课题背景：破骨细胞是惟一能吸收矿化骨的细胞，在骨发育、骨形成、骨吸收和骨量的调节方面起着关键作用。到目前为止，虽然还没有建立成熟的破骨细胞株，但近年来随着对破骨细胞分化调控机制的深入认识，破骨细胞的体外培养基础研究进展迅速。因此获取纯度高，利于分子生物学研究，有骨吸收功能的破骨细胞非常重要。

同行评价：本实验选择孔径0.4 μm聚碳酸酯膜的Transwell培养板，将基质细胞与破骨前体细胞分层共培养，成功建立了可形成成熟的、有骨吸收功能的多核破骨细胞的三维培养体系。文章立意明确，选题新颖，设计严谨，有较高的学术水平与创新性。

重要的概念：骨组织持续地进行骨重建，包含成骨细胞和破骨细胞主导的2个对立的统一过程：成骨细胞的主导骨形成过程及破骨细胞的主导骨吸收过程。破骨细胞的骨吸收与成骨细胞的骨形成之间的平衡是维持正常骨重建的基础。破骨细胞的数量及活性改变是导致各种代谢性骨病如骨质疏松症、Paget's骨病、类风湿性关节炎、癌症骨转移等的主要原因，也是代谢性骨病研究的热点和重点。

摘要
背景：建立基质细胞与骨髓细胞共培养模型能诱导破骨样细胞产生，但这种培养体系存在细胞不单纯，不适合基因转染等缺点。
目的：建立基质细胞MBA-1细胞与破骨前体细胞RAW264.7共培养形成不混杂基质细胞的成熟多核破骨细胞的新方法。
设计、时间及地点：开放性实验，于2006-10/2007-03在南华大学附属第一医院临床研究所实验室完成。
材料：小鼠源性破骨前体细胞RAW264.7细胞从ATCC引进，基质细胞MBA-1细胞由周厚德博士惠赠。
方法：RAW264.7 细胞以1×104/孔接种于Transwell三维培养板下层，将聚碳酸酯膜孔径0.4 μm的培养板上层先取出接种MBA-1细胞，待细胞贴壁胞体伸展后，将其重新与下层联合共培养，共培养体系细胞每3 d换液1次。
主要观察指标：①抗酒石酸酸性磷酸酶染色法观察染色阳性的破骨细胞形态特点。②扫描电镜观察骨吸收陷窝形态，评价共培养形成的破骨样细胞骨吸收活性。
结果：共培养体系中的RAW264.7细胞，第9天可见较多TRAP阳性单核细胞，出现TRAP阳性多核破骨样细胞。共培养14 d后，已分化成熟的破骨细胞，能形成骨吸收陷窝。
结论：MBA-1细胞与破骨前体细胞RAW264.7分层共培养可形成成熟的、有骨吸收功能的多核破骨细胞，且这种破骨细胞中不混杂基质细胞。
关键词：破骨细胞；共培养；基质细胞；组织构建

肖新华，廖二元，董源媛，赵向，刘江华，曹仁贤，文格波. 破骨细胞三维培养体系的建立[J].中国组织工程研究与临床康复，2008，12(20):3863-3866 [www.zglckf.com/zglckf/ejournal/upfiles/08-20/20k-3863(ps).pdf]

Inhibitory effect of valsartan on intimal hyperplasia in rabbit autogenous grafted veins

Abstract

BACKGROUND：Coculture model of stromal cells and bone marrow cells can induce production of osteoclast. However, the cells by this culture system are not purified and not suitable for gene transfection.
OBJECTIVE: To establish a coculture model of stromal cells MBA-1 and osteoclast precursor RAW264.7 in order to provide a new method for studying coupling mechanism of the osteoblasts and osteoclasts.
DESIGN, TIME AND SETTING: The open experiment was performed at the laboratory of Institute of Clinical Research, First Hospital OF Nanhua University from October 2006 to March 2007.
MATERIALS: MBA-1 cells were graciously provided by Dr. Zhou and RAW264.7 cells were brought from ATCC.
METHODS: RAW264.7 cells were seeded onto the lower layer of Transwell 3D culture plate with a density of 1×104 cells per hole and MBA-1 cells were seeded on the separated upper layer of the culture plate (polycarbonate membrane diameter, 0.4 μm). When the MBA-1 cells began to attach and expand, they were cocultured with RAW264.7 cells by combining the two layers of culture plate. The culture solution was replaced every three days.
MAIN OUTCOME MEASURES: The morphological features of tartrate-resistant acid phosphatase (TRAP) positive osteoclasts; The activity of bone absorption of osteoclast-like cells from coculture system by observing the absorption lacuna under a scanning electron microscopy.
RESULTS: There were many TRAP positive monocytes and a small quantity of TRAP positive multinucleated osteoclast-like cells in coculture system when cultured for 9 days. Mature osteoclasts and bone absorption lacunas appeared after coculture for 14 days.
CONCLUSION: Separate layer and co-culture of MBA-1 cells and RAW264.7 cells form mature multinucleated osteoclasts in vitro with bone absorption capacity but no stromal cells.

Xiao XH, Liao EY, Dong YY, Zhao X, Liu JH, Cao RX, Wen GB. Three-dimensional coculture system for osteoclasts.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(20):3863-3866(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-20/20k-3863(ps).pdf]