唑来膦酸对大鼠骨髓间充质干细胞增殖及成骨分化的作用研究

doi:10.7518/hxkq.2019.03.003

华西口腔医学杂志 ›› 2019, Vol. 37 ›› Issue (3): 242-247.doi: 10.7518/hxkq.2019.03.003

唑来膦酸对大鼠骨髓间充质干细胞增殖及成骨分化的作用研究

李颉颃,苏志飞,白璇,袁鹤,李继遥()

口腔疾病研究国家重点实验室国家口腔疾病临床医学研究中心四川大学华西口腔医院牙体牙髓科,成都 610041

收稿日期:2018-08-11 修回日期:2019-01-19 出版日期:2019-06-01 发布日期:2019-06-12
通讯作者: 李继遥 E-mail:jiyaoliscu@163.com
作者简介:李颉颃,硕士,E-mail:490375604@qq.com
基金资助:
国家自然科学基金(81701028);国家自然科学基金(81670977);四川省科技厅重点项目(2017SZ0030);四川大学华西口腔医院青年科学研究基金(WCHS-201614)

Effect of zoladronate on the proliferation and osteogenic differentiation of rat bone mesenchymal stem cells

Jiehang Li,Zhifei Su,Xuan Bai,He Yuan,Jiyao Li()

State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Dept. of Conservative Dentistry and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China

Received:2018-08-11 Revised:2019-01-19 Online:2019-06-01 Published:2019-06-12
Contact: Jiyao Li E-mail:jiyaoliscu@163.com
Supported by:
The National Natural Science Foundation of China(81701028);The National Natural Science Foundation of China(81670977);The Key Program of Science and Technology Department of Sichuan Province(2017SZ0030);The Youth Research Foundation of West China Hospital of Stomatology, Sichuan University(WCHS-201614)

摘要/Abstract

摘要：

目的研究唑来膦酸对大鼠骨髓间充质干细胞（BMSCs）增殖及成骨分化的作用。方法取大鼠BMSCs体外原代培养,使用含不同浓度（1、5、10、20 μmol·L ^-1）唑来膦酸的成骨诱导培养基干预细胞作为实验组,不含唑来膦酸的培养基干预细胞作为对照组。CCK-8检测各组细胞增殖活性;茜素红和碱性磷酸酶染色检测各组细胞成骨分化能力;实时荧光定量聚合酶链反应（qRT-PCR）检测各组细胞碱性磷酸酶（ALP）、骨形态发生蛋白2（BMP-2）、Ⅰ型胶原酶（COL-Ⅰ）、Runt相关转录因子2（Runx-2）、锌指结构转录因子（Osx）、骨钙蛋白（OCN）、骨桥蛋白（OPN）的mRNA表达量。结果 1 μmol·L ^-1唑来膦酸对BMSCs的增殖、成骨分化无影响,与对照组比较差异无统计学意义（P>0.05）。浓度大于1 μmol·L ^-1唑来膦酸抑制BMSCs的增殖和成骨分化,且抑制作用呈浓度依赖性,与对照组比较差异有统计学意义（P<0.05）。当唑来膦酸浓度为5 μmol·L ^-1时,ALP、BMP-2、COL-Ⅰ等成骨相关基因的表达量高于对照组（P<0.05）,而当唑来膦酸浓度大于5 μmol·L ^-1时,成骨相关基因的表达量低于对照组（P<0.05）。结论低浓度唑来膦酸对BMSCs增殖和成骨分化无影响,5 μmol·L ^-1唑来膦酸抑制BMSCs增殖但促进其成骨分化,高浓度唑来膦酸抑制BMSCs的增殖和成骨分化。

关键词: 双膦酸盐, 骨髓间充质干细胞, 增殖, 成骨分化

Abstract:

