中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (20): 3230-3235.doi: 10.12307/2023.488

• 骨组织构建 bone tissue construction • 上一篇    下一篇

三花接骨散对成骨细胞Wnt/β-Catenin信号通路的影响

段嘉豪1，谭旭仪1，2，卢  敏3，邝高艳3，方传龙4，肖  满4   

  1. 1湖南中医药大学，湖南省长沙市  410208；2湖南省中医药研究院附属医院骨伤科，湖南省长沙市  410006；3湖南中医药大学第一附属医院骨伤科，湖南省长沙市  410021；4湖南方盛制药股份有限公司，湖南省长沙市  410205
  • 收稿日期:2022-07-01 接受日期:2022-08-27 出版日期:2023-07-18 发布日期:2022-11-19
  • 通讯作者: 谭旭仪，博士后，副主任医师，湖南中医药大学，湖南省长沙市 410208；湖南省中医药研究院附属医院骨伤科，湖南省长沙市 410006
  • 作者简介:段嘉豪，男，1993年生，湖南省长沙市人，汉族，湖南中医药大学在读博士，医师，主要从事脊柱脊髓相关疾病的研究。
  • 基金资助:
    湖南中医药大学横向课题：三花接骨散促骨形成细胞/分子生物学机制研究

Effects of Sanhua Jiegu San on Wnt/beta-catenin signaling pathway in osteoblasts

Duan Jiahao1, Tan Xuyi1, 2, Lu Min3, Kuang Gaoyan3, Fang Chuanlong4, Xiao Man4   

  1. 1Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China; 2Department of Orthopedics, Affiliated Hospital of Hunan Academy of Chinese Medical Science, Changsha 410006, Hunan Province, China; 3Department of Orthopedics, The First Hospital of Hunan University of Chinese Medicine, Changsha 410021, Hunan Province, China; 4Hunan Fangsheng Pharmaceutical Co., Ltd., Changsha 410205, Hunan Province, China
  • Received:2022-07-01 Accepted:2022-08-27 Online:2023-07-18 Published:2022-11-19
  • Contact: Tan Xuyi, MD, Associate chief physician, Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China; Department of Orthopedics, Affiliated Hospital of Hunan Academy of Chinese Medical Science, Changsha 410006, Hunan Province, China
  • About author:Duan Jiahao, MD candidate, Physician, Hunan University of Chinese Medicine, Changsha 410208, Hunan Province, China
  • Supported by:
    the Horizontal Project of Hunan University of Chinese Medicine

PDF

36

摘要：


文题释义：

三花接骨散：是中药制剂，20世纪90年代初期由首都医科大学等多家医疗教研单位研发而成，由三七、西红花、当归、川芎、血竭、桂枝、大黄、地龙、马钱子粉、煅自然铜、土鳖虫、续断、牛膝、烫骨碎补、木香、沉香、冰片、白芷等中药组成，具有活血化瘀、消肿止痛、接骨续筋的作用。
成骨细胞：与成纤维细胞、脂肪细胞同源，均源于间充质始祖细胞分化而来。成骨细胞是骨形成的主要功能细胞，其主要功能是介导骨基质的合成、分泌和矿化，负责新骨生成，与破骨细胞共同负责骨重建修复。其在骨形成过程中分为成骨细胞增殖、细胞外基质成熟、细胞外基质矿化和成骨细胞凋亡等4个阶段。

