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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

27卷

期数:

2021年03期

页码:

217-221,228

栏目:

基础医学

出版日期:

2021-05-30

文章信息/Info

Title:

Effects of wnt classic signaling pathway on the inhibition of osteoblast function by acidic pH

文章编号:

1006-8147(2021)03-0217-05

作者:

张顺¹; 2; 田爱现²; 张杨²; 郭悦²; 王岩²; 董本超²; 马剑雄²; 马信龙¹; 2

1.天津医科大学总医院骨科，天津300052；2.天津市天津医院骨科研究所，天津大学天津医院骨科研究所，天津300050

Author(s):

ZHANG Shun¹; 2;  TIAN Ai-xian²;  ZHANG Yang²; GUO Yue²; WANG Yan²; DONG Ben-chao²; MA Jian-xiong²; MA Xin-long¹; 2

1.Department of Orthopedics， General Hospita，Tianjin Medical University，Tianjin 300052，China；2. Institute of Orthopedics， Tianjin Hospital of Tianjin，Tianjin University Tianjin Hospital，Tianjin 300050，China

关键词:

原代培养; 成骨细胞; 酸性环境; pH值; wnt经典信号通路

Keywords:

primary culture; osteoblasts; acidic environment; pH; wnt classic signaling pathway

分类号:

R681

DOI:

-

文献标志码:

A

摘要:

目的：探讨wnt经典通路在酸性pH抑制成骨细胞功能中的作用。方法：胶原酶消法提取新生Sprague-Dawley（SD）大鼠颅骨原代成骨细胞，碱性磷酸酶（ALP）和茜素红（ARS）染色鉴定。传代培养至第3代，分别用pH6.0、pH6.4、pH6.8、pH7.4（对照）的DMEM高糖培养基进行培养。CCK8法检测各组细胞增殖情况；Live/Dead染色观察凋亡情况；反转录聚合酶链反应（RT-PCR）检测各组细胞COL1A1、β-catenin、RUNX2、wnt3a的mRNA表达水平；Western印迹法分析各组细胞COL1A1、β-catenin、RUNX2、wnt3a蛋白的表达情况。结果：ALP和ARS染色结果均为阳性，证明提取的原代细胞为成骨细胞。CCK8结果显示随着pH值降低和时间延长，pH6.0、pH6.4、pH6.8组实验各组细胞增殖活力均有下降（F=42.800、93.700，均P<0.05）。Live/Dead染色结果显示，随着pH值降低，细胞凋亡百分比显著增加（F=63.74，均P<0.05）。RT-PCR结果显示,与对照组相比，pH6.0、pH6.4、pH6.8组mRNA的表达显著降低（F=20.32、17.76、7.809、11.16，均P<0.05）。Western 印迹结果显示,与对照组相比，pH6.0、pH6.4、pH6.8组蛋白表达水平均显著下降（F=20.02、15.67、22.11、42.11，均P<0.05）。结论：酸性pH可影响成骨细胞的增殖、凋亡和分化，其机制可能与wnt经典信号通路有关。

Abstract:

Objective： To explore the role of wnt classic pathway in inhibiting osteoblast function at acidic pH. Methods： The collagenase digestion method was used to extract primary osteoblasts from the skull of newborn Sprague-Dawley（SD） rats and stained with alkaline phosphatase（ALP） and alizarin red（ARS）. After subculture to the third generation，DMEM high-glucose medium of pH 6.0，pH 6.4，pH 6.8，and pH 7.4（control）was used for culture. CCK8 method was used to detect cell proliferation； Live/Dead staining was used to observe apoptosis； reverse transcription polymerase chain reaction（RT-PCR） was used to detect mRNA expression levels of COL1A1，β-catenin，RUNX2，wnt3a；Western blotting was used to analysis the protein expression levels of COL1A1，β-catenin，RUNX2，wnt3a in each group of cells. Results: The results of ALP and ARS staining were both positive，which proved that the extracted primary cells were osteoblasts.CCK8 results indicated that as the pH value decreased and time prolonged，the cell proliferation activity in the pH6.0，pH6.4 and pH6.8 groups decreased（F=42.800，93.700，both P<0.05）.Live/Dead staining results showed that as the pH value decreased，the percentage of apoptosis increased significantly（F=63.74，P<0.05）.RT-PCR results showed that compared with the control group，the expression of mRNA in the pH 6.0，pH 6.4，and pH 6.8 groups was significantly decreased（F=20.32，17.76，7.809，11.16，all P<0.05）. Western blot ting results indicated that compared with the control group，the protein expression level in the pH6.0，pH6.4 and pH6.8 groups decreased significantly（F=20.02，15.67，22.11，42.11，all P<0.05）. Conclusion: The proliferation，apoptosis and differentiation of osteoblasts are affected by acidic pH，and the mechanism may be related to the classic wnt signaling pathway.

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备注/Memo

备注/Memo:

基金项目 国家自然科学基金面上项目（81871777，11772226）
作者简介 张顺(1994-)，男，硕士在读，研究方向：骨质疏松；
通信作者：马信龙，E-mail：maxinlong5566@163.com。

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更新日期/Last Update: 2021-05-30