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乳鼠成骨细胞的培养及鉴定
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卓丽玲1，顾建红1，赵瑞英1，刘宗平1,张春岭2
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（1.扬州大学 兽医学院，江苏 扬州225009；2.泰安牧神饲料有限公司，山东 泰安271000）
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摘要：
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无菌采取5日龄SD大鼠的颅盖骨，用胰蛋白酶预消化后，漂洗，剪碎，再用Ⅱ型胶原酶消化获得成骨细胞。通过倒置相差显微镜观察，并用碱性磷酸酶和矿化结节染色鉴定，利用微量滴定（MTT）法测定一个培养周期的成骨细胞活性分布，探讨一种经济、高效的原代成骨细胞培养方法。倒置相差显微镜形态观察结果显示，未贴壁的细胞呈圆形，贴壁生长的细胞呈不规则梭形、三角形或多角形，单核，有1~3个核仁；碱性磷酸酶和钙化结节染色均呈阳性（阳性率≥98.06%）；MTT法结合形态学观察结果显示，成骨细胞培养至第10d时活性最强。证实用改良的二次酶消化法能够在短时间内获得大量成骨细胞，且所培养的细胞具有典型的成骨细胞形态和功能。 |
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关键词：
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乳鼠；成骨细胞；鉴定；矿化结节 |
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中图分类号：Q 813.11 文献标识码：
A 文章编号：1673-4696(2007)10-0901-04 |
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Cultivation and identification of osteoblasts in sucking rat |
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ZHUO Li-ling1，GU Jian-hong1，ZHAO
Rui-ying1，LIU Zong-ping1，ZHANG Chun-ling2 |
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(1.College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;
2.Tai’an Mushen Feed Company Ltd.,Tai’an 271000,China) |
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Abstract: To investigate an economical and efficient cultivation method for primary osteoblasts,the cranial bones were harvested from 5-day-old SD rat under sterile conditions,digested by trypsin,rinsed and cut to shiver,and digested byⅡcollagenase,and then the osteoblast cells were harvested.The cells were identified by invert phase contrast microscopy and alkaline phosphatase staining and mineralized nodules staining.The activity distribution of osteoblasts was determined by microtitration (MTT) during a culture cycle.The cells were round before adherence,and anomalism fusiform,triangle and polygon when adhered,monocaryon,1-3 nucleole by invert phase contrast microscopy observation.Alkaline phosphatase staining and mineralized nodules staining were positive(≥98.06%).The osteoblastic activity was the highest on day 10,which was observed by MTT and morphological method.It was concluded that the modified twice-enzyme isolation method was an ideal technique to obtain and culture osteoblasts with typical characteristics. |
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Key words: sucking rat；osteoblast；identification；mineralized nodule |
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