不同引经药对股骨头坏死模型兔骨髓干细胞归巢的影响

目的:观察比较活骨Ⅱ方配伍不同引经药对液氮冷冻性股骨头坏死(ONFH)兔股骨头内骨髓干细胞归巢的影响,探讨其防治ONFH的作用机制。方法:84只兔经液氮冷冻法建立股骨头坏死模型后,随机分为模型、活骨Ⅱ方及活骨Ⅱ方分别加牛膝、细辛、独活和桔梗(简称活骨、牛膝、细辛、独活和桔梗)等6组,另14只设为假手术组。在造模同时所有动物均皮下注射rhG-CSF(30μg.kg-1.d-1,连续7 d),并分别灌胃生理盐水和相应中药。于注射rhG-CSF前后分别进行外周血WBC计数。分别于给药2周和4周后,股骨头经HE染色观察组织病理学改变,血管墨汁灌注观察血管形态,免疫组织化学法检测BrdU及SDF-1的表达。结果:与假手术组相比,模型组股骨头空骨陷窝率明显升高,血管面积和SDF-1蛋白表达水平显著降低;与模型组相比,活骨Ⅱ方组股骨头空骨陷窝率降低,血管面积增大、BrdU阳性细胞数和SDF-1表达升高;与活骨Ⅱ方组比较,牛膝组给药4周时股骨头内空骨陷窝率降低、血管面积增大,2周时SDF-1表达增高;桔梗组股骨头内空骨陷窝率有增高趋势,4周时BrdU阳性细胞数明显减少;细辛和独活组无明显变化。结论:活骨Ⅱ方能通过促进动员起来的骨髓干细胞定向归巢从而防治股骨头坏死;引经药牛膝可进一步提高活骨Ⅱ方的上述作用。     

活骨Ⅱ方对股骨头坏死模型兔血液流变学的影响

目的 观察活骨Ⅱ方对股骨头坏死模型兔血液流变学的影响,并探讨其治疗股骨头坏死的作用机制.方法 将48只雄性日本大耳白兔随机分为空白组、模型组和活骨组.除空白组外,采用液氮冷冻方法复制股骨头坏死模型,造模后活骨组给予活骨Ⅱ方水煎剂灌胃治疗,模型组及空白组子等体积蒸馏水灌胃.2、4周分别行HE染色、血管墨汁灌注观察股骨头组织病理形态及其血管变化;检测血液流变学指标及血脂.结果 与空白组相比,模型组2周时可见股骨头组织骨小梁稀疏变细,空骨陷窝增多,脂肪变性,血管变细、减少,全血黏度、血浆黏度和红细胞压积升高(P＜0.05);4周时血小板聚集、纤维蛋白原也明显升高(P＜0.05).与模型组相比,2周时活骨组除了能明显改善股骨头局部的病理组织结构、促进血管形成外,对血黏度和三酰甘油等也有较明显的降低作用(P＜0.05或P＜0.01),4周时尚能降低总胆固醇和低密度脂蛋白胆固醇(P＜0.05).结论 活骨Ⅱ方能有效改善股骨头坏死模型兔血液流变异常,这可能是其促进骨修复、治疗股骨头坏死的作用机制之一.     

