摘要
目的建立小鼠成骨细胞和破骨细胞共培养模型,为探讨成骨细胞和破骨细胞之间的相互调控作用奠定基础。方法利用24h内新生小鼠颅骨和4~8周龄成年小鼠四肢长骨分别分离、培养成骨细胞和破骨细胞,采用间接接触的培养模式将接种了骨髓单核细胞的玻片或骨片置入提前24h接种了成骨细胞的培养皿中进行共培养。共培养一定时间后进行破骨细胞抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色鉴定和骨吸收功能检测,并进一步采用RT-PCR对破骨细胞标志酶基因基质金属蛋白酶-9(MMP-9)、TRAP和组织蛋白酶K(CathepsinK)的表达进行检测。结果共培养5天后可见TRAP(+)多核细胞形成,13天础(+)多核细胞数目达到高峰;骨吸收陷窝在共培养7天后开始出现,随着培养时间的延长,陷窝面积呈增加趋势;破骨细胞标志基因TRAP在共培养3天时开始表达,而MMP-9和CathepsinK则在共培养5天后表达。结论共培养体系中成骨细胞对破骨细胞调控作用显著,诱导的破骨细胞具有噬骨能力,该共培养模型可用于成骨细胞和破骨细胞之间的相互调控作用研究。
Objective To establish a co-culture system of mouse osteoblasts and osteoclasts for further investigating the interaction function of them. Methods Primary osteoblasts and osteoclasts are obtained respectively from the calvarias of newborn mice and long bones (femur and tibia) of adult mice (4-8 weeks old). An indirect contact co-culture system is set up by inserting a glass sheet sticking bone marrow mononuclear phagocytes into culture dish which had inoculated osteoblasts for 24h. The resistant acid phosphatase (TRAP) staining and bone resorption essay were used to identify the osteoclasts after 3 days of co-culture. The marked genes of osteoclast, MMP-9, TRAP and Cathepsin K, were detected by RT-PCR. Results The TRAP positive multinuclear cells emerged after 5 days of co-culture and the number of TRAP positive multinuclear cells reached the maximum after 13 days. Bone resorption essay indicated that resorption pits formed on bone slices after 7 days of co-culture and the area of pits gradually increased with the period of co-culture extending. The marked gene of osteoclast of TRAP expressed after 3 days of co-culture, but the MMP-9 and Cathepsin K were detected after 5 days. Conclusions The co-culture system can induce osteoclasts which degraded bone actively. This model can provide foundation for the research on the interaction function of osteoblasts and osteoclasts.
出处
《中国骨质疏松杂志》
CAS
CSCD
2008年第3期159-163,共5页
Chinese Journal of Osteoporosis
基金
国家重点基础研究发展规划项目(“973”项目)(2005CB522604)
国家自然科学基金杰出青年基金项目(30425023)
国家自然科学基金重点项目(30530410)