Objective:To compared the activity and yield rate of osteoblast obtained by different collagenase digestion methods,to find a better way to extract osteoblast for the experimental researches of osteoporosis. Methods:Ten 24-hour-old SD rats were were euthanized. The cranium of rats were removed and cuted into blocks of 1 mm×1 mm size. After digested by trypsin for 15 min,all the cranium were divided into two equal parts,and randomly divided into two groups which would be digested by type Ⅰ collagenase and type Ⅱ collagenase separately for two times. The rat cells of the two groups were cultured in thermostat incubator with 5% CO2 under the condition of 37 ℃. The primary culture osteoblasts were counted by using a haemacytometer after digestion and 72 hours later. The second generation osteoblasts cultured 48 h were dyed by NBT/BCIP staining solution, and were detected by quantitative measurement with PNPP. Results: The cells had irregular shapes. The results of cell counting showed that the cell number of type Ⅰ group was larger than type Ⅱ group. Alkaline phosphatase dyeing were positive. Detecting of alkaline phosphatase using the method of PNPP showed that the absorbance value in type Ⅰ group were higher than type Ⅱ group (P<0.05). Conclusion: Two types of collagenase are both suitable for the in vitro culture of rat osteoblasts. The activity and yield rate of osteoblasts in type I group are higher which could provide more stable seed cells for the treatment of osteoporosis.%目的:比较Ⅰ、Ⅱ型胶原酶对新生大鼠颅盖骨的消化效果,选择效率较高的胶原酶,为日后从细胞水平研究中药治疗骨质疏松症的机制提供大量种子细胞.方法:新生24 h SD大鼠10只,雌雄不限,脱颈处死后取出颅盖骨,剪成约1 mm×1 mm的碎片,采用胰酶预消化15 min后将骨片按重量平分成两等份,随机分为Ⅰ型组、Ⅱ型组,分别用0.1％Ⅰ型、0.1％Ⅱ型胶原酶37℃消化1h×2次,所得细胞置于5％CO2培养箱中37 ℃恒温培养.原代细胞分别于培养0h、72 h时采用血球计数板进行计数,第2代细胞于培养48 h后采用NBT/BCIP染色液进行染色,并通过对硝基苯磷酸盐法(PNPP)进行ALP定量检测.结果:镜下观察所获得的细胞形态不规则,细胞计数显示Ⅰ型组细胞数量大于Ⅱ型组；ALP染色为阳性；PNPP法测得两组吸光度均值分别为0.427和0.262,Ⅰ型组显著高于Ⅱ型组,差异具有统计学意义(P＜0.05).结论:两种胶原酶均适用于成骨细胞的原代消化,但采用Ⅰ型胶原酶获得的细胞数量更多、活性更好,可为日后骨质疏松症的机制研究提供优质的种子细胞.
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