脉络宁复合液治疗大鼠激素性股骨头坏死机理的实验研究
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摘要
1研究背景:
股骨头缺血性坏死(Avascular Necrosis of the Femoral Head简ANFH)是因创伤或非创伤等诸多原因导致股骨头的骨细胞、骨髓、造血细胞及脂肪细胞因缺血坏死,骨组织营养中断,最终造成股骨头密度增高或减低,骨囊性改变,骨小梁断裂,股骨头塌陷畸形等一系列病变,称为股骨头缺血性坏死。又因骨坏死并非细菌感染所致,所以又称为股骨头无菌性坏死是骨科常见病,由于多种病因破坏股骨头的血供,使骨的活性成分(包括骨细胞、骨髓细胞和脂肪细胞)死亡所引起的一个病理过程。由于发病原因复杂,病变导致关节软骨破坏,股骨头坏死性改变,使关节功能丧失,致残率很高。股骨头缺血性坏死发病以30-40岁青中年为主,40-80%患者为双侧发病;根据对其自然病程的研究表明,约80%未经治疗的股骨头坏死在1-3年内会发生股骨头塌陷。股骨头一旦塌陷,就会出现不同程度的疼痛及功能障碍,严重者不得不接受人工髋关节置换,极大地影响了患者的生活质量。
股骨头坏死的发生既有全身性的基础因素,更有其局部容易发病的特殊性。骨坏死发生后机体存在自身修复过程,表现为活骨部分的间充质细胞及毛细血管增生,侵入死骨间替代坏死的骨髓组织,未分化的间充质细胞附于死骨表面分化为成骨细胞形成新骨。对本病治疗的策略和目标应该是在积极治疗原发病,改善全身失衡状态的同时,解除局部供血不足,改善微循环,减轻静脉瘀阻,降低骨内压防止不良生物应力因素影响,并进行恰当功能锻炼,使其病理过程停止或逆转,诱导和促进新骨的形成和重建,重新修复股骨头,恢复髋关节功能。在中医看来该病主要因恶血内阻,一方面阻滞卫气运行于肌肉之间,丧失其卫外功能:另一方面阻滞气血运行于局部,引起局部失养,容易引起外邪侵袭,恶血与外邪交接发为骨痹。正如《灵枢.贼风》所言:“若有所堕坠,恶血在内而不去”。
目前国内外对于股骨头坏死的治疗临床多以手术疗法为主,其近期效果明显,但远期效果不理想。中医药综合疗法以保留骨结构及其功能为目的,从整体观念出发,内外并治,筋骨并重,以动静结合为原则而进行辨证论治,弥补了手术疗法之不足。我导师根据传统中医药理论,并结合临床实践创建了脉络宁复合液神经阻滞疗法治疗股骨头缺血性坏死,取得了满意的疗效。脉络宁复合液由脉络宁、2%利多卡因、5%碳酸氢钠按一定比例配伍而成。脉络宁为纯中药制剂,由牛膝、玄参、金银花、石斛组成,具有活血化瘀、滋阴养血、清热解毒的功效。主要用于治疗血栓闭塞性脉管炎、脑血栓形成及后遗症等病。脉络宁注射液组方中虽然未见活血化瘀中药,但其化学有效成分为绿原酸、阿牙品、滨蒿内脂和香豆素类化合物,能扩张血管,改善微循环,提高纤溶活性,降低纤维蛋白原含量,降低血液粘稠度,增加血流量,具有抗凝血、溶血栓作用,说明其确实有活血化瘀的功效。利多卡因局部神经阻滞可以切断疼痛的恶性循环,解除肌肉的痉挛,降低了髋关节内部的压力;同时利多卡因阻滞股动脉附近的交感神经,可以舒张血管,增加了股骨头周围的血供。此法真正从中医血瘀认识该病,并积极从改善股骨头局部的血供入手。中药制剂脉络宁的组方具有活血化瘀与滋阴养血互补,清热解毒与抗炎止痛并施特点,诸药配伍攻中寓补,补中有攻,既活血化瘀又不伤阴血,瘀祛新生,使局部病灶炎症消散,疼痛消失,经络气血运行正常。实验研究表明,脉络宁复合液对于神经根炎症引起的根性痛具有确切的疗效。脉络宁能够益气活血行血,疏通经络改善局部微循环。
2研究目的
通过小剂量激素长期应用制备股骨头坏死模型,应用脉络宁复合液疗法进行干预,观察治疗前后的变化。应用HE染色的方法,从组织形态学方面观察股骨头坏死的病理变化;应用免疫组织化学的方法,观察各组大鼠股骨头组织中影响骨生长和修复的多种生长因子的变化情况。检验脉络宁复合液治疗大鼠激素性股骨头坏死的疗效,从组织形态学和分子生物学进一步认识脉络宁复合液治疗该病的机理,为临床治疗提供理论基础。
3研究方法
3.1材料及方法
选用26只Wistar雄性成年大白鼠(贰级,由中国中医研究院实验动物中心提供,动物合格证号为SCXK(军)2002-001),体重290±20g。随机分为激素模型组(以下称模型组)10只,脉络宁复合液治疗组(以下称治疗组)10只,空白对照组(以下称空白组)6只。其中模型组和治疗组,腹腔注射醋酸强的松龙6.7mg/kg,3次/周,连续三周。造模成功后,治疗组从第4周开始在闭孔神经注射脉络宁复合液2ml/kg,3次/周,共8周:定位选用贝朗神经定位仪进行定位,定位前于大鼠右侧腹股沟处局部备皮,利多卡因凝胶局部均匀涂抹,以利于定位笔更好的与局部接触,定位笔发出电刺激后,发现股内侧肌群有节律跳动,调节刺激电量,当电量达到50mA时,股内收肌群仍然继续跳动,即为闭孔神经在体表的准确位置。治疗组、模型组和空白组大鼠一起于治疗给药后第9周的第一天取材,用0.3%戊巴比妥钠腹腔注射麻醉(1ml/100g),剖开腹腔,从腹主动脉采血5ml,采集前禁食12h,分别放于各管中,4℃离心3500rpm,15分钟,分离血清,4℃冰箱保存,送望京医院生化检验室测定总胆固醇、甘油三酯。同时对取下的股骨头制备石蜡切片,进行HE染色,光镜下观察坏死股骨头组织结构的病理变化,并通过图像分析仪对HE染色切片的空骨陷窝率、骨陷窝面积、骨小梁面积进行定量分析;进行血管内皮细胞生长因子(Vessel endothelial cell growth factor,VEGF)、骨发生蛋白(Bone morphogeneticproteins,BMPs)、转化生长因子β1(Transformng growth factor-β1,TGF-β1)、Ⅷ因子相关抗原(Ⅷfactor fos-related antigen,Ⅷ-R Ag)的免疫组织化学SP法染色,利用图像分析仪分析VEGF、BMPs、TGF-β1的阳性面积比、阳性细胞数目比,Ⅷ-R Ag所表达的微血管密度。
3.2统计方法
