骨巨细胞瘤中多核巨细胞的纯化及其性质的探讨

目的 :利用酶消化方法对骨巨细胞瘤中多核巨细胞进行纯化 ,以弥补破骨细胞获取量少的问题 ,为骨质疏松的体外研究提供丰富的细胞来源。并用免疫组化的方法对骨巨细胞瘤中多核巨细胞的性质和来源进行探讨。方法 :体外分离培养 8例骨巨细胞瘤的多核巨细胞 ,在培养 2 0h后 ,用 0 .5 g·L-1胰蛋白酶 / 0 .2 g·L-1EDTA联合消化的方法去除贴壁能力较弱的单核基质细胞。将分离培养的骨巨细胞瘤多核巨细胞与牙磨片共培养 ,观察骨巨细胞中多核巨细胞的噬骨能力。并用破骨细胞特异表达的空泡型质子泵、Ⅱ型碳酸酐酶、组织蛋白酶K、基质金属蛋白酶 9对多核巨细胞进行免疫组化染色和TRAP染色。结果 :利用酶联合消化的方法可以获得较高纯度的多核巨细胞 ,纯化率达 85 %。骨巨细胞瘤中的多核巨细胞对破骨细胞所特异表达的空泡型质子泵、Ⅱ型碳酸酐酶、组织蛋白酶K、基质金属蛋白酶 9呈强阳性表达 ,TRAP染色阳性 ,体外培养具有噬骨能力。结论 :利用 0 .5 g·L-1胰蛋白酶 / 0 .2g·L-1EDTA消化的方法可获得较高纯度的多核巨细胞 ,可为破骨细胞体外研究提供丰富的细胞来源。骨巨细胞瘤中的多核巨细胞在功能表达上与破骨细胞相似 ,它可能来源于病变中圆形单核基质细胞的融合

Purification and study of the characteristics of the multinucleated giant cells of the giant cell tumor

OBJECTIVE: To harvest high purified osteoclast-like giant cells from the giant cell tumor by using trypsin and EDTA(ethylenediamineteraacetic acid) digestion. And explore the role and the origin of the multinucleated giant cells of the giant cell tumor. METHODS: Multinucleated giant cells were isolated and cultured from 8 cases of giant cell tumor of the long bone. After 20 hours of culture, 0.5 g.L-1 trypsin/0.2 g.L-1 EDTA was applied to detach the mononuclear stromal cells. The isolated multinucleated giant cells were inoculated on dentine slice to observe bone resorption ability in vitro. Immunohistochemistry study with V-ATPase, CA II (carbonic anhydrate II), cathepsin K, MMP-9 (matrix metalloproteinase-9), CD68 and TRAP(tartrate-resistant acid phosphatase) staining were carried out in the purified multinucleated giant cells. RESULTS: We obtained 85% purified multinucleated giant cells by trypsin/EDTA digestion. Multinucleated giant cells formed resorption lacunae on dentine slice in vitro and positively stained for V-ATPase, CA II, cathepsin K and MMP-9. The TRAP staining was also positive. CONCLUSION: Using 0.5 g.L-1 trypsin/0.2 g.L-1 EDTA digestion can get high purified multinucleated giant cells from the giant cell tumor. The multinucleated giant cells match all the character of the osteoclast and may originate from the fusion of the mononuclear stromal cells of the lesion.