共育体系中成骨细胞和破骨细胞生物学特性观察

The biological characteristics of osteoblasts and osteoclasts in co culture system

目的建立成骨细胞和破骨细胞的体外共育体系,观察在此体系中成骨细胞和破骨细胞生物学特性的变化,探讨成骨细胞和破骨细胞间的相互作用。方法取髂骨松质骨,Ⅱ型胶原酶消化,分次获得成骨细胞和破骨细胞。建立培养上清相通但二者互不接触的成骨细胞-破骨细胞共育模型。以细胞增殖(MTT法)、碱性磷酸酶(ALP)活性代表成骨细胞的成骨活性,以抗酒石酸酸性磷酸酶(TRAP)活性、骨吸收陷窝面积代表破骨细胞的破骨能力,检测共育对成骨细胞和破骨细胞生物学特性的影响。结果成骨细胞呈饱满的梭形,ALP染色阳性;破骨细胞呈多核,TRAP染色阳性,可以吸收骨质形成骨陷窝。当成骨细胞与破骨细胞共育后,其MTT法OD值(0.60±0.08)较单独培养时(0.36±0.03)明显提高(P=0.000);其ALP活性(23.37±2.48)u/mg较单独培养时(18.33±0.34)u/mg明显提高(P=0.000)。破骨细胞与成骨细胞共育后,形成骨吸收陷窝的平均面积犤(6.55±0.34)×10-2犦μm2较单独培养时犤(5.15±0.17)×10-2犦μm2明显增大(P=0.000)。结论共育体系中成骨细胞和破骨细胞的功能相互促进,为骨组织代谢的体外研究提供了可靠的模型。

Objective To establish osteoblasts osteoclasts co culture system in vitro for the study of the biological characteristics of osteoblasts and osteoclasts and also to analyze the interactions between osteoblasts and osteoclasts. Methods The osteoblasts and osteoclasts were isolated from cancellous bone in illium. Osteoblasts osteoclasts co culture system was built to prevent these two kinds of cells from contact with each other although with the facility of their medium exchange. The osteogenety of osteoblasts was evaluated by MTT and ALP activity; the resorption activity of osteoclasts was investigated by TRAP activity and the area of absorption lacuna. Results Osteoblasts are full fusiform, ALP stain positive cells, while osteoclasts are multinuclear and TRAP stain positive, with the ability of forming bone lacuna; while in the co culture system, the proliferation of osteoblasts in co culture (0.60±0.08)OD is quicker(P=0.000)than that in mono culture (0.36±0.03)OD, and the ALP activity increased significantly (P=0.000)from (18.33±0.34)u/mg to (23.37±2.48) u/mg; while the average area of bone resorption formed by osteoclasts [(6.55±0.34)×10-2] μm2 was also increased obviously(P=0.000)from [(5.15±0.17)×10-2] μm2. Conclusion In co culture system, osteoclasts were able to enhance ossification effects of osteoblasts by accelerating their proliferation and differentiation; at the same time, osteoblasts upregulated the ability of bone resorption of osteoclasts. This implied that osteoblasts and osteoclasts can promote each others function. This study provide reliable model for bone metabolism research.