采用SPSS13.0软件数据进行统计分析,组间计量资料数据比较采用单因素方差分析,计量资料数据以均数±标准差((?)±S)表示,P＜0.05为有统计学意义。
4研究结果
4.1病理组织学观察
4.1.1 HE染色光镜观察
(1)空白组
①骨小梁整齐清楚、排列规则、致密饱满。②骨皮质及骨小梁中骨细胞清晰可见,核较大,位于中央,偶可见骨陷窝空虚。③潮线完整,钙化带与软骨下骨小梁连接完好。④髓腔内造血细胞丰富,形态正常。软骨下血管较丰富,管径较大的血管壁结构清晰。⑤骨小梁边缘成骨细胞数量较多,髓腔中破骨细胞数量较少。
(2)模型组
①骨小梁稀疏,变细,甚至有断裂现象,结构紊乱,部分有碎片出现。②骨小梁中骨细胞坏死,空骨陷窝数较正常组增多。③髓腔内可见部分脂肪细胞体积增大,有的甚至融合成泡状,造血细胞数量较正常组明显减少。④坏死骨小梁间隙可见有明显纤维肉芽组织增生,在坏死骨小梁周围包绕。⑤骨小梁周围可见少量成骨细胞排列,但数量很少,骨小梁周围无明显新骨沉积性生长。⑥髓腔内多核破骨细胞数量增多。⑦软骨下小血管数量明显减少。⑧潮线明显出现了不延续,有的部分甚至消失。
(3)治疗组
①骨小梁较模型组变粗,数量明显增多,骨小梁结构大体清晰。②骨小梁中较多正常骨细胞,但仍可见少量坏死骨细胞,空骨陷窝较模型组明显减少。③髓腔内脂肪空泡较模型组减少,造血细胞数量较模型组增多。④成骨细胞数量较模型组增多。⑤髓腔内多核破骨细胞数量较模型组明显减少。⑥软骨下小血管数量较模型组明显增多。
4.1.2图像分析仪定量分析
①空骨陷窝率:模型组空骨陷窝率增高明显,与治疗组及空白组比较,组间差异显著(P＜0.05);治疗组空骨陷窝率较模型组明显降低,但与空白组比较组间差异仍显著(P＜0.05)。
②骨陷窝面积:模型组骨陷窝面积明显增高,与空白组比较,组间差异显著(P＜0.05);治疗组骨陷窝面积明显降低,与模型组比较,组间差异显著(P＜0.05)。
③骨小梁面积:模型组骨小梁面积明显降低,与空白组比较,组间差异显著(P＜0.05);治疗组骨小梁面积在数值上高于模型组,但是二者组间比较无显著性差异(P＞0.05)。
4.2大鼠股骨头组织内VEGF、BMPs、TGFβ1、Ⅷ-R Ag的表达
(1)在VEGF的表达方面:①阳性面积比:治疗组表达高于模型组,且差异显著p＜0.05;但治疗组的表达低于空白组,且差异显著p＜0.05;②阳性细胞数目比:治疗组表达高于模型组,且差异显著p＜0.05;但治疗组的表达低于空白组,差异显著p＜0.05。
(2)在BMPs的表达方面:①阳性面积比:治疗组表达高于模型组,且差异显著p＜0.05;模型组表达低于空白组,且差异显著p＜0.05:②阳性细胞数目比:治疗组表达高于模型组,且差异显著p＜0.05;模型组表达低于空白组,且差异显著p＜0.05。
(3)在TGF-β1的表达方面:①阳性面积比:治疗组表达高于模型组,且差异显著p＜0.05;模型组表达低于空白组差异显著p＜0.05;治疗组与空白组比较差异显著p＜0.05;②阳性细胞数目比:治疗组表达高于模型组,且差异显著p＜0.05,治疗组与空白组比较差异显著p＜0.05;模型组表达低于空白组两组间差异显著p＜0.05。
(4)Ⅷ-R Ag:微血管密度,治疗组的表达高于模型组,低于空白组,与模型组比较差异显著p＜0.05,与空白组比较差异显著p＜0.05。
4.3血脂
总胆固醇及甘油三酯水平比较可见,治疗组低于模型组,差异显著p＜0.05;模型组高于空白组,差异显著p＜0.05。
5结语
在本研究中,我们采用小剂量激素长期应用的方法,在用药的第3周就成功诱导出了激素性股骨头坏死模型,表现为骨髓内脂肪细胞增生、肥大,骨小梁稀疏,空骨陷窝率明显增多,造血细胞数量明显减少,成骨细胞数量明显减少,骨小梁周围无明显新骨沉积性生长,髓腔内多核破骨细胞数量增多,软骨下小血管数量明显减少。
在实验过程中,我们发现激素性股骨头坏死的发生可能与TGFβ_1、BMPs、VEGF、的表达下调和新生微血管数量的减少有关;同时发现模型组血清甘油三酯及胆固醇明显升高,脂质代谢紊乱。
脉络宁复合液能通过其活血化瘀、清热解毒、滋阴养血的作用预防及治疗激素性股骨头坏死。该治疗方法不仅能纠正脂肪代谢紊乱,并能上调VEGF、BMPs、TGFβ1、的表达;且该疗法能明显增加坏死股骨头的血供。
6.结论
(1)采用单纯小剂量激素长期应用的方法,可以成功的建立激素性股骨头坏死模型,能较好反应临床激素性股骨头坏死的病理过程。
(2)激素性股骨头坏死的发生与激素所致的脂肪代谢紊乱及组织内骨相关基因蛋白表达改变相关。
(3)通过脉络宁复合液治疗可以使与微血管及骨细胞生成密切相关的生长因子表达上调,促进骨修复,从而治疗激素性股骨头坏死。
(4)运用脉络宁复合液治疗可以改善激素引起的高血脂状况,恢复全身的脂代谢平衡,减少髓腔内的脂肪堆积,有效的降低了骨内压,使坏死区血供增加。
(5)脉络宁注射液具有活血化瘀、滋阴养血、清热解毒的功效。本实验结果说明脉络宁注射液与利多卡因、碳酸氢钠以一定比例配合,在治疗激素性股骨头坏死方面,疗效确切;通过增加与微血管及骨细胞生成密切相关的生长因子的生成,以及改善激素引起的高血脂状态,促进坏死区组织的修复是该疗法的主要治疗机理。
1 Background:
