【作者】 陈光友；

【导师】 邓友章；

【作者基本信息】 成都中医药大学 ， 中医骨伤科学， 2009， 博士

【摘要】 目的本研究通过测定BMP-2、bFGF、VEGF、Bcl-2、甘油三脂、血浆粘度在激素造摸致股骨头坏死的兔股骨头组织及血清中的表达,研究中药川骨片对激素造摸致股骨头坏死中的影响,深入探讨川骨片对激素性股骨头坏死中成骨细胞、微血管修复、抗凋亡、血脂、血流变学的影响,及其对上述影响产生的信号传导途径和调节系统机制的分子作用机制的探索。方法采用Miyanishi方法造模,甲强龙20mg/kg体重臀部肌注,每周2次,同时肌注青霉素16万U和链霉素15万U预防感染。将日本大耳兔分为5组,正常组(12只)、模型组(12只)、阳性对照组(12只)、高剂量组(12只)、低剂量组(12只)；除正常组外,其余各组给予甲基强的松龙20mg/kg体重臀部肌注,每周2次,同时肌注青霉素16万U和链霉素15万U预防感染,川骨片高剂量组每天0.9g／1kg体重.ml／d灌胃；川骨片低剂量组为每天0.3g／1kg体重.ml／d灌胃。对照组左归丸每组给药为1.5g／1kg体重.ml／d灌胃。模型组给予蒸馏水1ml/1kg体重／d灌胃。42天后处死动物,采用组织病理学观察和免疫组织化学方法检测,分析BMP-2、bFGF、VEGF、Bcl-2的表达。同时送检血清甘油三脂、血浆粘度。结果一般状态：42天末正常组同实验前,模型组结束时兔毛干枯脱落蓬乱,活动摄饮均减少,川骨片高剂量和阳性对照组状态较模型组好。HE染色结果：模型组呈现骨细胞核固缩,部分核消失,空骨陷窝数较多,坏死骨小梁变细,骨小梁数目减少,毛细血管扩张、淤血,脂肪细胞增大增多,细胞间质出现大量胶原纤维,并有断裂。川骨片高剂量和阳性对照组骨细胞核固缩减少,空骨陷窝数少,部分骨小梁周围成骨细胞缺失,毛细血管扩张、造血细胞增多,脂肪细胞增多明显,细胞间质出现胶原纤维。股骨头组织免疫组化测定结果：BMP-2、bFGF、VEGF、Bcl-2的免疫组化染色阳性物质均呈棕黄色,显微镜下观察：Bcl-2的阳性物质从川骨片高剂量组、阳性对照组、川骨片低剂量组、模型组、正常组有逐渐减少的侵蚀；BMP-2、bFGF、VEGF的阳性物质从模型组、川骨片低剂量组、阳性对照组、川骨片高剂量组、正常组有逐渐增加的趋势。免疫组化染色的阳性物质经经Image-Pro Plus 6.0专业图象分析软件分析量化：川骨片高剂量组、阳性对照组、川骨片低剂量组、模型组与正常组(BMP-2、bFGF、VEGF、Bcl-2)比较,有显著差异(P<0.05)；川骨片高剂量组、阳性对照组、川骨片低剂量组与模型组(BMP-2、bFGF、VEGF、Bcl-2)比较,有显著差异(P<0.05),川骨片高剂量组、阳性对照组与川骨片低剂量组(BMP-2、bFGF、VEGF、Bcl-2)比较,有显著差异(P<0.05)；川骨片高剂量组与阳性对照组(BMP-2、bFGF、VEGF、Bcl-2)比较,无统计学意义(P>0.05)。甘油三脂和血浆粘度检测结果；与模型组比较,血清VP、S-TG的表达明显下降(P<0.05)；川骨片高剂量组、阳性对照组、川骨片低剂量组、模型组与正常组比较血清VP、S-TG的表达有差异(P<0.05),川骨片高剂量组、阳性对照组川与川骨片低剂量组比较血清VP、S-TG的表达有差异(P<0.05)；川骨片高剂量组与阳性对照组川比较VP、S-TG的表达无统计学意义(P>0.05)。结论本研究在成功建立甲强龙诱导日本大耳白兔的股骨头坏死模型的基础上,探讨了中药川骨片对该模型的影响。研究发现：中药川骨片促进成骨细胞的分化增殖、促进微血管的生长、保护骨细胞、促进骨的修复重建。其作用机制可能与川骨片的以下作用有关：通过调节BMP-2、bFGF的表达,促进成骨细胞的分化增殖；调控VEGF的表达,促进骨组织的微血管增生,促进Bcl-2的表达,保护骨细胞。降低甘油三脂和血浆粘度,可增加股骨头血供。更多还原

