| 不同胶原酶消化对原代成骨细胞获得率及活性的比较 |
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Received:July 17, 2012
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| 作者 | Author | 单位 | Unit | E-Mail |
| 李玲慧 |
LI Ling-hui |
上海中医药大学附属曙光医院石氏伤科医学中心, 上海 201203 |
Shi's Center of Orthopedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of TCM, Shanghai 201203, China |
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| 丁道芳 |
DING Dao-fang |
上海中医药大学附属曙光医院石氏伤科医学中心, 上海 201203 |
Shi's Center of Orthopedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of TCM, Shanghai 201203, China |
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| 杜国庆 |
DU Guo-qing |
上海中医药大学附属曙光医院石氏伤科医学中心, 上海 201203 |
Shi's Center of Orthopedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of TCM, Shanghai 201203, China |
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| 王辉昊 |
WANG Hui-hao |
上海中医药大学附属曙光医院石氏伤科医学中心, 上海 201203 |
Shi's Center of Orthopedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of TCM, Shanghai 201203, China |
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| 詹红生 |
ZHAN Hong-sheng |
上海中医药大学附属曙光医院石氏伤科医学中心, 上海 201203 |
Shi's Center of Orthopedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of TCM, Shanghai 201203, China |
zhanhongsheng2010@163.com |
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| 期刊信息：《中国骨伤》2013年26卷，第4期，第328-331页 |
| DOI：10.3969/j.issn.1003-0034.2013.04.016 |
| 基金项目:“中医骨伤科学”国家重点学科(编号:100508);上海领军人才项目(编号:041);上海市科委重点项目(编号:09dZ1973800);国家自然基金项目(编号:81073114,81001527,81102603, 81173621);上海市科委科技人才计划项目(编号:11XD1404600) |
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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1 h×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0 h、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型组显着高于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。 |
| [关键词]:成骨细胞 原代培养 胶原酶类 大鼠 |
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| Comparison of the activity and yield rate of osteoblast obtained by different digestion methods |
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Abstract:
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 typeⅠcollagenase and typeⅡcollagenase 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 typeⅠgroup was larger than typeⅡgroup. Alkaline phosphatase dyeing were positive. Detecting of alkaline phosphatase using the method of PNPP showed that the absorbance value in typeⅠgroup were higher than typeⅡgroup(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 typeⅠgroup are higher which could provide more stable seed cells for the treatment of osteoporosis. |
| KEYWORDS:Osteoblasts Primary culture Collagenases Rats |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 李玲慧,丁道芳,杜国庆,王辉昊,詹红生.不同胶原酶消化对原代成骨细胞获得率及活性的比较[J].中国骨伤,2013,26(4):328~331 |
| 英文格式: | LI Ling-hui,DING Dao-fang,DU Guo-qing,WANG Hui-hao,ZHAN Hong-sheng.Comparison of the activity and yield rate of osteoblast obtained by different digestion methods[J].zhongguo gu shang / China J Orthop Trauma ,2013,26(4):328~331 |
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