CAJ | 학술논문

目的探讨胰岛素样生长因子Ⅰ (IGF-Ⅰ)对破骨细胞骨吸收的促进作用是否需要成骨细胞的协同.方法体外培养MC3T3小鼠成骨细胞及NF-κB受体的配体(receptor activator of NF-κB ligand,RANKL)诱导分化成熟的破骨细胞,分别给予重组人胰岛素样生长因子Ⅰ(rhIGF-Ⅰ)进行干预,Western印迹检测IGF-Ⅰ受体的活化情况.以0、10 ng/ml的rhIGF-Ⅰ直接干预破骨细胞或与成骨细胞在Transwell双层培养板中共培养的破骨细胞,MTT法测定破骨细胞增殖；流式细胞仪检测破骨细胞凋亡率；实时PCR检测组织蛋白酶K基因的表达.将不同方式干预的破骨细胞接种在骨磨片上,光镜观察骨吸收陷窝的形成.结果在成骨细胞、破骨细胞表面均检测到可被IGF-Ⅰ激活的IGF-Ⅰ受体.仅在成骨细胞、破骨细胞共培养时rhIGF-Ⅰ能够促进破骨细胞活细胞的增殖(P＜0.05)；抑制破骨细胞的凋亡(P＜0.05)；上调组织蛋白酶K基因的表达(P＜0.05)；增加骨吸收陷窝的数量及面积.IGF-Ⅰ对单独培养的破骨细胞则作用不明显.结论 IGF-Ⅰ对破骨细胞骨吸收的促进作用需要成骨细胞的协同.
목적 탐토이도소양생장인자Ⅰ (IGF-Ⅰ)대파골세포골흡수적촉진작용시부수요성골세포적협동.방법 체외배양MC3T3소서성골세포급NF-κB수체적배체(receptor activator of NF-κB ligand,RANKL)유도분화성숙적파골세포,분별급여중조인이도소양생장인자Ⅰ(rhIGF-Ⅰ)진행간예,Western인적검측IGF-Ⅰ수체적활화정황.이0、10 ng/ml적rhIGF-Ⅰ직접간예파골세포혹여성골세포재Transwell쌍층배양판중공배양적파골세포,MTT법측정파골세포증식；류식세포의검측파골세포조망솔；실시PCR검측조직단백매K기인적표체.장불동방식간예적파골세포접충재골마편상,광경관찰골흡수함와적형성.결과 재성골세포、파골세포표면균검측도가피IGF-Ⅰ격활적IGF-Ⅰ수체.부재성골세포、파골세포공배양시rhIGF-Ⅰ능구촉진파골세포활세포적증식(P＜0.05)；억제파골세포적조망(P＜0.05)；상조조직단백매K기인적표체(P＜0.05)；증가골흡수함와적수량급면적.IGF-Ⅰ대단독배양적파골세포칙작용불명현.결론 IGF-Ⅰ대파골세포골흡수적촉진작용수요성골세포적협동.
Objective To study whether osteoblast is necessary for IGF-Ⅰ to promote bone resorption by osteoclast.Methods Mouse MC3T3 osteoblast cells and mature osteoclasts induced by RANKL were cultured in vitro.These osteoblasts and osteoclasts were subjected to treatment with recombinant human insulin-like growth factor-1 (rhIGF-Ⅰ),and the activation of IGF-Ⅰ receptor was verified by Western blotting.Thereafter,osteoclasts were cultured individually or co-cultured with osteoblast,in the absence or presence of rhIGF-Ⅰ.Osteoclast proliferation and apoptosis were observed by MTT colorimetric assay and flow cytometry.Cathepsin K gene expression was detected by real-time PCR; bone adsorption activity of osteoclast was determined by resorption pits formation on calf cortex slice with toluidine blue staining.Results Western blotting result confirmed that rhIGF-Ⅰ could effectively activate IGF-Ⅰ receptors either in osteoblast or osteoclast.In co-cultured group,in the presence of rhIGF-Ⅰ osteoclast showed inhibited apoptosis,enhanced proliferation and up-regulated cathepsin K expression (P ＜ 0.05).The functional experiment revealed that osteoclasts collected from IGF-Ⅰ treated co-cultured group resulted in more resorption pits formation (P ＜ 0.05); rhIGF-Ⅰ did not show any significant effect on the individually cultured osteoclasts.Conclusion Osteoblast is necessary for osteoclast induced bone resorption resulting from IGF-Ⅰ treatment.