Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (2): 551-558.DOI: 10.1016/j.jia.2022.08.051

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JIA-2021-1478 肉鸡胫骨成骨细胞原代培养模型的构建与评价

  

  • 收稿日期:2021-08-24 接受日期:2022-01-05 出版日期:2023-02-20 发布日期:2022-01-05

Establishment and evaluation of the primary cultured tibial osteoblast model of broiler chicks

CAO Su-mei1, 2, 3*, LI Ting-ting1*, SHAO Yu-xin2, ZHAO Yu-zhen4, ZHANG Li-yang2, LU Lin2, ZHANG Ri-jun3, HOU Shui-sheng2, LIAO Xiu-dong2, LUO Xu-gang1, WANG Run-lian4   

  1. 1 Poultry Mineral Nutrition Laboratory, College of Animal Science and Technology, Yangzhou University, Yangzhou 225000, P.R.China

    2 Mineral Nutrition Research Division, State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

    3 Laboratory of Feed Biotechnology, State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P.R.China

    4 Department of Animal Science, Guangdong Ocean University, Zhanjiang 524088, P.R.China

  • Received:2021-08-24 Accepted:2022-01-05 Online:2023-02-20 Published:2022-01-05
  • About author:Correspondence LUO Xu-gang, E-mail: wlysz@263.net; WANG Run-lian, E-mail: wangrunlian2005@163.com *These authors contributed equally to the present study.
  • Supported by:

    The present study was supported by the Key Program of the National Natural Science Foundation of China (31630073), the Initiation Funds of Yangzhou University for Distinguished Scientists, China, and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (ASTIP-IAS09).


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摘要：

成骨细胞在骨骼发育和矿化中扮演重要角色。然而，对肉仔鸡胫骨成骨细胞原代培养模型的建立和评价研究很少。因此，在目前研究中通试验1采用组织块法从1日龄AA肉公鸡胫骨中分离成骨细胞，通过细胞形态、碱性磷酸酶(ALP)染色和茜素红染色进行鉴定；试验2分别在成骨细胞持续培养第4、8、12、16、20、24、28和32天对原代培养的肉仔鸡胫骨成骨细胞的活力和矿化进行评价。

试验1结果表明，肉仔鸡胫骨原代成骨细胞呈梭形、三角形或多边形。ALP染色后95%以上细胞呈蓝黑色，连续培养4天后形成矿化结节；试验2结果表明，在整个培养过程中，虽然培养时间对乳酸脱氢酶(LDH)活性有影响(P=0.0012)，但LDH活性保持在相对稳定的水平。另外，培养时间显著影响(P≤0.0001)矿化结节的数量和面积比例，且随着培养时间的增加，其矿化结节数量呈线性和二次曲线增长(P<0.04)，并在24-32天内保持稳定。根据矿化结节数量和面积比例的最佳拟合断线模型或二次曲线模型(P<0.0001)评价最佳培养时间分别为17和26天。

结果表明，采用组织块法成功构建了肉仔鸡胫骨成骨细胞原代培养模型，其具有典型的成骨细胞形态、ALP活性和矿化特征，在持续培养4-32天内能保持相对稳定的活力，胫骨原代成骨细胞的最佳培养时间为17-26天。因此，该方法建立的肉鸡胫骨成骨细胞原代培养模型可用于进一步研究肉鸡骨骼发育和矿化的潜在机理。

本研究成功构建了稳定、可靠的肉鸡胫骨成骨细胞原代培养模型。

Abstract:

Osteoblasts are considered as a major factor contributing to bone development and mineralization, however, few studies have been done to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.  Therefore, in the present study, two experiments were conducted to establish and evaluate the primary cultured tibial osteoblast model of broiler chicks.  In experiment 1, osteoblasts were isolated from the tibia of one-day-old Arbor Acre male broiler chicks using the explant method and identified through the cell morphology, alkaline phosphatase (ALP) and alizarin red staining.  Experiment 2 was carried out to evaluate the vitality and mineralization of primary cultured tibial osteoblasts of broilers on days 4, 8, 12, 16, 20, 24, 28 and 32 after incubation, respectively.  The results from experiment 1 demonstrated that primary cultured tibial osteoblasts of broilers showed a spindle-shaped, triangular or polygonal morphology.  More than 95% of the cells were stained blue-black after ALP staining, and mineralized nodules were formed after 4 days of continuous incubation.  in experiment 2, lactate dehydrogenase (LDH) activity stayed at a relatively stabilized level although incubation time affected (P=0.0012) it during the whole culture period.  Additionally, incubation time affected (P≤0.0001) the number and proportion of the area of mineralized nodules.  They increased linearly and quadratically (P<0.04) with the increase of incubation time, and remained at a stabilized level from 24 to 32 days of incubation.  The estimates of the optimal incubation time were 17 and 26 days based on the best fitted broken-line or quadratic models (P<0.0001) of the number and proportion of the area of mineralized nodules, respectively.  These results indicate that the primary cultured tibial osteoblast model of broilers has been established successfully by the explant method, and it showed typical osteoblast morphology and characteristics of ALP activity and mineralization, and could maintain a relatively stabilized vitality from 4 to 32 days of incubation; and the optimal incubation time of primary tibial osteoblasts was 17 to 26 days.  Therefore, it could be used to further study the underlying mechanisms of bone development and mineralization of broiler chicks.

Key words: broiler , tibial osteoblast , primary culture , vitality , mineralization