Avascular necrosis(Avascular Necrosis of the Femoral Head SR ANFH) is the result of trauma or injury,and many other reasons for avascular necrosis of the bone cells of femoral head,bone marrow,blood cells and fat cells,bone nutrition interruption,finally result in Increase or reduce the density of the femoral head,bone cystic changes,bone fracture,the femoral head collapse deformity and a series of diseases,known as femoral head avascular necrosis.Because of bone necrosis is not caused by bacterial infection,also known as axenic avascular necrosis of the femoral head.It is the orthopedic common diseases,due to a variety of causes destructing the blood supply of the femoral head,the active component of bone (including bone cells,bone marrow Cell and fat cell) death caused by a pathological process.As the incidence reasons are complicated,leading to lesions of cartilage damage, osteonecrosis of the femoral head of change,lose joint function and has a high morbidity.Avascular necrosis in the incidence of middle-aged mainly 30 to 40-year-old Green,40-80%of the patients for bilateral disease,according to their natural illness course of the study showed that about 80 percent of untreated ANFH Femoral head collapse will occur during the 1-3years.Once the femoral head collapse,there will be varying degrees of pain and dysfunction,serious patient had to accept artificial hip replacement,It has greatly affected the quality of patients life.
Necrosis of the femoral head happens not only for the basis of systemic factors,but also incidence of local particularity,the body repair its own existence after Osteonecrosis, performance on mesenchymal cells on live bone and capillary proliferation invasive necrosis of bone and replace marrow,undifferentiated mesenchymal cells attached to the surface necrosis bone differentiation into skeletogenous cell and form new bone.The strategy and the goal on the treatment of this disease on should be active in the treatment of the original disease, improve the general state of imbalance at the same time,partial lifting of insufficient blood supply,improve microcirculation and reduce vein Stagnation and reduce pressure within the bone to prevent adverse biological stress factors influence and take appropriate Functional training,to stop or reverse pathological process,induction and promotion new bone formation and reconstruction,the femoral head to repair and restore the hip joint function,in Chinese medicine ANFH occurs mainly because of bad blood disease internal resistance,on the one hand block Weiqi through the muscle and skin their health,loss of protected function:On the other hand block of qi and blood running in local,caused partial loss support,and lead to invasion evil easily,evil blood and invasion evil maxed for the Bone Bi.As《Lingshu.Thief wind》said:If the falling something,the worsen blood will store.
