01,二者有负相关关系；血清OPG水平与股骨头坏死率的相关系数是-0.673,P=0.00<0.01,二者有负相关关系；血清RANKL水平与股骨头坏死面积的相关系数是0.142,P=0.479>0.05,无显著性差异；OPG/RANKL与股骨头坏死率的相关系数是-0.638,P=0.000<0.01,二者有负相关关系。4研究结论4.1单纯肌注大剂量甲基波尼松龙造家兔SONFH模型,方法简单易行,形成周期短,动物死亡率低。4.2激素可导致兔血脂代谢紊乱,血液流变学异常,可能导致兔OPG、OPG/RANKL降低,上调破骨细胞的活性,促进骨吸收,导致骨小梁断裂,发生激素性股骨头坏死。4.3补肾活血祛痰中药组方健骨方可预防兔激素诱导的股骨头坏死的发生。4.4补肾活血祛痰组方健骨方高剂量组、低剂量对应用激素的兔的血脂代谢紊乱和血液流变学异常、血液粘稠度增加,具有明显改善作用,高剂量组的作用更明显。该方药可能通过改善激素引起的血脂代谢紊乱、血流变学异常来预防SONFH。4.5补肾活血健脾祛痰组方健骨方对激素导致的血OPG水平下降、OPG/RANKL降低有改善作用,有助于抑制破骨细胞功能,促进成骨,增加骨强度,防治骨坏死和塌陷的发生。更多还原

【Abstract】 1 Objective:With the mass use of clinical application of hormone, the trend of SONFH is increasing. SONFH is a difficult and complicate disease as its pathogenesis is not clear and lack of certain effective treatment approach for SONFH. Traditional Chinese medicine shows superiority in terms of prevention and treatment. But to the advanced SONFH, it’s unsatisfactory when simply uses traditional Chinese medicine. To study the therapeutic effects and mechanism of the prescription includes supplement kidney, invigorate blood and dispel phlegm and prescription of Jian Gu on rabbit’s SONFH prevention.2 Methods30 healthy SPF New Zealand rabbits were randomly allotted to 4 groups by weight:normal group (6 rabbits), the model group (8 rabbits), the prescription includes supplement kidney, invigorate blood and dispel phlegm and prescription of Jian Gu group (8 rabbits High-dosage group group for short), and the prescription of Jian Gu group (8 rabbits Low-dosage group group for short).The normal control group was intragastrically perfused with saline of the same volume as 10 ml per kilogram at the same time. Low-dosage group were intragastrically perfused with suspension of prescription of Jian Gu with same volume (effective components is 3.6g/kg/d); and High-dosage group is 7.19g/kg/d. After gavage for 3 weeks, the model group, Low-dosage group and High-dosage group were injected sodium succinate methylprednisolone on the internal side of left leg 20mg/kg each time for another 3weeks. After the experiment for 3 weeks, physical examination such as TC, TG, HDL and hemorrheological changes were collected for detection. Part of the centrifugal blood kept on the -70℃would be test for OPG and RANKL. After injected by sodium succinate methylprednisolone for 3 weeks, TC, TG, HDL and hemorrheological changes were be measured again. All the blood samples were determined for OPG and RANKL by enzyme linked immunosorbent assay (ELISA) especially include the sample before modeling; and killed the rabbits, took the right posterior femoral head, which fixed formalin then observed pathologically with frost slice techniques and Sudan staining. It was observed under light microscopy from lower power to higher power according to Matsui grades to measure if the marrow and bone trabeculae were necorsis.Date was tested with Image-Pro Plus 6.0 software to calculate the rate and square of necrosis of femoral head. The rest of the date was tested with SPSS18.0 software. The results were measured by ((?)±S). mean comparison in groups was conducted with single factor variance analysis, and the pairwise comparison was performed with S-N-K method. Correlation between the variable was deal with Spearman. There is statistical significance when P<0.05, and a very significant difference when P<0.01.3 Results3.1 The general result:One rabbit was died as faulty intragastrically method to block the tracheobronchial and made acute asphyxia in the Low-dosage group group. And 2 rabbits were died after modling.3.2 Compare between blood lipids level:HDL、TCHO and TG blood level before the experiment is F=0.733, P=0.542>0.05;there is no statistical significance in the pairwise comparison with S-N-K method (P>0.05), which shows comparable. After the experiment:①compare between each group in the level of HDL:F=2.534, P=0.082>0.05; the pairwise comparison with S-N-K method shows that:the HDL blood level in the model group was lower than normal group and had a statistical significance (P=0.042<0.05); the HDL blood level in the model group was lower than High-dosage group and had a statistical significance (P=0.018<0.05).②compare between each group in the level of TCHO, F=0.458, P=0.714>0.05; there is no statistical significance in the pairwise comparison with S-N-K method (P>0.05).③compare between each group in the level of TRIG:F=5.041, P=0.008<0.01; the pairwise comparison with S-N-K method shows that:the TRIG blood level in the model group was higher than normal group and had a very statistical significance (P=0.001<0.01); higher than low-dosage group (P=0.019<0.05) and high-dosage group (P=0.013 <0.05).3.3 Compare between hemorrheological changes:index of hemorrheological changes before the experiment is P>0.05, there is no statistical significance in the pairwise comparison with S-N-K methodwhich shows comparable. After the experiment:①compare between each group in the level of whole blood viscosity: there was no statistical significance(P>0.05) between low-dosage group and model group;model group was much higher than normal group (P<0.01); blood viscosity 200 of model group was higher than High-dosage group (P<0.05); blood viscosity. (30、5、1) of model group was much higher than High-dosage (P<0.01);blood viscosity 200 of Low-dosage group was higher than normal group (P<0.05);blood viscosity (30、5、1) of Low-dosage group was much higher than normal group (P<0.01).