两种方法分离新生大鼠颅骨成骨细胞纯度和活性的比较

目的比较预消化组织块培养法与传统组织块培养法分离的新生大鼠颅骨成骨细胞纯度和活性的差异。方法无菌条件下取新生大鼠颅骨骨片,分传统法和预消化法进行培养。预消化法先以0.25%胰蛋白酶/0.02%EDTA预先消化,再按传统组织块培养法培养,观察细胞移出时间,采用Gomori钙钴法对原代细胞及纯化后细胞进行鉴定,对比两种方法获得的成骨细胞纯度。并测定第1、2代成骨细胞培养第10d胞浆内碱性磷酸酶活性。结果预消化法约第3d开始细胞自骨片移出,传统法约第5d开始细胞移出。两种方法分离的原代细胞碱性磷酸酶染色阳性率分别为(87.83±2.34)%以及(82.88±3.14)%,差异有统计学意义(P<0.05)。纯化后成骨细胞染色阳性率分别为(90.71±3.15)%以及(90.17±2.97)%,两组比较差异无统计学意义(P>0.05)。两种方法获得的第1、2代成骨细胞培养第10d碱性磷酸酶活性比较差异无统计学意义(P>0.05)。结论预消化组织块培养法细胞移出时间早,且所分离的原代细胞中成骨细胞纯度高。经纯化后成骨细胞纯度和活性与传统组织块培养法相似。

Comparison of Purity and Activity of Osteoblasts Cultured from Newborn Rat Calvarias with Two Different Methods

Objective To compare the difference of purity and activity of osteoblasts isolated from neonatal rat calvarias with pre-digestion tissue culture method and traditional tissue culture. Methods Newborn rat calvarias were harvested under sterile condition and cultured with pre-digestion tissue culture method and traditional tissue culture. In pre-digestion tissue culture method, the bone chips were digested with 0.25% trypsin/0.02% EDTA for 20 minutes and followed by traditional tissue culture. The cultures were observed under microscope, emergence times of cells were recorded. Osteoblasts purity was compared using Gomori staining for primary cells and purified cells. Alkaline phosphatase activity of the first and second generation osteoblasts was measured after 10 days culture. Results Cells transfered from the bone chips about 3 days in pre-digestion method group and about 5 days in the traditional method group. The alkaline phosphatase staining positive rates of primary cells isolated with two methods were (87.83 ± 2.34) % and (82.88 ± 3.14)%, with statistically significance (P <0.05). The staining positive rate of osteoblasts after purification were (90.71 ± 3.15) % and (90.17 ± 2.97) %, without statistically significance (P >0.05). The alkaline phosphatase activity of the first and second generation osteoblasts was not statistically different after 10 days culture between two methods (P >0.05). Conclusions The pre-digestion tissue culture method can harvest higher purity primary cells earlier. After purification, the purity and activity of osteoblasts are similar between two methods.