For the current therapy to osteonecrosis of the femoral head mainly depend on the surgery in china,the recent results is clearly,but not an ideal long-term effect.Chinese medicine combination therapy reserve bone structure and function proceeding from the whole concept focus on both internal and external,ax activity and still,bones and tendons take with the principle of a Differential Treatment,made complete deficiency surgical therapy.My teacher accroding on the basis of traditional Chinese medicine theory and clinical practice,created a Mailuoning composite liquid therapy avascular necrosis by nerve block, and achieved satisfactory results.Mailuoning composite liquid make uo with Mailouning,2% lidocaine and 5%sodium bicarbonate by a certain percentage from compatibility.Mailouning is pure Chinese medicines combine with Achyranthes,Scrophulariaceae,honeysuckle, Dendrobium composition,with function of Promoting Blood Circulation and Relieving Blood Stasis,nourishing yin and blood,Qingrejiedu,Mainly use for the treatment of thrombosis obliterans,and sequelae of cerebral thrombosis and other diseases.There is no moving blood medicine in Mailuoning composit but it has active ingredients for chemical chlorogenic acid, Azerbaijan dental materials,and the fat within the foreshore Artemisia coumarin compounds, to expand blood vessels,improve microcirculation,Improve fibrinolytic activity,reduce fibrinogen levels,lower blood viscosity,increasing blood flow,with an anticoagulant, thrombolytic effect.it prove that have the effect of promoting blood circulation,lidocaine can cut off the vicious cycle of pain by Local nerve block,lift muscle cramps and lower the pressure within the hip joint,while lidocaine block femoral artery near the sympathetic, diastolic blood vessels,increase blood supply of the femoral head around.This method really understands the disease from Chinese medicine blood stasis,and start with improving the local blood supply of femoral head actively,This study's main purpose is observing the effect of Mailuoning compound in the treatment of hormone-ANFH mechanism by nerve block. theoretical basis for clinical treatment.To this end,the topics prepare to de research in three areas from the morphology,immunohistochemistry,blood lipid changes.
2 Purpose of the study
Through the application of ANFH model in Long-term low-dose hormone of Mailuoning composite therapy to intervene observing before and after treatment changes.Application HE staining methods,observed ANFH the pathological changes from the morphology in the application of immunohistochemistry methods to observe the rats' in the femoral head impact on bone growth and repair of a variety of growth factor's in the changes.Mailuoning test compound in the treatment of hormone-necrosis of the femoral head,and further understanding Mailuoning compound in the treatment of the disease mechanism from morphology and molecular biology,to provide a theoretical basis for clinical treatment.
3 Research Methods
3.1 Materials and Methods
Selection of 26 adult male Wistar rats(second degree by the China Institute of Traditional Chinese Medicine Centre for laboratory animals,animal qualified for the No.SCXK(Army) 2002-001) weight 290±20g,Select 20 copies of the hormone necrosis of the femoral head animal model:intraperitoneal injection of acetic acid prednisolone 6.7mg/kg,3 times/ week,for three weeks,after the completion of the model group were randomly divided into hormone(for short hormone group),10,Mailuoning compound in the treatment group(for short the treatment group) 10,and the remaining six is the blank group(for short blank group).The treatment group,hormone group and blank group were taken the rat model in the first day after nine weeks.with 0.3 percent sodium pentobarbital intraperitoneal injection anesthesia(1ml/100g),paunch and take abdominal aorta blood 5 ml,fasting 12 h before Acquisition,were put in the tube apartly.4℃centrifugal 3500 rpm,15 minutes,separation of serum,4℃refrigerator preservation,sent to the WanNing hospital biochemical laboratory tests total cholesterol,triglycerides.At the same time to remove the paraffin sections of the femoral head,HE stain,Observed the femoral head of the pathological changes in organizational structure by light microscopy and,through image analysis instrument analysis HE staining biopsy rate of lacunae,bone lacunae area,Trabecular bone area of quantitative analysis for vascular endothelial growth factor(Vessel endothelial cell growth factor,VEGF),bone protein(Bone morphogenetic proteins,BMPs),transforming growth factorβ1(Transforming growth factor-β1,TGF-β1),Ⅷ-related antigen(Ⅷfactor fos-related antigen,Ⅷ-R Ag) Immunohistochemical staining SP method,using image analysis instrument analysis VEGF,BMPs,TGF-β1,the positive area ratio,the number of positive cells,Ⅷ-RAg of the MVD.
3.2 statistical methods
SPSS13.0 software used for statistical analysis,the measurement data group compared the single-factor analysis of variance,the measurement data to mean±standard deviation(x±S) said,P＜0.05 for statistical significance.
4 Conclusion
(1).A simple small dose of the hormone long-term approach can be successful establishment of hormone-ANFH model,to better clinical response the pathological process of the hormone ANFH.
(2).Necrosis of the femoral head of hormones in hormone-induced fat metabolic disorders in the bone-related gene expression changes were related.
(3).Through Mailuoning compound treatment can microvascular and growth factor generate expression closely related to bone cells growth increased and promote bone repair,then cure hormone osteonecrosis of the femoral head.
(4).Mailuoning compound used in the treatment can improve by high blood lipid situation caused the hormone and restore balance the body lipid metabolism and reduce fat accumulation in the marrow,effectively reduce the pressure within the bone,increase the blood supply of necrosis.
(5).Mailuoning injection has functions of Promoting Blood Circulation and Relieving Blood Stasis,nourishing yin and blood,Qingrejiedu.The experimental results show that Mailuoning injection with lidocaine,sodium bicarbonate according a certain percentage with a nerve block approach in the treatment of hormone necrosis of the femoral head,is effective through increased and microvascular and the growth factor of production which closely related bone cells generate,and to improve high blood lipid status caused by the hormone,It is the main mechanism of that therapy.