②compare between each group in the level of TCHO: F=0.458, P=0.714>0.05; there is no statistical significance in the pairwise comparison with S-N-K method (P>0.05).③compare between each group in the level of TRIG:F=5.041, P=0.008<0.01; the pairwise comparison with S-N-K method shows that:the TRIG blood level in the model group was higher than normal group and had a very statistical significance (P=0.001<0.01); and higher than low-dosage group (P=0.019<0.05) and high-dosage group (P=0.013<0.05); The low-dosage group was lower than normal group in hemorrheological changes, there was a very statistical significance between two groups.3.4 Compare between OPG、RANKL、OPG/RANKL:OPG、RANKL、OPG/RANKL blood level before the experiment is F=0.046, P>0.05; there is no statistical significance in the pairwise comparison with S-N-K method (P> 0.05), which shows comparable. After the experiment:①compare between each group in the level of OPG:F=6.667, P=0.002<0.01; the pairwise comparison with S-N-K method shows that:the OPG blood level in the model group was much lower than normal group and had a very statistical significance (P=0.000<0.01); was lower than Low-dosage group and had a statistical significance (P=0.026<0.05); and was lower than High-dosage group and had a statistical significance (P=0.034< 0.05); the OPG blood level in Low-dosage group and High-dosage group was lower than normal group and had a statistical significance (P<0.05).②compare between each group in the level of RANKL:F=0.616, P=0.612>0.05; there is no statistical significance in the pairwise comparison with S-N-K method (P> 0.05), which shows comparable.③ompare between each group in the level of OPG/RANKL:F=14.146, P=0.000<0.01;the pairwise comparison with S-N-K method shows that:the OPG/RANKL in the model group was much lower than normal group、Low-dosage group and High-dosage group and had a very statistical significance (P<0.01); the OPG/RANKL level in Low-dosage group and High-dosage group was lower than normal group and had a very statistical significance (P<0.01)3.5 Pathology of femoral head:①ormal group:all of the 6 femoral head pathology were normal with 0 grade pathology;model group:2 rabbits were died after modling, the other 6 femoral head pathology were 2 grade pathology;Low-dosage group:1 femoral head pathology was 2 grade pathology and 6 femoral head pathology were 1 grade; pathology high-dosage group:1 femoral head pathology was 2 grade pathology and 7 femoral head pathology were 1 grade.②the necrotic area and necrotic rate of normal group were much higher than the other three groups and had a very statistical significance (P<0.01).3.6 Correlation analysis between OPG、RANKL、OPG/RANKL in blood and pathology of femoral head:there is negative correlation between OPG and necrotic area (R=-0.638, P=0.00<0.01) and has much statistical significance; there is no correlation between RANKL and necrotic area (R=0.131, P=0.516>0.05) and has no statistical significance;there is negative correlation between OPG/RANKL and necrotic area (R=-0.636, P=0.00< 0.01) and has much statistical significance;there is negative correlation between OPG and necrotic rate (R=-0.673, P=0.00<0.01) and has much statistical significance; there is no correlation between RANKL and necrotic rate (R=0.142, P=0.479>0.05) and has no statistical significance; there is negative correlation between OPG/RANKL and necrotic rate (R=-0.638, P=0.000< 0.01) and has much statistical significance.4 Conclusion4.1 Model of SONFH by injected sodium succinate methylprednisolone on the rabbit is an easy way. Because it could shorter the production cycle and decrease the mortality of the rabbit.4.2 Hormone would cause abnormal blood lipid metabolisom and blood rheology, which lead to decrease the level of OPG. OPG/RANKL in rabbit. Also it could increase the activity of osteoclasts and bring down the activity of the osteoblast-like cells so that cause SONFH happen.4.3 Prescription includes supplement kidney, invigorate blood and dispel phlegm and prescription of Jian Gu can prevent rabbit’s SONFH happened.4.4 In the experiment, both the high-dosage group and low-dosage group can obviously improve the abnormal blood lipid, hemorrheological changes and blood viscosity. And the hight dosage, the more effect in the experiment. Maybe it would prevent SONFH through improving the abnormal blood lipid, hemorrheological changes cause by hormone.4.5 Prescription includes supplement kidney, invigorate blood and dispel phlegm and prescription of Jian Gu can improve the OPG, low level of OPG/RANKL in the blood which was caused by hormone. It could help to bring down the activity of osteoclast and increase the osteoblast in order to strength the density of the bone and prevent the bone collapsed and degraded.更多还原