1,25(OH)2维生素D3诱导大鼠骨髓单核细胞形成破骨细胞

目的 观察1,25二羟维生素D31,25(OH)2 D3]诱导成年大鼠骨髓来源的单核细胞形成多核破骨细胞的作用.方法 实验分为单核细胞诱导组和全骨髓诱导组,每组各10只大鼠.密度梯度离心法分离大鼠骨髓中的单核细胞,α-MEM培养液中加入10-8 mol/L的1,25(OH)2 D3诱导单核细胞向破骨细胞分化.利用抗酒石酸酸性磷酸酶(TRAP)染色、倒置相差显微镜和扫描电镜,观察诱导不同时相TRAP阳性(TRAP )多核细胞的形态学特征、特异性酶表达、以及骨吸收陷窝.进而计数诱导的破骨细胞数量,鉴定其功能活性,并与全骨髓诱导方法比较.结果 骨髓单核细胞诱导组在培养第7 d出现TRAP 多核细胞,第14 d TRAP 多核细胞数量达峰值,第21 d TRAP 多核细胞数量开始减少.从培养第14 d到第23 d,单核细胞诱导组TRAP 多核细胞数量多于全骨髓诱导组(P＜0.05);培养第10 d到第23 d单核细胞诱导组骨吸收陷窝数量多于全骨髓诱导组 (P＜0.05).单核细胞诱导组破骨细胞数量高峰期可持续7 d,全骨髓诱导法仅持续3 d.结论 1,25(OH)2 D3能够诱导成年大鼠骨髓来源的单核细胞形成破骨细胞;所诱导的破骨细胞的数量和峰值持续时间、以及骨吸收活性均高于全骨髓诱导方法.

1,25(OH)2 Dihydroxyvitamin D3 Induces the Formation of Osteoclasts from Mononuclear Cells of Rat Marrow

OBJECTIVE: To investigate the effect of 1,25 (OH)2 dihydroxyvitamin D3 on the generation of osteoclasts from mononuclear cells of adult rats. METHODS: With density gradient centrifugation, the mononuclear cells were isolated from rat bone marrow and cultured in the alpha-MEM with 10(-8) mol/L 1,25 (OH)2 dihydroxyvitamin D3. The induced cells were fixed and stained with tartrate-resistant acid phosphatase (TRAP). The bone resorption pits were examined by scanning electron microscope, and the morphology of osteoclasts were examined with inverted phase contrast microscope. RESULTS: The TRAP+ multinucleated cells could be observed on the seventh culturing day of mononuclear cell group. The number of TRAP+ multinucleated cells went up to a peak on the fourteenth culturing day, and then began to decrease on the twenty-first culturing day. The number of osteoclasts induced from mononuclear cell group was more than that from bone marrow group (P < 0.05) when it was during the fourteenth culturing day to the twenty-third culturing day. The number of bone resorption pits in the mononuclear cell group was much more than that in the bone marrow group (P < 0.05) when it was in period from the tenth culturing day to the twenty-third culturing day. The number of osteoclasts induced from mononuclear cell group could keep the peak value of lasting 7 days, but that induced from bone marrow group could only last its peak value for 3 days. CONCLUSION: The method with 1,25(OH)2 dihydroxyvitamin D3 inducing the formation of osteoclasts from the marrow mononuclear cells is better than that from the whole bone marrow.