股骨头缺血性坏死(Avascular Necrosis of the Femoral Head简ANFH)是因创伤或非创伤等诸多原因导致股骨头的骨细胞、骨髓、造血细胞及脂肪细胞因缺血坏死,骨组织营养中断,最终造成股骨头密度增高或减低,骨囊性改变,骨小梁断裂,股骨头塌陷畸形等一系列病变,称为股骨头缺血性坏死。又因骨坏死并非细菌感染所致,所以又称为股骨头无菌性坏死是骨科常见病,由于多种病因破坏股骨头的血供,使骨的活性成分(包括骨细胞、骨髓细胞和脂肪细胞)死亡所引起的一个病理过程。由于发病原因复杂,病变导致关节软骨破坏,股骨头坏死性改变,使关节功能丧失,致残率很高。股骨头缺血性坏死发病以30-40岁青中年为主,40-80%患者为双侧发病;根据对其自然病程的研究表明,约80%未经治疗的股骨头坏死在1-3年内会发生股骨头塌陷。股骨头一旦塌陷,就会出现不同程度的疼痛及功能障碍,严重者不得不接受人工髋关节置换,极大地影响了患者的生活质量。
股骨头坏死的发生既有全身性的基础因素,更有其局部容易发病的特殊性。骨坏死发生后机体存在自身修复过程,表现为活骨部分的间充质细胞及毛细血管增生,侵入死骨间替代坏死的骨髓组织,未分化的间充质细胞附于死骨表面分化为成骨细胞形成新骨。对本病治疗的策略和目标应该是在积极治疗原发病,改善全身失衡状态的同时,解除局部供血不足,改善微循环,减轻静脉瘀阻,降低骨内压防止不良生物应力因素影响,并进行恰当功能锻炼,使其病理过程停止或逆转,诱导和促进新骨的形成和重建,重新修复股骨头,恢复髋关节功能。在中医看来该病主要因恶血内阻,一方面阻滞卫气运行于肌肉之间,丧失其卫外功能:另一方面阻滞气血运行于局部,引起局部失养,容易引起外邪侵袭,恶血与外邪交接发为骨痹。正如《灵枢.贼风》所言:“若有所堕坠,恶血在内而不去”。
目前国内外对于股骨头坏死的治疗临床多以手术疗法为主,其近期效果明显,但远期效果不理想。中医药综合疗法以保留骨结构及其功能为目的,从整体观念出发,内外并治,筋骨并重,以动静结合为原则而进行辨证论治,弥补了手术疗法之不足。我导师根据传统中医药理论,并结合临床实践创建了脉络宁复合液神经阻滞疗法治疗股骨头缺血性坏死,取得了满意的疗效。脉络宁复合液由脉络宁、2%利多卡因、5%碳酸氢钠按一定比例配伍而成。脉络宁为纯中药制剂,由牛膝、玄参、金银花、石斛组成,具有活血化瘀、滋阴养血、清热解毒的功效。主要用于治疗血栓闭塞性脉管炎、脑血栓形成及后遗症等病。脉络宁注射液组方中虽然未见活血化瘀中药,但其化学有效成分为绿原酸、阿牙品、滨蒿内脂和香豆素类化合物,能扩张血管,改善微循环,提高纤溶活性,降低纤维蛋白原含量,降低血液粘稠度,增加血流量,具有抗凝血、溶血栓作用,说明其确实有活血化瘀的功效。利多卡因局部神经阻滞可以切断疼痛的恶性循环,解除肌肉的痉挛,降低了髋关节内部的压力;同时利多卡因阻滞股动脉附近的交感神经,可以舒张血管,增加了股骨头周围的血供。此法真正从中医血瘀认识该病,并积极从改善股骨头局部的血供入手。中药制剂脉络宁的组方具有活血化瘀与滋阴养血互补,清热解毒与抗炎止痛并施特点,诸药配伍攻中寓补,补中有攻,既活血化瘀又不伤阴血,瘀祛新生,使局部病灶炎症消散,疼痛消失,经络气血运行正常。实验研究表明,脉络宁复合液对于神经根炎症引起的根性痛具有确切的疗效。脉络宁能够益气活血行血,疏通经络改善局部微循环。
2研究目的
通过小剂量激素长期应用制备股骨头坏死模型,应用脉络宁复合液疗法进行干预,观察治疗前后的变化。应用HE染色的方法,从组织形态学方面观察股骨头坏死的病理变化;应用免疫组织化学的方法,观察各组大鼠股骨头组织中影响骨生长和修复的多种生长因子的变化情况。检验脉络宁复合液治疗大鼠激素性股骨头坏死的疗效,从组织形态学和分子生物学进一步认识脉络宁复合液治疗该病的机理,为临床治疗提供理论基础。
3研究方法
3.1材料及方法
选用26只Wistar雄性成年大白鼠(贰级,由中国中医研究院实验动物中心提供,动物合格证号为SCXK(军)2002-001),体重290±20g。随机分为激素模型组(以下称模型组)10只,脉络宁复合液治疗组(以下称治疗组)10只,空白对照组(以下称空白组)6只。其中模型组和治疗组,腹腔注射醋酸强的松龙6.7mg/kg,3次/周,连续三周。造模成功后,治疗组从第4周开始在闭孔神经注射脉络宁复合液2ml/kg,3次/周,共8周:定位选用贝朗神经定位仪进行定位,定位前于大鼠右侧腹股沟处局部备皮,利多卡因凝胶局部均匀涂抹,以利于定位笔更好的与局部接触,定位笔发出电刺激后,发现股内侧肌群有节律跳动,调节刺激电量,当电量达到50mA时,股内收肌群仍然继续跳动,即为闭孔神经在体表的准确位置。治疗组、模型组和空白组大鼠一起于治疗给药后第9周的第一天取材,用0.3%戊巴比妥钠腹腔注射麻醉(1ml/100g),剖开腹腔,从腹主动脉采血5ml,采集前禁食12h,分别放于各管中,4℃离心3500rpm,15分钟,分离血清,4℃冰箱保存,送望京医院生化检验室测定总胆固醇、甘油三酯。同时对取下的股骨头制备石蜡切片,进行HE染色,光镜下观察坏死股骨头组织结构的病理变化,并通过图像分析仪对HE染色切片的空骨陷窝率、骨陷窝面积、骨小梁面积进行定量分析;进行血管内皮细胞生长因子(Vessel endothelial cell growth factor,VEGF)、骨发生蛋白(Bone morphogeneticproteins,BMPs)、转化生长因子β1(Transformng growth factor-β1,TGF-β1)、Ⅷ因子相关抗原(Ⅷfactor fos-related antigen,Ⅷ-R Ag)的免疫组织化学SP法染色,利用图像分析仪分析VEGF、BMPs、TGF-β1的阳性面积比、阳性细胞数目比,Ⅷ-R Ag所表达的微血管密度。
3.2统计方法
采用SPSS13.0软件数据进行统计分析,组间计量资料数据比较采用单因素方差分析,计量资料数据以均数±标准差((?)±S)表示,P＜0.05为有统计学意义。