活血通络方加不同引经药对股骨头坏死兔成骨相关因子的影响

目的:观察活血通络方配伍不同引经药对股骨头坏死兔血清骨钙素(BGP)、骨形态发生蛋白-2(BPM-2)含量及股骨头BMP-2mRNA表达的影响,探讨其防治股骨头坏死的作用机制。方法:健康雄性日本大耳白兔98只,体重2.2～2.8kg。84只兔经液氮冷冻左侧股骨头法建立股骨头坏死模型后,随机分为6组,每组14只:模型组,活血通络方组,以及活血通络方分别加牛膝、独活、细辛、桔梗组(分别简称活骨、牛膝、独活、细辛、桔梗组)。另外14只设为伪手术组。造模后药物组分别给予相应的中药,模型和伪手术组给予生理盐水灌胃,所有动物皮下注射重组人粒细胞集落刺激因子(30μg·kg-1·d-1,连续7d)。实验2、4周时分别取材,放射免疫法和酶联免疫法分别检测血清BGP和BMP-2含量,左侧股骨头行苏木精-伊红染色观察组织病理学改变,原位杂交法检测BMP-2mRNA表达。结果:与伪手术组相比,模型组股骨头空骨陷窝率显著增多,2周时血清BGP升高,4周时血清中BMP-2含量及股骨头BMP-2mRNA降低。与模型组相比,活骨组空骨陷窝率降低,2、4周时血清BGP、BMP-2含量及股骨头BMP-2mRNA的表达均升高。与活骨组比较:牛膝组4周时空骨陷窝率显著减少,血清BGP、BMP-2及股骨头BMP-2mRNA升高;桔梗组空骨陷窝有降低趋势,且2、4时周血清BGP降低,2周时血清BMP-2降低,4周时股骨头BMP-2mRNA降低;独活组2周时血清BMP-2含量和股骨头BMP-2mRNA的表达升高;细辛组各项指标未见明显改变。结论:引经药牛膝可以进一步提高活血通络方上调血清BGP、BMP-2含量和股骨头内BMP-2mRNA表达的作用,这可能是其促进骨成有效防治股骨头坏死的机制之一。     

Effect of HuoguⅡFormula(活骨Ⅱ方) with Medicinal Guide Radix Achyranthis Bidentatae on Bone Marrow Stem Cells Directional Homing to Necrosis Area after Osteonecrosis of the Femoral Head in Rabbit

<正>Objective:To investigate the effect of HuoguⅡFormula(活骨Ⅱ方) with medicinal guide Radix Achyranthis Bidentatae(Ach) on bone marrow stem cells(BMSCs) homing to necrosis area after osteonecrosis of the femoral head(ONFH) frozen by liquid nitrogen in rabbit as well as to explore the mechanism of prevention and treatment for ONFH.Methods:The animal model of ONFH was established by liquid nitrogen frozen on the rabbit left hind leg.Forty-eight Japanese White rabbits were randomly assigned to sham-operated group,model group, HuoguⅡgroup,and HuoguⅡplus Ach group,with 12 rabbits in each.During the course of ONFH animal model establishment,all rabbits were subcutaneously injected with recombinant human granulocyte colony-stimulating factor[rhG-CSF,30μg/(kg·day) for continuous 7 days].Meanwhile,normal saline and decoction of the two formulae were administrated by gavage,respectively.White blood cells(WBC) were counted in peripheral blood before and after injection of rhG-CSF.Materials were drawn on the 2nd and 4th weeks after model built;bone glutamine protein(BGP) and bone morphogenetic protein 2(BMP2) levels in serum were tested.Histopathologic changes were observed by hematoxylin and eosin(HE) staining.BMP2 mRNA levels were detected with in situ hybridization(ISH) staining.5-Bromo-2'-deoxyuridine(BrdU) and stromal cell derived factor 1(SDF-1) were measured by immunohistochemical assay in femoral head of the left hind leg.Results:Compared with the shamoperated group,the ratio of empty lacuna,serum BGP,and SDF-1 level in the model group increased significantly, and BMP2 in both serum and femoral head decreased significantly.However,in comparison with the model group, the empty lacuna ratio of HuoguⅡgroup and HuoguⅡplus Ach group decreased obviously in addition to the levels of serum BGP and BMP2,and the expressions of BMP2 mRNA,BrdU,and SDF-1 increased significantly. Above changes were particularly obvious in HuoguⅡplus Ach group.BGP and SDF-1 on the 2nd week and empty lacuna rate and serum BMP2 level on the 4th week in HuoguⅡgroup significantly exceeded their counterparts.On the 2nd week,only in Huogu U plus Ach group that the BrdU counting rose significantly.On the 4th week,empty lacuna rate and serum BMP2 level in HuoguⅡplus Ach group exceeded those in HuoguⅡgroup distinctively. Conclusions:To a certain extent,the medicinal guide Ach improves the preventive and therapeutic effects of HuoguⅡFormula on experimental ONFH model.The possible mechanism of this is related to its promoting effect on directional homing of BMSCs to the necrosis area.     

