Four to six week-old mice were killed and humerus, radius, tibia and femur were removed aseptically and washed two times with Hank's solution containing penicillin-streptomycin and then soft tissue were removed. Bone marrow cells were collected by 22 gauge needle. Cells were cultured in Hank's solution containing 1 mg/ml type II collagenase, 0.05% trypsin, 4mM EDTA. Supernatant solution was removed 5 times after 15 minutes of digestion with above mentioned enzyme solution, and remained bone particles were maintained in alpha-MEM for 15 minutes and 4℃ temperature. Bone particles were agitated for 1 minute and supernatant solution containing osteoclast precursor cells were filtrated with cell stainer. These separated osteoclast precursor cells were dispensed with 100-mm culture dish by 1×10⁷ cells unit and cultured in α-MEM containing 20 ng/ml recombinant human M-CSF, 30 ng/ml recombinant human soluble osteoclast differentiation factor and 10% fetal calf serum for 2 and 7 days. Total RNA of osteoclast precursor cells were extracted using RNeasy kit. One µg of total RNA was reverse transcribed in 42℃ for 30 minutes using SuperScriptII reverse transcriptase. Expression of transcribed receptors of each hormone and cytokine were traced with 1 µl of cDNA solution by PCR amplification.

Vitamin D receptor was found in cells cultured for 7 days. TNF-αreceptor was found in cells cultured for 2 days and amount of receptors were increased by 7 days. IL-1 type I receptor was not found in cells cultured 2 and 7 days. But, IL-1 receptor type II was found in cells cultured for 2 days. TGF-α,βtype I receptor was found in cells cultured 2 and 7 days, and amount of receptors were increased by 7 days of culture. These results implies Vitamin D and cytokines can affect osteoclasts directly, and affecting period in differentiation cycle of osteoclasts is different by Vitamin D and cytokines.