预防用药对激素性股骨头坏死骨细胞凋亡的影响

背景:由于临床长程使用糖皮质激素的情况日益增多,激素性股骨头坏死的发病率出现增高趋势。骨细胞凋亡在早期激素性股骨头坏死中起着重要作用。目的:应用类固醇激素诱导兔股骨头坏死模型,观察分析采取中西药物并用预防激素性股骨头骨细胞凋亡的效果。设计:随机对照动物实验。单位:韶关学院医学院药学教研室和解剖学教研室,广东省粤北人民医院病理科。材料:实验于2005-04/07在韶关学院医学院中心实验室完成。选取成年新西兰大白兔30只,随机分为空白对照组、激素组、预防用药组,10只/组。血塞通片(主要成分为三七总皂甙,玉溪市维和制药有限公司,生产批号200410290,25mg/片);脂必妥片(主要成分为山楂、白术、红曲,成都地奥九泓制药厂,产品批号0410063,350mg/片);阿仑膦酸钠片(主要成分为4-氨基-1-羟基亚丁基-1,海南曼克星制药厂生产,批号040501,5mg/片);地塞米松磷酸钠注射液(河南天康制药有限公司生产,生产批号040905,5g/L)。方法:①动物适应实验环境1周后,激素组和预防用药组动物均肌注地塞米松磷酸钠注射液1mL,2次/周,建立激素性股骨头坏死动物模型。空白对照组不造模,在相同时间点肌注等量生理盐水。②预防用药组注射地塞米松的同时,每天灌喂给予血塞通25mg、脂必妥350mg和阿仑膦酸钠5mg,连续给药8周。激素组和空白对照组不给药。各组动物均肌注青霉素预防感染,5万u/只,2次/周。③停药1周后采用原位末端标记法对各组动物进行股骨头细胞凋亡检测,每张玻片上随机选择10个区域,每个区域计数100个骨细胞,统计每100个细胞中的细胞凋亡数。采用醋酸铀及柠檬酸铅双染色观察股骨头细胞的超微病理结构变化。主要观察指标:①各组股骨头骨细胞凋亡检测结果。②各组股骨头骨细胞超微病理结构电子显微镜观察结果。结果:实验选取成年新西兰大白兔30只,预防用药组实验过程中死亡1只,共29只兔进入结果分析。①各组股骨头细胞凋亡检测结果:激素组的骨标本可见细胞核中出现棕黄色颗粒的阳性细胞,即凋亡细胞。激素组每100个骨细胞中的凋亡细胞数均明显多于空白对照组和预防用药组[(24.14±5.46),(2.47±0.76),(8.12±3.23)个,q=8.76,q=13.45,P均<0.01];空白对照组和预防用药组基本相似(P>0.05)。②各组股骨头骨细胞超微病理结构电子显微镜观察结果:激素组骨细胞核体积缩小,染色质呈边聚现象,部分染色质更浓密,电子密度增加,结构模糊不清,具有凋亡细胞特征。预防用药组骨细胞电镜下核膜完整,染色质均匀,核占细胞的1/3左右。结论:采取类固醇激素诱导免股骨头坏死后,血塞通、脂必妥和阿仑膦酸钠联合应用能够抑制兔激素性股骨头细胞的凋亡。

Effect of preventive drugs on steroid-induced osteonecrosis of the femoral head

BACKGROUND: Due to glucocorticoid (GG) is used wildly on clinic, incidence rate of steroid-induced osteonecrosis of femoral head is increased.Apoptosis of bone cells plays a key role in steroid-induced osteonecrosis of femoral head during earlier period.OBJECTIVE: To induce models of femoral head necrosis of rabbits by steroid steroid so as to observe the effect of integrated Chinese and western medicine on preventing apoptosis of steroid-induced osteonecrosis of femoral head.DESIGN: Randomized controlled animal study.SETTING: Pharmacological Department and Anatomy Department of Medical College of Shaoguan University, Pathological Department of Yuebei People's Hospital of Guangdong Province.MATERIALS: The experiment was carried out at the Central Laboratory of Medical College of Shaoguan University from April to July 2005. A total of 30 adult New Zealand white rabbits were selected and randomly divided into blank control group, steroid group and preventive medicine group with 10 in each group.Xuesai tongpian, main component of panax notoginseng saponin, was provided by Yuxi Weihe Pharmaceutical Company Limited (batch number: 200410290; 25 mg/pill); zhibituo pian, main component of shanzha, baizhu, hongqu, was provided by Chengdu Di 'ao Jiuhong Pharmaceutical Factory (batch number: 0410063, 350 mg/pill); alendronate, main component of 4-amido-1-hydroxy butylidene-1, was provided by Hainan Mankexing Pharmaceutical Factory (batch number: 040501, 5 mg/pill); dexamethasone sodium phosphate injection was provided by Henan Tiankang Pharmaceutical Company Limited (batch number: 040905, 5 g/L).METHODS: ① One week later, rabbits in steroid group and preventive medicine group were injected with 1 mL dexamethasone sodium phosphate solution twice a week to establish models of steroid-induced osteonecrosis of femoral head. Animals in blank control group did not model and were injected with the same volume of saline at the same time point. ② Rabbits in preventive medicine group were prefused with 25 mg xuesai tong, 350 mg zhibituo and 5 mg alendronate at the same time for 8 weeks. Animals in steroid group and blank control group did not treat with any medicine. Benzylpenicillin was injected into muscle of animals in each group for infection prevention with the dosage of 50 000 u each rabbit twice a week. ③ One week after discontinue, apoptosis of femoral head was detected with in situ end-labeling (ISEL) method. Ten areas were selected from each carry sheet glass to calculate numbers of apoptosis in every 100 cells. Pathological changes of ultrastructure were observed with double staining of uranium acetic acid and lead citromalic acid.MAIN OUTCOME MEASURES: ① Results of apoptosis of femoral head; ② Results of pathological changes of ultrastructure of femoral head under electron microscope.RESULTS: A total of 30 adult New Zealand white rabbits were selected in this study. Because one rabbit in preventive medicine group died during process, 29 rabbits were involved in the final analysis. ① Results of apoptosis of femoral head: Buffy granulosum positive cells, i.e. apoptosis cells,were observed in nucleus in steroid group. Numbers of apoptosis every 100cells were more in steroid group than those in blank control group and preventive medicine group (24.14±5.46, 2.47±0.76, 8.12±3.23, q=8.76, q=13.45, P ＜ 0.01), and numbers in blank control group were similar to those in preventive medicine group (P ＞ 0.05). ② Results of pathological changes of ultrastructure of femoral head under electron microscope: Volume of nucleus in steroid group was decreased; chromatin was aggregated at edge; part of chromatin was dark and thick; density of electron was increased; structure was unclear; characteristics of apoptosis were shown. Nuclear membrane in preventive medicine group was complete under electron microscope, chromatin was steady, and the nucleus was accounted for one third of cell.CONCLUSION: The integrated application of xuesai tong,, zhibituo and alendronate can inhibit hormonal-induced apoptosis of femoral head after necrosis of femoral head induced by steroid steroid.