4研究结果
4.1病理组织学观察
4.1.1 HE染色光镜观察
(1)空白组
①骨小梁整齐清楚、排列规则、致密饱满。②骨皮质及骨小梁中骨细胞清晰可见,核较大,位于中央,偶可见骨陷窝空虚。③潮线完整,钙化带与软骨下骨小梁连接完好。④髓腔内造血细胞丰富,形态正常。软骨下血管较丰富,管径较大的血管壁结构清晰。⑤骨小梁边缘成骨细胞数量较多,髓腔中破骨细胞数量较少。
(2)模型组
①骨小梁稀疏,变细,甚至有断裂现象,结构紊乱,部分有碎片出现。②骨小梁中骨细胞坏死,空骨陷窝数较正常组增多。③髓腔内可见部分脂肪细胞体积增大,有的甚至融合成泡状,造血细胞数量较正常组明显减少。④坏死骨小梁间隙可见有明显纤维肉芽组织增生,在坏死骨小梁周围包绕。⑤骨小梁周围可见少量成骨细胞排列,但数量很少,骨小梁周围无明显新骨沉积性生长。⑥髓腔内多核破骨细胞数量增多。⑦软骨下小血管数量明显减少。⑧潮线明显出现了不延续,有的部分甚至消失。
(3)治疗组
①骨小梁较模型组变粗,数量明显增多,骨小梁结构大体清晰。②骨小梁中较多正常骨细胞,但仍可见少量坏死骨细胞,空骨陷窝较模型组明显减少。③髓腔内脂肪空泡较模型组减少,造血细胞数量较模型组增多。④成骨细胞数量较模型组增多。⑤髓腔内多核破骨细胞数量较模型组明显减少。⑥软骨下小血管数量较模型组明显增多。
4.1.2图像分析仪定量分析
①空骨陷窝率:模型组空骨陷窝率增高明显,与治疗组及空白组比较,组间差异显著(P＜0.05);治疗组空骨陷窝率较模型组明显降低,但与空白组比较组间差异仍显著(P＜0.05)。
②骨陷窝面积:模型组骨陷窝面积明显增高,与空白组比较,组间差异显著(P＜0.05);治疗组骨陷窝面积明显降低,与模型组比较,组间差异显著(P＜0.05)。
③骨小梁面积:模型组骨小梁面积明显降低,与空白组比较,组间差异显著(P＜0.05);治疗组骨小梁面积在数值上高于模型组,但是二者组间比较无显著性差异(P＞0.05)。
4.2大鼠股骨头组织内VEGF、BMPs、TGFβ1、Ⅷ-R Ag的表达
(1)在VEGF的表达方面:①阳性面积比:治疗组表达高于模型组,且差异显著p＜0.05;但治疗组的表达低于空白组,且差异显著p＜0.05;②阳性细胞数目比:治疗组表达高于模型组,且差异显著p＜0.05;但治疗组的表达低于空白组,差异显著p＜0.05。
(2)在BMPs的表达方面:①阳性面积比:治疗组表达高于模型组,且差异显著p＜0.05;模型组表达低于空白组,且差异显著p＜0.05:②阳性细胞数目比:治疗组表达高于模型组,且差异显著p＜0.05;模型组表达低于空白组,且差异显著p＜0.05。
(3)在TGF-β1的表达方面:①阳性面积比:治疗组表达高于模型组,且差异显著p＜0.05;模型组表达低于空白组差异显著p＜0.05;治疗组与空白组比较差异显著p＜0.05;②阳性细胞数目比:治疗组表达高于模型组,且差异显著p＜0.05,治疗组与空白组比较差异显著p＜0.05;模型组表达低于空白组两组间差异显著p＜0.05。
(4)Ⅷ-R Ag:微血管密度,治疗组的表达高于模型组,低于空白组,与模型组比较差异显著p＜0.05,与空白组比较差异显著p＜0.05。
4.3血脂
总胆固醇及甘油三酯水平比较可见,治疗组低于模型组,差异显著p＜0.05;模型组高于空白组,差异显著p＜0.05。
5结语
在本研究中,我们采用小剂量激素长期应用的方法,在用药的第3周就成功诱导出了激素性股骨头坏死模型,表现为骨髓内脂肪细胞增生、肥大,骨小梁稀疏,空骨陷窝率明显增多,造血细胞数量明显减少,成骨细胞数量明显减少,骨小梁周围无明显新骨沉积性生长,髓腔内多核破骨细胞数量增多,软骨下小血管数量明显减少。
在实验过程中,我们发现激素性股骨头坏死的发生可能与TGFβ_1、BMPs、VEGF、的表达下调和新生微血管数量的减少有关;同时发现模型组血清甘油三酯及胆固醇明显升高,脂质代谢紊乱。
脉络宁复合液能通过其活血化瘀、清热解毒、滋阴养血的作用预防及治疗激素性股骨头坏死。该治疗方法不仅能纠正脂肪代谢紊乱,并能上调VEGF、BMPs、TGFβ1、的表达;且该疗法能明显增加坏死股骨头的血供。
6.结论
(1)采用单纯小剂量激素长期应用的方法,可以成功的建立激素性股骨头坏死模型,能较好反应临床激素性股骨头坏死的病理过程。
(2)激素性股骨头坏死的发生与激素所致的脂肪代谢紊乱及组织内骨相关基因蛋白表达改变相关。
(3)通过脉络宁复合液治疗可以使与微血管及骨细胞生成密切相关的生长因子表达上调,促进骨修复,从而治疗激素性股骨头坏死。
(4)运用脉络宁复合液治疗可以改善激素引起的高血脂状况,恢复全身的脂代谢平衡,减少髓腔内的脂肪堆积,有效的降低了骨内压,使坏死区血供增加。
(5)脉络宁注射液具有活血化瘀、滋阴养血、清热解毒的功效。本实验结果说明脉络宁注射液与利多卡因、碳酸氢钠以一定比例配合,在治疗激素性股骨头坏死方面,疗效确切;通过增加与微血管及骨细胞生成密切相关的生长因子的生成,以及改善激素引起的高血脂状态,促进坏死区组织的修复是该疗法的主要治疗机理。
1 Background:
