新生鼠股骨破骨细胞存在位置的确定及其噬骨能力验证

Identification of the location and resorption ability of femoral osteoclasts in newborn rats

目的:确定新生鼠体内破骨细胞的存在位置,观察分离纯化后的破骨细胞形态结构,并检测其噬骨能力。方法:实验于2006-07/11在沈阳医学院中心实验室完成。①市售新鲜成年牛股骨皮质,临用前锯成1cm宽条,磨片机磨成厚50μm的1cm×1cm骨片,乙醇浸泡,晾干,紫外线照射消毒骨片的两面,放于DMEM培养基中4℃备用。②选取新生大鼠10只,处死取其后肢股骨,于中间位置截成两段,多聚甲醛固定,甲苯胺蓝染色确定体内破骨细胞的存在位置,并进行形态结构观察。③另选取新生大鼠6只,处死取其四肢长骨,刮取骨髓腔内及干骺端表面直至骨干成为极微小的碎片,机械分离破骨细胞,胰蛋白酶+乙二胺四乙酸联合消化。④吉姆莎染色观察细胞形态结构,抗酒石酸酸性磷酸酶染色检测破骨细胞酶活性,扫描电镜观察破骨细胞形态结构,通过破骨细胞在骨片上的陷凹情况验证噬骨能力。结果:①鼠后肢股骨行甲苯胺兰染色,大量破骨细胞位于靠近干骺端骨小粱的骨陷凹中,核呈深蓝色,核大且多。②分离培养的破骨细胞数量较少,体积大,胞浆丰满,细胞突起延展,细胞核呈圆形或椭圆形,积聚在细胞中央或排列在细胞周边。胰蛋白酶+乙二胺四乙酸联合消化后可使混杂的单核基质细胞脱落,培养皿上大部分为多核巨细胞,破骨细胞纯化率达80%。③抗酒石酸酸性磷酸酶染色后的破骨细胞呈阳性表达,胞质内酶活性部位为红橙色。④成熟破骨细胞体积大,细胞突起延展,细胞周围出现伪足。破骨细胞在牛皮质骨片上培养时产生骨陷凹,吸收陷窝大部分呈梅花瓣形或腊肠形。结论:新生鼠体内破骨细胞多位于靠近干骺端骨小粱的骨陷凹中,具有噬骨能力,可使牛皮质骨片产生骨陷凹。为体外分析破骨细胞生物特性、骨吸收功能及骨组织工程应用提供丰富的细胞来源。

AIM：To identify the location of osteoclasts in newborn rats, observe the structure of isolated and purified osteoclasts, and test the resorption ability. METHODS： The experiment was carried out in the canter laboratory of Shenyang Medical College from July to November 2006. （1）Fresh adult cow femur bones ware saw into 1 cm strips, and grinded to 1 cmxl cm bone slices of 50 μm in thickness, soaked in alcohol, dried and sterilized by ultraviolet radiation, then put in DMEM nutrient medium at 4 ℃ for following use.（2）Ten newborn rats were selected and executed to harvest the posterior limb femur, which was cut into two halves, formalin fixed, and stained by toluidine blue to identify the location of osteoclasts and observe the structure. （3）Another 6 newborn rats ware selected and executed to take the posterior limb long bone, and scrape the internal surface into tiny pieces. The osteoclasts ware isolated and digested by trypsin plus ethylenediamine tetraacetic acid （EDTA）.（4）The osteoclasts ware Giemsa stained to observe the structure and tartrate-resistant acid phosphates （TRAP） to identify the enzymatic activity of osteoclasts; the osteoclasts ware also observed by scanning electron microscope. The ability of bone resorption was checked according to the resorption lacuna on bone slice. RESULTS： （1） After the posterior limbs of rats were stained by toluidine blue, plenty osteoclasts ware found in the lacuna near the metaphysis with big and blue stained nucleus. （2）The isolated and cultured osteoclasts ware big but few with much kytoplasm, stretched call process, round or ellipse nucleus, which accumulated in the middle or surroundings of call. After trypsin and EDTA digestion, the mononuclear matrix cells ware scaled with multinucleated giant calls left on the culture dish, and the purified rate of osteoclasts reached 80%. （3）The osteoclasts ware positive after TRAP staining and the active enzyme was in reddish orange color. （4）The mature osteoclasts ware big, and had call process and pseudopod. The osteoclasts formed resorption lacuna on femoral cortex slice from calf and most resorption lacuna ware in plum petal or salami shape. CONCLUSION： Osteoclasts of newborn rats mostly locate in the lacuna near the metaphysis, possess the resorption ability and form lacuna on the calf cortex slice. Osteoclasts provide abundant cell sources for in vitro analysis of biological characteristics, bone resorption ability and application of osteoclasts in bone tissue engineering.