体外分离培养成骨细胞的一种新方法

A New Method of Isolating and Culturing Rabbit Osteoblasts in Vitro

为了建立易引起基质钙化的成骨细胞体外实验模型,取新生兔仔,无菌取其颅盖骨,去掉骨缝间的结缔组织,随意剪成不大于1mm2的碎块,组织块法提取成骨细胞,倒置显微镜观察原代成骨细胞以及传代后成骨细胞的生长状况;取第3代成骨细胞行酶化学染色和茜素红染色检测成骨细胞的碱性磷酸酶(ALP)表达和钙化结节。结果显示:原代成骨细胞1周后即有钙化结节或钙化灶形成,传代后细胞仍有钙化发生,连续培养后钙化结节增多,成骨细胞质膜高表达ALP,钙化结节染色呈红色。因此,本方法提取的成骨细胞容易发生钙化,可作为一种新的细胞实验模型。

To establish an experimental model of osteoblasts to easily cause calcification of bone matrix in vitro, we took cranium of a newborn rabbit out under an aseptic condition, removed the connective tissue of the bony suture, and cut the cranium freely into the fragments of not more than 1 mm2. The we isolated and cultured the osteohlasts using tissue explant method. We observed growth status of primary osteobtasts and subcultured osteoblasts using in- verted microscope. Then we conducted enzyme staining and alizarin red staining for the third generation of osteo- blasts to detect the alkaline phosphatase （ALP） expression and calcified nodules. The result showed that there were calcified nodules or calcification formed after the primary osteoblasts climbing out from the bone for 1 week, and each generation of osteoblasts had the similar calcification with the primary osteoblasts, and there was an increase in calci- fied nodules after the continuous culture. There was a strong expression of ALP in the plasma membrane of osteo- blasts. The calcified nodules were red with alizarin red staining. It is well concluded that osteoblasts isolated with this method easily cause calcification, and can be used as a new experimental model.