Objective To evaluate the effect of zoledronate acid (ZA) on the proliferation and osteogenic differentiation of rat mesenchymal stem cells (BMSCs). Methods The BMSCs isolated from the SD rats were cultured with different con-centrations of ZA (1, 5, 10, and 20 μmol·L ^-1), and the contro1 group received the same volume of culture medium but without ZA. Cell counting kit-8 was used to detect proliferation activity in each group. Alkaline phosphatase (ALP) staining and alizarin red staining were used to detect the osteogenic differentiation ability in each group. The gene expression levels of ALP, bone morphogenetic protein-2 (BMP-2), typeⅠcollagenase (COL-Ⅰ), runt-related transcription factor-2 (Runx-2), zinc finger structure transcription factor (Osx), osteocalcin (OCN), and osteopontin (OPN) were evaluated by real-time quantitative polymerase chain reaction (qRT-PCR). Results Zoledronate at 1 μmol·L ^-1concentration had no effect on the proliferation and osteogenic differentiation of BMSCs. No significant difference was observed between this group and the control group (P>0.05). When the ZA concentration was more than 1 μmol·L ^-1, ZA inhibited the proliferation and osteogenic differentiation of BMSCs, and the effect was concentration dependent. The difference between each group and the control group was statistically significant (P<0.05). At ZA concentration of 5 μmol·L ^-1, ZA enhanced the expression of ALP, BMP-2, COL-Ⅰ, Runx-2, Osx, OCN, and OPN (P<0.05). However, at ZA concentration of more than 5 μmol·L ^-1, the expression levels of osteogenicrelated genes in each group was lower than those of the control group (P<0.05). Conclusion Low ZA concen-tration has no effect on the proliferation and osteogenic differentiation of BMSCs. ZA at 5 μmol·L ^-1 concentration inhibits the proliferation but promotes the osteogenic differentiation of BMSCs. High ZA concentration inhibits the proliferation and osteogenic differentiation of BMSCs.

Key words: bisphosphonates, bone mesenchymal stem cells, proliferation, osteogenic differentiation

中图分类号:

Q254

李颉颃,苏志飞,白璇,袁鹤,李继遥. 唑来膦酸对大鼠骨髓间充质干细胞增殖及成骨分化的作用研究[J]. 华西口腔医学杂志, 2019, 37(3): 242-247.

Jiehang Li,Zhifei Su,Xuan Bai,He Yuan,Jiyao Li. Effect of zoladronate on the proliferation and osteogenic differentiation of rat bone mesenchymal stem cells[J]. West China Journal of Stomatology, 2019, 37(3): 242-247.

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参考文献 20

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基因名称	引物序列
GAPDH	F:5’-GCCTTCCGTGTTCCTACC-3’
	R:5’-AGAGTGGGAGTTGCTGTTG-3’
ALP	F:5’-CCAACTCTTTTGTGCCAGAGA-3’
	R:5’-GGCTACATTGGTGTTGAGCTTTT-3’
BMP-2	F:5’-ACATGCAGCGTGAAATCCTG-3’
	R:5’-CGTGGTATAGGTCCAACATGAAG-3’
COL-Ⅰ	F:5’-ACGTCCTGGTGAAGTTG-3’
	R:5’-CAGGGAAGCCTCTTTCTCCT-3’
Runx-2	F:5’-GACTGTGGTTACCGTCATGGC-3’
	R:5’-ACTTGGTTTTTCATAACAGCGGA-3’
Osx	F:5’-CCACCAAGAATTACTCAGAACCC-3’
	R:5’-AAGGACCGACTCACTCAGTCT-3’
OCN	F:5’-CTGACCTCACAGATCCCAAGC-3’
	R:5’-TGGTCTGATAGCTCGTCACAAG-3’
OPN	F:5’-GGGGACTATGCACCTGAGC-3’
	R:5’-GACTGTCGAAATGGGCTACCT-3’

唑来膦酸对大鼠骨髓间充质干细胞增殖及成骨分化的作用研究

Effect of zoladronate on the proliferation and osteogenic differentiation of rat bone mesenchymal stem cells

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