[1]张 瑞,柳围堤,程自超,等.恒古骨伤愈合剂对人骨髓间充质干细胞生长、分化的影响及机制研究[J].陕西医学杂志,2021,50(12):1477-1481,1486.[doi:DOI:10.3969/j.issn.1000-7377.2021.12.003]
　ZHANG Rui,LIU Weidi,CHENG Zichao,et al.Effect and mechanism of Osteoking on growth and differentiation of human bone mesenchymal stem cells[J].,2021,50(12):1477-1481,1486.[doi:DOI:10.3969/j.issn.1000-7377.2021.12.003]
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恒古骨伤愈合剂对人骨髓间充质干细胞生长、分化的影响及机制研究
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《陕西医学杂志》[ISSN:1000-7377/CN:61-1281/TN]

卷:
50
期数:
2021年12期
页码:
1477-1481,1486
栏目:
基础研究
出版日期:
2021-12-05

文章信息/Info

Title:
Effect and mechanism of Osteoking on growth and differentiation of human bone mesenchymal stem cells
作者:
张 瑞; 柳围堤; 程自超; 杜怀峰
(西安交通大学医学院附属3201医院康复医学科,陕西 汉中 723000)
Author(s):
ZHANG Rui; LIU Weidi; CHENG Zichao; DU Huaifeng
(Department of Rehabilitation Medicine,3201 Hospital,School of Medicine,Xi'an Jiaotong University,Hanzhong 723000,China)
关键词:
恒古骨伤愈合剂;  人骨髓间充质干细胞;  细胞增殖活性;  细胞分化;  TGF-β1/Smads信号通路
Keywords:
Osteoking;  Human bone marrow mesenchymal stem cells;  Cell proliferation activity;  Cell differentiation;  TGF-β1/Smads signaling pathway
分类号:
R 285.5
DOI:
DOI:10.3969/j.issn.1000-7377.2021.12.003
文献标志码:
A
摘要:
目的:探讨恒古骨伤愈合剂对人骨髓间充质干细胞(hBMSCs)生长、分化的影响及机制。方法:全骨髓贴壁法培养hBMSCs,取第3代以含恒古骨伤愈合剂血清培养基培养(研究组),并以完全培养基为对照组。观察两组hBMSCs形态和碱性磷酸酶(ALP)染色结果。采用四氮唑盐比色法(MTT)检测两组hBMSCs细胞增殖活性。采用实时荧光定量PCR(RT-PCR)测定两组成骨分化特异性标志物转化生长因子-β1(TGF-β1)、人Ⅰ型胶原(Col Ⅰ)、骨钙素(OCN),破骨分化标志物核因子-κB活化因子受体配体(RANKL),破骨细胞抑制因子(OPG)相对表达量。采用Western blot检测Smad2/3蛋白表达。结果:研究组培养48 h后细胞形态逐渐变为圆形、多边形,细胞界限模糊,呈现聚集特点,类似成骨细胞形态,可见数量不均微小颗粒物质,部分见发育成熟矿化物; 对照组培养48 h后细胞形态呈梭形,突起变长,分散生长,细胞两极朝向不规律,可见少量微小颗粒物质。研究组hBMSCs含药血清培养1周后ALP染色程度高、显色范围广,对照组ALP着色弱、强度低,表明研究组hBMSCs分化潜能高。研究组hBMSCs培养不同时间增殖活性高于对照组(均P<0.05)。研究组hBMSCs培养1周TGF-β1、Col Ⅰ、OCN、OPG mRNA相对表达量高于对照组,RANKL mRNA相对表达量低于对照组(均P<0.05)。研究组hBMSCs培养1周p-Smad2/3蛋白相对表达量高于对照组(均P<0.05)。结论:恒古骨伤愈合剂可上调TGF-β1/Smads信号通路相关促骨形成靶基因表达,促进骨形成,抑制骨吸收,进而促进hBMSCs生长及分化。
Abstract:
Objective:To investigate the effect and mechanism of Osteoking on the growth and differentiation of human bone marrow mesenchymal stem cells(hBMSCs).Methods:The hBMSCs were cultured by whole bone marrow adherence method.The third generation of hBMSCs were cultured in serum medium containing Osteoking(study group),and the complete medium was used as the control group.The cell morphology and alkaline phosphatase(ALP)staining results of the two groups of hBMSCs were observed.The proliferation activity of the two groups of hBMSCs was detected by MTT.The expression of specific markers of osteogenic differentiation transforming growth factor-β1(TGF-β1),human type Ⅰ collagen(Col Ⅰ)and osteocalcin(OCN),markers of osteoclast differentiation the ligand for receptor activator of nuclear factor-κB(RANKL)and osteoclast inhibitor(OPG)in each group was determined by RT-PCR.The expression of Smad2/3 proteins were detected by Western blot.Results:After 48 hours of culture in the study group,the morphology of the cells gradually became round and polygonal,and the cell boundaries were blurred,showing the characteristics of aggregation,similar to the shape of osteoblasts,with uneven amounts of small particulate matter and some mature minerals; after 48 hours of culture in the control group,the cell morphology was fusiform and the protrusions was elongated,the growth was scattered,the cell poles were oriented irregularly,and a small amount of small particulate matter could be seen. The hBMSCs-containing serum of the study group was cultured for 1 week with high degree of ALP staining and a wide range of color development,while the control group had weak ALP staining and low intensity,indicating that the study group had a high differentiation potential for hBMSCs.The proliferation activities of hBMSCs in study group were higher than those in the control group after different time of culture(all P<0.05).The relative expression levels of TGF-β1,ColⅠ,OCN and OPG mRNA in hBMSCs in study group were higher than those in the control group after 1 week of culture,and the relative expression level of RANKL mRNA was lower than that in the control group(all P<0.05).The expression levels of p-Smad2/3 proteins in study group were higher than those in the control group after 1 week of culture(all P<0.05).Conclusion:Osteoking can up-regulate the expression of TGF-β1/Smads signaling pathway associated osteotropic formation target genes,promote bone formation,inhibit bone resorption,and promote the growth and differentiation of hBMSCs.

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备注/Memo

备注/Memo:
基金项目:国家中医药管理局中医药科学技术研究专项课题(CZY-KJS-2021-003)
更新日期/Last Update: 2021-12-07