Avascular necrosis(Avascular Necrosis of the Femoral Head SR ANFH) is the result of trauma or injury,and many other reasons for avascular necrosis of the bone cells of femoral head,bone marrow,blood cells and fat cells,bone nutrition interruption,finally result in Increase or reduce the density of the femoral head,bone cystic changes,bone fracture,the femoral head collapse deformity and a series of diseases,known as femoral head avascular necrosis.Because of bone necrosis is not caused by bacterial infection,also known as axenic avascular necrosis of the femoral head.It is the orthopedic common diseases,due to a variety of causes destructing the blood supply of the femoral head,the active component of bone (including bone cells,bone marrow Cell and fat cell) death caused by a pathological process.As the incidence reasons are complicated,leading to lesions of cartilage damage, osteonecrosis of the femoral head of change,lose joint function and has a high morbidity.Avascular necrosis in the incidence of middle-aged mainly 30 to 40-year-old Green,40-80%of the patients for bilateral disease,according to their natural illness course of the study showed that about 80 percent of untreated ANFH Femoral head collapse will occur during the 1-3years.Once the femoral head collapse,there will be varying degrees of pain and dysfunction,serious patient had to accept artificial hip replacement,It has greatly affected the quality of patients life.
Necrosis of the femoral head happens not only for the basis of systemic factors,but also incidence of local particularity,the body repair its own existence after Osteonecrosis, performance on mesenchymal cells on live bone and capillary proliferation invasive necrosis of bone and replace marrow,undifferentiated mesenchymal cells attached to the surface necrosis bone differentiation into skeletogenous cell and form new bone.The strategy and the goal on the treatment of this disease on should be active in the treatment of the original disease, improve the general state of imbalance at the same time,partial lifting of insufficient blood supply,improve microcirculation and reduce vein Stagnation and reduce pressure within the bone to prevent adverse biological stress factors influence and take appropriate Functional training,to stop or reverse pathological process,induction and promotion new bone formation and reconstruction,the femoral head to repair and restore the hip joint function,in Chinese medicine ANFH occurs mainly because of bad blood disease internal resistance,on the one hand block Weiqi through the muscle and skin their health,loss of protected function:On the other hand block of qi and blood running in local,caused partial loss support,and lead to invasion evil easily,evil blood and invasion evil maxed for the Bone Bi.As《Lingshu.Thief wind》said:If the falling something,the worsen blood will store.
