摘要
目的:采用腹腔注射链脲佐菌素+切除双侧卵巢复合方法建立绝经后糖尿病骨质疏松大鼠模型,观察糖尿病骨质疏松大鼠破骨细胞的改变。方法:实验于2003-12/2004-08在河北医科大学第三医院中心实验室完成。①实验材料:选用2.5～3个月龄清洁级雌性Wistar大鼠60只。②实验分组:完全随机设计分为6组:正常对照组、正常假手术组及正常双侧卵巢切除组均为8只;糖尿病对照组、糖尿病假手术组及糖尿病双侧卵巢切除组均为12只。③实验干预:采用链脲佐菌素诱导制备糖尿病大鼠模型1周后,将大鼠麻醉结扎输卵管切除卵巢。假手术组大鼠不作卵巢切除,余操作同去卵巢组。卵巢切除后0,2,4,8周随机选择各组大鼠1只,收集骨髓,进行体外骨髓破骨细胞样细胞的培养。④实验评估:切除双侧卵巢后2,4,8周时测量各组大鼠体质量、血糖;细胞培养7d后,进行细胞固定和抗酒石酸酸性磷酸酶染色(抗酒石酸酸性磷酸酶染色阳性且细胞核≥3个的细胞认定为破骨细胞样细胞);倒置显微镜计数破骨细胞样细胞数量。结果:制成糖尿病模型及切除卵巢后,3组糖尿病大鼠死亡7只,进入结果分析53只。①各组大鼠血糖和体质量的变化:切除卵巢0,2,4,8周,3组糖尿病大鼠血糖均高于正常组(P<0.01),体质量均低于正常组(P<0.01);4周时,正常双侧卵巢切除组体质量高于同期正常对照组和正常假手术组;糖尿病双侧卵巢切除组体质量在2,4,8周时均高于同期糖尿病对照组和糖尿病假手术组(4周P<0.05,8周P<0.01)。②切除卵巢后各组大鼠体外培养的破骨细胞样细胞形成的变化:糖尿病双侧卵巢切除大鼠破骨细胞多于糖尿病大鼠及正常去卵巢大鼠,且破骨细胞数量随着去卵巢的时间和糖尿病病程延长而增加。③骨髓来源的体外破骨细胞样细胞的形成与血糖、糖尿病病程及去卵巢时间的相关性分析:破骨细胞数量与糖尿病病程呈正相关,而与血糖增高的程度无关(r=-0.537;P=0.109)。结论:①卵巢切除对大鼠的血糖无显著影响,对大鼠体质量的影响被糖尿病减弱,在糖尿病早期破骨细胞样细胞的形成已有增加。②破骨细胞形成增加可能是绝经后糖尿病骨质疏松的原因之一。
AIM： To establish the rat model of postmenopausal diabetic osteoporosis by the ovariectomy plus streptozotocin （STZ） injection, and observe the change of osteoclasts. METHODS： The experiment was carried out in the central laboratory of the Third Hospital of Hebei Medical University from December 2003 to August 2004. （1）Sixty 2.5- to 3-month-old female Wistar rats of clean grade were selected and randomly divided into 6 groups： normal control group, normal sham-opeartion group, and normal ovariectomy group with 8 rats in each group; diabetes control group, diabetes sham-operation group, diabetes ovariectomy group with 12 rats in each group. （2）The rats were injected with STZ to induce diabetes. One week later, under anesthetized condition, the oviduct was ligated and the ovary was excised. The treatment in the sham-operation group was the same as ovariectomy group but the ovary was not excised. One rat of each group was randomly selected 0, 2, 4, and 8 weeks after ovariectomy, and the bone marrow was collected to culture the osteoclast-like cells in vitro. （3）The levels of blood glucose, and body mass of rats in each group were determined 2, 4, 8 weeks days after ovariectomy; after 7 days culture, the cells were fixed and stained with tartaric-resistant （cells with positive staining and ≥3 nuclei were regarded as osteoclast-like cells）. The number of osteoclast-like cells was measured under inverted microscopy. RESULTS： After modeling and ovariectomy, 7 rats of 3 diabetic groups died, and finally 53 rats were involved in the result analysis. （1）The blood glucose of 3 diabetic groups were higher than that of normal groups 0, 2, 4, and 8 weeks after ovariectomy （P 〈 0.01）, but the body mass was lower than that of normal groups （P 〈 0.01 ）; at 4 weeks, the body mass of normal ovariectomy group was higher than that of normal control group and normal sham operation group; the body mass of diabetes ovariectomy group was higher than that of diabetes control group and diabetes sham-operation group at 2, 4 and 8 weeks （4 weeks： P 〈 0.05, 8 weeks： P 〈 0.01 ）. （2）The number of osteoclast-like cells in diabetes ovariectomy group was higher than that in diabetic rats and normal ovariectomized rats, and the number was increased with time of ovariectomy and duration of diabetes. （3）The number of osteoclasts was positively related with duration of diabetes, but not correlated with the increased of blood glucose （r =-0.537; P =0.109）. CONCLUSION： （1）Ovariectomy has no significant influence on the blood glucose of rats, and its effect on body mass is weakened by diabetes. In the early stage of diabetes, the number of osteoclast-like cells begins to increase. （2）The increased number of osteoclast-like cell is one of the reasons for postmenopausal diabetic osteoporosis.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第27期5341-5345,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
河北省自然科学基金资助项目(303496)~~
