酶消化法联合组织块法培养大鼠原代成骨细胞

发布时间：2018-09-15 07:23

【摘要】：背景:获取高纯度、高活性的成骨细胞是进行骨代谢研究的基础。目的:探索一种简便、高效的原代成骨细胞培养方法。方法:将新生24 h内的SD大鼠,分离获取颅骨的顶骨和额骨,预先采取胰酶和胶原酶消化,再进行组织块培养的方法提取成骨细胞。通过倒置相差显微镜和透射电镜观察细胞形态,细胞计数绘制细胞生长曲线,采用碱性磷酸酶BCIP/NBT染色和茜素红矿化结节染色进行成骨细胞鉴定。结果与结论:(1)细胞形态呈长梭形、三角形、不规则多边形,有两至三个细胞突起;(2)透射电镜下线粒体和内质网在成骨细胞中非常丰富,并且有较多细胞突起,呈现出典型成骨细胞特点;(3)细胞刚接种时生长缓慢,3 d后增殖加快,第7天达高峰;(4)碱性磷酸酶BCIP/NBT染色显著阳性,茜素红钙结节染色橘红色;(5)采用酶消化法联合组织块法提取的细胞具有典型的成骨细胞特征和功能,是一种理想的原代成骨细胞分离培养方法。
[Abstract]:Background: the acquisition of high purity and high activity osteoblasts is the basis of bone metabolism. Objective: to explore a simple and effective method for primary osteoblast culture. Methods: the parietal and frontal bones of the skull were isolated from SD rats within 24 hours. The osteoblasts were extracted from the parietal and frontal bones by trypsin and collagenase digestion. Cell morphology was observed by inverted phase contrast microscope and transmission electron microscope. Cell growth curve was plotted by cell count. Osteoblasts were identified by alkaline phosphatase BCIP/NBT staining and alizarin red mineralized nodule staining. Results and conclusions: (1) the shape of cells was long fusiform, triangular, irregular polygon, with two to three cell processes, (2) mitochondria and endoplasmic reticulum were abundant in osteoblasts under transmission electron microscope, and there were many cell processes in osteoblasts. There were typical osteoblasts, (3) the proliferation of the cells increased after 3 days of inoculation, and reached the peak on the 7th day; (4) the alkaline phosphatase BCIP/NBT staining was significantly positive. Alizarin red calcium nodules staining orange; (5) the cells extracted by enzyme digestion combined with tissue block method have typical characteristics and functions of osteoblasts, which is an ideal method of primary osteoblast isolation and culture.
【作者单位】：广西医科大学第一附属医院;广西医科大学第一附属医院内分泌科;
【基金】：国家自然科学基金资助项目(81260142)~~
【分类号】：R68

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