摘要
[目的]探讨不同方法培养下的大鼠破骨细胞(osteoclast,OC)骨吸收功能的差异,以及骨吸收关键基因ATPase a3的mRNA表达水平的差异,为体外实验奠定基础。[方法]机械分离和诱导培养,即从新生24h的大鼠长干骨骨髓腔内壁机械分离成熟OC和1,25(OH)2 D3诱导大鼠骨髓细胞形成破骨样细胞(osteoclast like cell,OLC),对获得的OC进行形态和骨吸收功能观察,并测定OC骨吸收关键基因ATPase a3的mRNA表达水平的差异。[结果]OC和OLC均为抗酒石酸酸性磷酸酶(tartrate resist-ant acid phosphatase,TRAP)染色阳性的多核巨细胞,与细胞共培养骨片上可形成骨吸收陷窝;诱导法培养出的OLC数目多于机械分离法,但诱导早期陷窝较之小而浅,诱导后期基本接近OC;OC骨吸收关键基因ATPase a3的mRNA,在机械分离8h与诱导培养6天的细胞表达量无显著差异,但都远少于培养8天的表达量。[结论]诱导法可以培育出大量的OLC,优于机械分离法,但早期骨吸收功能较弱,OC骨吸收功能与其核数相关。后期的OCL与机械分离OC接近,可以用于各类实验。
[Objective]To investigate the difference of bone absorption function of the rat osteoelast（OC） in different cultural methods, and the difference in the levels of mRNA expression of bone absorption key gene, ATPase a3 gene, and provide the basis for further investigation on the OC in vitro. [Methods] with mechanical separation method, mature osteoclast was separated from inner wall of 24 hours newborn rat long bones medullary cavity; and induction culture methods, bone marrow cell was induced to osteoclast like cell（OLC） by using 1,25 （OH）2D3. The morphological and functional change of osteoclast was observed and the difference in the level of ATPase a3 mRNA expression was determined. [ Result]OC and OLC are multinucleated giant cells, which can be stained positive by potartrate resistant acid phosphatase（TRAP）, and can form bone absorption lacuna. The number of OLC in induction method is more than in mechanical separation method, but bone absorption lacuna is smaller and shallower than the early stage of induction. The late stage of OCL is extremely similar to OC. There is no significant difference of ATPase a3 mRNA expression from the cells between mechanical separation 8 hour and induction culture 6 days, but either of them is significantly less than inducing 8 days. [Conclusion]Induction method can produce a large number of OLC which is superior to mechanical separation method, but its bone resorption function is weaker in the earlier stage, because bone resorption function is associated with the number of nucleus. The late stage of OCL is extremely similar to OC of mechanical separation method. And it can be used in any experiments.
出处
《浙江中医药大学学报》
CAS
2009年第1期28-32,共5页
Journal of Zhejiang Chinese Medical University
基金
浙江省自然科学基金资助项目(No:Z303656)
Item supported by Zhejiang Provincial Natural Science Foundation(No:Z303656)