背景：三花接骨散为临床骨折筋伤常用药，使用效果良好，但缺少相关实验学依据，其作用机制尚不明确。
目的：观察三花接骨散对成骨细胞Wnt/β-Catenin信号通路的影响。
方法：首先预实验筛选三花接骨散干预成骨细胞的最佳浓度，之后采用CCK-8法进行成骨细胞增殖实验，碱性磷酸酶检测成骨细胞成熟度，RT-PCR检测碱性磷酸酶、Runt相关转录因子2、骨钙蛋白、骨桥蛋白、胶原酶Ⅰ、Axin2、Dkk4、Lef-1、Tcf-1 mRNA表达，Western blot法检测β-Catenin、P-β-Catenin蛋白表达。
结果与结论：①成骨细胞培养5 d后，10 mg/L质量浓度时成骨细胞数量最多；②成骨细胞培养21 d后，三花接骨散不同浓度组的细胞增殖数量均有增加，以10 mg/L组明显，但20 mg/L已有减少的趋势；③成骨细胞培养7 d后，三花接骨散低、中、高剂量组碱性磷酸酶表达均有增加，以高剂量组(20 mg/L)明显；④成骨细胞培养7 d后，三花接骨散低、中、高剂量组碱性磷酸酶、Runt相关转录因子2、骨钙蛋白，骨桥蛋白，胶原酶ⅠmRNA表达均有增加，以高剂量组(20 mg/L)明显；⑤成骨细胞培养21 d后，三花接骨散低、中、高剂量组Axin2、Dkk4 mRNA表达均有降低，Lef-1、Tcf-1 mRNA表达增加，以高剂量组(20 mg/L)明显；⑥成骨细胞培养21 d后，三花接骨散低、中、高剂量组P-β-Catenin蛋白表达均有降低，β-Catenin蛋白表达增加，均以高剂量组(20 mg/L)明显；⑦提示三花接骨散可调控成骨细胞Wnt/β-Catenin信号通路，促进碱性磷酸酶、Runt相关转录因子2、骨钙蛋白、骨桥蛋白、胶原酶Ⅰ、Lef-1、Tcf-1 mRNA以及β-Catenin蛋白表达，降低Axin2、Dkk4 mRNA以及P-β-Catenin蛋白表达，促进成骨细胞的增殖，这可能是其促进骨折愈合的作用机制。

https://orcid.org/0000-0002-1537-3323(段嘉豪)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 三花接骨散, 成骨细胞, 细胞增殖, 骨折愈合, Wnt/β-Catenin信号通路, 分子机制

Abstract: BACKGROUND: Sanhua Jiegu San is a common drug for clinical fracture and tendon injury, which have good effect. However, there is a lack of relevant experimental evidence, and its mechanism of action remains unclear. 
OBJECTIVE: To observe the effect of Sanhua Jiegu San on Wnt/β-catenin signaling pathway in osteoblasts. 
METHODS: Firstly, the optimal concentration of Sanhua Jiegu San for osteoblast intervention was screened by pre-experiment. Then, the proliferation of osteoblasts was tested by cell counting kit-8 method. The maturity of osteoblasts was detected by alkaline phosphatase assay. The mRNA expressions of alkaline phosphatase, Runt-related transcription factor 2, osteocalcin, osteopontin, collagenase I, Axin2, Dkk4, Lef-1, and Tcf-1 were detected by PCR. The protein expressions of β-Catenin and P-β-Catenin were detected by western blot method. 
RESULTS AND CONCLUSION: After 5 days of osteoblast culture, the number of osteoblasts reached the maximum at the concentration of 10 mg/L. After 21 days of osteoblast culture, the proliferation of cells in different concentration groups of Sanhua Jiegu San increased, which was obvious in the 10 mg/L group, but tended to decrease at 20 mg/L. After 7 days of osteoblast culture, alkaline phosphatase expression was increased in the low, medium and high concentration groups, especially in the high concentration group (20 mg/L). After 7 days of osteoblast culture, mRNA expressions of alkaline phosphatase, Runt-related transcription factor 2, osteocalcin, osteopontin, collagenase I were increased in the low, medium and high concentration groups, especially in the high concentration group (20 mg/L). After 21 days of osteoblast culture, the mRNA expressions of Axin2 and Dkk4 were decreased in the low, medium and high concentration groups, while the mRNA expressions of Lef-1 and Tcf-1 were increased, especially in the high concentration group (20 mg/L). After 21 days of osteoblast culture, the expression of P-β-Catenin protein was decreased in the low, medium and high concentration groups, while the expression of β-Catenin protein was increased, especially in the high concentration group (20 mg/L). To conclude, Sanhua Jiegu San can regulate the Wnt/β-Catenin signaling pathway in osteoblasts, promote the mRNA expression of alkaline phosphatase, Runt-related transcription factor 2, osteocalcin, osteopontin, collagenase I, Lef-1, and Tcf-1 and the protein expression of β-Catenin, and decrease the mRNA expression of Axin2 and Dkk4 and the protein expression of P-β-Catenin, thereby promoting osteoblast proliferation. This may be the mechanism by which Sanhua Jiegu San promotes fracture healing.

Key words: Sanhua Jiegu San, osteoblast, cell proliferation, fracture healing, Wnt/β-catenin signaling pathway, molecular mechanism

中图分类号: