成年比格犬骨髓间充质干细胞体外定向成骨诱导实验研究  

An in vitro experimental study on the induction of bone marrow mesenchymal stem cells of adult beagle dogs into osteoblasts

作　　者：许蕾[1] 李家锋[2] 唐巍[2] 韩建国[1] 孙晋虎[1] 

机构地区：[1]徐州医学院口腔医学院,江苏徐州221004 [2]徐州医学院附属医院口腔颌面外科,江苏徐州221002

出　　处：《徐州医学院学报》2015年第10期684-689,共6页Acta Academiae Medicinae Xuzhou

摘　　要：目的：通过将成年比格犬骨髓间充质干细胞（BMSCs）建立体外培养体系，观察及掌握细胞生长形态，运用诱导剂体外定向诱导分化为成骨细胞，为后期建立骨组织工程提供种子细胞。方法提取成年比格犬的BMSCs，运用半骨髓直接贴壁法进行体外分离与培养。将第2代BMSCs分成2组，实验组用加入等量诱导剂1（地塞米松、β－磷酸甘油钠、L－抗坏血酸）和诱导剂2（骨形态发生蛋白－2即BMP－2）的含胎牛血清培养液进行联合定向成骨诱导培养，对照组只用含血清的培养液进行培养，并于诱导的3、7、14、21天分别行碱性磷酸酶（ ALP）活性检测，并于3周后进一步行茜素红染色、ALP染色，4周后行Von－Kossa 染色来鉴定诱导分化的成骨细胞。结果诱导3、7、14、21天，实验组细胞的ALP活性呈先增强后减弱的状态，对照组无明显变化。实验组细胞诱导3周后行茜素红染色、ALP染色及诱导4周后行Von－Kossa染色都呈阳性，对照组均为阴性。以上结果均符合成骨细胞的特性，说明实验组有大量成骨细胞生成。结论体外分离培养的成年比格犬BMSCs在一定诱导剂的作用下可以向成骨细胞定向分化。Objective To cultivate the bone marrow mesenchmyal stem cells （ BMSCs） of adult beagle dogs in vitro and observe their morphology.These cells were then induced into osteoblasts, facilitating the development of bone tissue engineering in the future.Methods The BMSCs were first collected from adult beagle dogs and then cultivated in vitro using the semi-marrow adherence method.The second generation of BMSCs were divided into the following two groups：an experimental group with addition of fetal bovine serum （ FBS） as well as equivalent amounts of Inducer 1 （ dexametha-sone,β-glycerol phosphate disodium salt pentahydrate, and L-ascorbic acid） and Inducer 2 （ BMP-2） , and a con-trol group cultured in media containing FBS alone.Then, on Days 3, 7, 14, and 21 of induction, both groups were de-termined for alkaline phosphatase （ ALP） activity, followed by alizarin red staining and ALP staining three weeks later. The differentiation of induced osteoblasts was identified by Von-Kossa staining.Results On Days 3, 7, 14, and 21 of induction, the activity of ALP in the experimental group became weakened, despite after initial enhancement.In con-trast, no significant changes were found in the control group.After three weeks of induction, positive results were shown for alizarin red staining, ALP staining and Von-Kossa staining in the experimental group, in comparison with negative staining in the control group.These findings were consistent with the characteristics of osteoblasts, indicating the forma-tion of osteoblasts in the experimental group.Conclusion The BMSCs of adult beagle dogs can directionally differentiate into osteoblasts in the presence of an inducing agent.

关 键 词：成骨细胞 定向诱导 比格犬 骨组织工程 

分 类 号：R782[医药卫生—口腔医学]