【Abstract】 ObjectiveThe steroid-induced necrosis of the femoral head was high in the whole world.One of the results of necrosis of the femoral head is osteoarthritis if it isn’t treat effectively. The necrosis of the femoral head was reversible through appropriate treatment in the early.This experiment studied the effect of the Traditional Chinese Medicine called Chuangu tablets on the expression of BMP-2,FGF,VEGF,Bcl-2, VP、TGin the bony tissue of necrosis of the femoral head and serum of the rabbits which were induced by subcutaneous injectin of methylprednisolone sodium succinate.To investigate the effects of Traditional Chinese Medicine Chuangu tablets on the experimental necrosis of the femoral head induced by methylprednisolone sodium succinate in the rabbits.Further to evaluate the prosthetic effects of Chuangu tablets on the bony tissue of necrosis of the femoral head, remodel the femoral head,and explore its possible bone remodeling molecular mechhanisma on both Signal Transduction Pathway.MethodsNecrosis of the femoral head was induced in rabitts by subcutaneous injectin of methylprednisolone sodium succinate.Sixty rabitts were randomly divided into 5 group; normal control group(N group,12 rabitts), model control group(M group,12 rabitts),positive control group(P group,12 rabitts),high dose of Chuangu Tablet(H group,12 rabitts), low dose of Chuangu Tablet(L group,12 rabitts),The rabbits of M,P,H and L group were disposed with injection of solumedrol injection 20mg/kg,once 3 days,at the time H and L groups were treated with Chuangu tablets,once Iday,and Pgroup was treated with Zhuogui pills,once 1 day.42 days later,all the rabbits were sacrificed.The pathological change of the femoral head were observed by HE staining under microscope.The protein expression of VEGF and BMP-2 were determinged by immunohistochemical staining method.Serum levels of VP and TG were dectected..Results:General status:At the end of 42 days normal control group was unchanged.The fur color pattern inmodel control group got parched unkempt,the activity and cibation reduced. The status between the high dose of Chuangu tablets group and positive control group were better than that of model control group.HE stain for the bony tissue:In the model control group, Empty lacunaes increased Liposome was found in the cytoplasm and nuclei of the osteocyte in early period.Pyknosis,karyolysis and karyorrhexis were observed in the osteocyte..There was obviously lipid droplet.The structure of plasma menbrans was not complete, obvious screvice could be found in collagenous fiber.But, in the high dose of Chuangu tablets group and positive control group, Empty lacunaes increasing wan in slight extent. Pyknosis,karyolysis and karyorrhexis were not obviously observed in the osteocyte.Immunohistochemisty stain for the bony tissue:Buffy represents positive result.. The normal control group is the most obvious by the positive mass of BMP-2,FGF and VEGF.The positive mass of BMP-2,FGF and VEGF increased gradually in the order of model control group, low dose of Chuangu Tablet group, positive control group(P group,high dose of Chuangu Tablet, normal control group.But, the positive mass of Bcl-2 decreased gradually in the order of model control group, low dose of Chuangu Tablet group, positive control group(P group,high dose of Chuangu Tablet, normal control group.The data from immunohistochemistry are puantitied by Image-Pro Plus 6.0 analysis program. BMP-2,FGF and VEGF of the model control group, low dose of Chuangu Tablet group, positive control group,high dose of Chuangu Tablet is significantly different from the normal control group (P<0.05).And those masses of the low dose of Chuangu Tablet group, positive control group,high dose of Chuangu Tablet is significantly different from the model control group (P<0.05). those masses of positive control group,high dose of Chuangu Tablet is significantly different from low dose of Chuangu Tablet group.Regarding of the high dose of Chuangu Tablet and positive control group,there is not significantly different between (P>0.05).VP and TG:Compared with the model control group,,serumVPA and TG (P< 0.05) were significantly different from low dose of Chuangu Tablet group, positive control group,high dose of Chuangu Tablet, normal control group. Serum TG and VP of low dose of Chuangu Tablet group, positive control group,high dose of Chuangu Tablet is significantly different from the normal control group (P<0.05). Serum TG and VP of low dose of Chuangu Tablet group is significantly different from positive control group,high dose of Chuangu Tablet (P< 0.05). Regarding of the high dose of Chuangu Tablet and positive control group,there is not significantly different between (P>0.05).ConclusionThis experiment researches the effects of Traditional Chinses Medicine Chuangu Tablets on the prevention and cure of steroid-induced necrosis of the femoral head in rabitts.The study reveals:Traditional Chinses Medicine Chuangu Tablets not only have a protective effect on steroid-induced necrosis of the femoral head and can definitely inhibit development byt necrosis of bony tissue,but also promote the prosthetic effects of Chuangu tablets on the bony tissue of necrosis of the femoral head, remodeling the femoral head.The possible remodeling the femoral head molecular mechanisms of the effects of Chuangu Tablets decoction may due to up-regulating expression of BMP-2、bFGF、VEGF、Bcl-2; up-regulating Expression of BMP-2、bFGF Signal Transduction Pathway can promote osteoblasts multiplication and differentiation;VEGF Signal Transduction Pathway can promote differentiation of epithelial cells in micrangium of the femoral head; Bcl-2 Signal Transduction Pathway is anti-apoptosis of bony cell. Moreover, Chuangu Tablets can regulate fat metabolism and rheology of the blood. Chuangu tablets can promote the expression level of VEGF and BMP-2 of callus tissue in steroid-induced necrosis of the femoral head, This may be one of the mechanisms of Chuangu tablets to prevention and cure of steroid-induced necrosis of the femoral head. protein更多还原