For the current therapy to osteonecrosis of the femoral head mainly depend on the surgery in china,the recent results is clearly,but not an ideal long-term effect.Chinese medicine combination therapy reserve bone structure and function proceeding from the whole concept focus on both internal and external,ax activity and still,bones and tendons take with the principle of a Differential Treatment,made complete deficiency surgical therapy.My teacher accroding on the basis of traditional Chinese medicine theory and clinical practice,created a Mailuoning composite liquid therapy avascular necrosis by nerve block, and achieved satisfactory results.Mailuoning composite liquid make uo with Mailouning,2% lidocaine and 5%sodium bicarbonate by a certain percentage from compatibility.Mailouning is pure Chinese medicines combine with Achyranthes,Scrophulariaceae,honeysuckle, Dendrobium composition,with function of Promoting Blood Circulation and Relieving Blood Stasis,nourishing yin and blood,Qingrejiedu,Mainly use for the treatment of thrombosis obliterans,and sequelae of cerebral thrombosis and other diseases.There is no moving blood medicine in Mailuoning composit but it has active ingredients for chemical chlorogenic acid, Azerbaijan dental materials,and the fat within the foreshore Artemisia coumarin compounds, to expand blood vessels,improve microcirculation,Improve fibrinolytic activity,reduce fibrinogen levels,lower blood viscosity,increasing blood flow,with an anticoagulant, thrombolytic effect.it prove that have the effect of promoting blood circulation,lidocaine can cut off the vicious cycle of pain by Local nerve block,lift muscle cramps and lower the pressure within the hip joint,while lidocaine block femoral artery near the sympathetic, diastolic blood vessels,increase blood supply of the femoral head around.This method really understands the disease from Chinese medicine blood stasis,and start with improving the local blood supply of femoral head actively,This study's main purpose is observing the effect of Mailuoning compound in the treatment of hormone-ANFH mechanism by nerve block. theoretical basis for clinical treatment.To this end,the topics prepare to de research in three areas from the morphology,immunohistochemistry,blood lipid changes.
2 Purpose of the study
Through the application of ANFH model in Long-term low-dose hormone of Mailuoning composite therapy to intervene observing before and after treatment changes.Application HE staining methods,observed ANFH the pathological changes from the morphology in the application of immunohistochemistry methods to observe the rats' in the femoral head impact on bone growth and repair of a variety of growth factor's in the changes.Mailuoning test compound in the treatment of hormone-necrosis of the femoral head,and further understanding Mailuoning compound in the treatment of the disease mechanism from morphology and molecular biology,to provide a theoretical basis for clinical treatment.
3 Research Methods
3.1 Materials and Methods
Selection of 26 adult male Wistar rats(second degree by the China Institute of Traditional Chinese Medicine Centre for laboratory animals,animal qualified for the No.SCXK(Army) 2002-001) weight 290±20g,Select 20 copies of the hormone necrosis of the femoral head animal model:intraperitoneal injection of acetic acid prednisolone 6.7mg/kg,3 times/ week,for three weeks,after the completion of the model group were randomly divided into hormone(for short hormone group),10,Mailuoning compound in the treatment group(for short the treatment group) 10,and the remaining six is the blank group(for short blank group).The treatment group,hormone group and blank group were taken the rat model in the first day after nine weeks.with 0.3 percent sodium pentobarbital intraperitoneal injection anesthesia(1ml/100g),paunch and take abdominal aorta blood 5 ml,fasting 12 h before Acquisition,were put in the tube apartly.4℃centrifugal 3500 rpm,15 minutes,separation of serum,4℃refrigerator preservation,sent to the WanNing hospital biochemical laboratory tests total cholesterol,triglycerides.At the same time to remove the paraffin sections of the femoral head,HE stain,Observed the femoral head of the pathological changes in organizational structure by light microscopy and,through image analysis instrument analysis HE staining biopsy rate of lacunae,bone lacunae area,Trabecular bone area of quantitative analysis for vascular endothelial growth factor(Vessel endothelial cell growth factor,VEGF),bone protein(Bone morphogenetic proteins,BMPs),transforming growth factorβ1(Transforming growth factor-β1,TGF-β1),Ⅷ-related antigen(Ⅷfactor fos-related antigen,Ⅷ-R Ag) Immunohistochemical staining SP method,using image analysis instrument analysis VEGF,BMPs,TGF-β1,the positive area ratio,the number of positive cells,Ⅷ-RAg of the MVD.
3.2 statistical methods
SPSS13.0 software used for statistical analysis,the measurement data group compared the single-factor analysis of variance,the measurement data to mean±standard deviation(x±S) said,P＜0.05 for statistical significance.
4 Conclusion
(1).A simple small dose of the hormone long-term approach can be successful establishment of hormone-ANFH model,to better clinical response the pathological process of the hormone ANFH.
(2).Necrosis of the femoral head of hormones in hormone-induced fat metabolic disorders in the bone-related gene expression changes were related.
(3).Through Mailuoning compound treatment can microvascular and growth factor generate expression closely related to bone cells growth increased and promote bone repair,then cure hormone osteonecrosis of the femoral head.
(4).Mailuoning compound used in the treatment can improve by high blood lipid situation caused the hormone and restore balance the body lipid metabolism and reduce fat accumulation in the marrow,effectively reduce the pressure within the bone,increase the blood supply of necrosis.
(5).Mailuoning injection has functions of Promoting Blood Circulation and Relieving Blood Stasis,nourishing yin and blood,Qingrejiedu.The experimental results show that Mailuoning injection with lidocaine,sodium bicarbonate according a certain percentage with a nerve block approach in the treatment of hormone necrosis of the femoral head,is effective through increased and microvascular and the growth factor of production which closely related bone cells generate,and to improve high blood lipid status caused by the hormone,It is the main mechanism of that therapy.
引文
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