活骨Ⅱ方加不同引经药对股骨头坏死兔股骨头内血管内皮生长因子表达的影响

目的:观察比较活骨Ⅱ方配伍不同引经药对股骨头坏死(0NFH)兔股骨头内血管内皮生长因子(VEGF)表达的影响,探讨其防治ONFH的作用机制.方法:84只兔经液氮冷冻法建立股骨头坏死模型后,随机分为以下6组:模型组活骨Ⅱ方组、活骨Ⅱ方加不同引经药组(牛膝、独活、细辛和桔梗),另14只设为伪手术组.造模后中药组立即分别给予活骨Ⅱ方(6 g·kg-1·d-1)、活骨Ⅱ方(6 g·kg-1·d-1)加引经药(牛膝0.75 g·kg-1·d-1,细辛0.15 g·kg-1·d-1,独活0.75 g·kg-1·d-1,桔梗0.75 g·kg-1·d-1,分别相当于临床等效量)灌胃,连续4周;模型和伪手术组给予生理盐水灌胃,所有动物均皮下注射重组人粒细胞集落刺激因子(rhG-CSF) 30 μg· kg-1 ·d-1,连续7d.实验2周和4周时取左侧股骨头,分别行苏木精-伊红(HE)染色观察其组织病理学改变和墨汁灌注染色观察股骨头内血管形态变化,并经免疫组织化学法检测股骨头内VEGF的表达水平.结果:与伪手术组相比,模型组实验2周和4周时股骨头空骨陷窝率显著增多,血管面积及VEGF表达明显减少(P ＜0.01或P ＜0.05).活骨Ⅱ方能显著降低股骨头空骨陷窝率,并提高血管面积及VEGF的表达(P＜0.01或P＜0.05).与活骨Ⅱ组相比,牛膝组4周时空骨陷窝率显著减少,血管面积明显增多(P＜0.01或P＜0.05),2周和4周VEGF表达均明显增多(P＜0.01);桔梗组4周时VEGF表达显著降低;细辛和独活组无显著变化.结论:引经药牛膝具有进一步提高活骨Ⅱ方上调股骨头内VEGF表达的作用,这可能是其有效改善股骨头血循环、防止坏死的机制之一.     

健脾活血方对激素性股骨头坏死血管内皮生长因子表达的影响

目的:通过观察健脾活血方对激素性股骨头坏死(SONFH)鸡股骨头内血管内皮生长因子(VEGF)表达的影响,探讨其防治SONFH的作用机制。方法:胸肌注射甲基氢化泼尼松建立SONFH模型,64只来航鸡随机平均分为正常组、模型组、洛伐他汀组和健脾活血组;实验8周和16周翼下静脉采血测定血清总胆固醇(CHO)和甘油三酯(TG)的含量,并取双侧股骨头行HE染色观察其组织病理学改变,用免疫组织化学法检测股骨头内VEGF的表达水平。结果:实验8周和16周,模型组股骨头出现空骨陷窝率和脂肪面积明显增多等病理改变,与正常组相比,股骨头内VEGF表达明显减少(P0.05),血清CHO、TG水平明显升高;洛伐他汀与健脾活血方能不同程度改善由激素引起的股骨头坏死组织形态学改变,升高股骨头内VEGF的表达,减低血清TG、CHO水平(与模型组比较P0.05)。结论:健脾活血方可上调股骨头内VEGF的表达,这可能是其有效改善股骨头微循环障碍,防治SONFH的作用机制之一。