当归多糖对高糖状态下大鼠骨髓间充质干细胞成骨向分化的影响

doi:10.7518/hxkq.2019.02.012

华西口腔医学杂志 ›› 2019, Vol. 37 ›› Issue (2): 193-199.doi: 10.7518/hxkq.2019.02.012

当归多糖对高糖状态下大鼠骨髓间充质干细胞成骨向分化的影响

廖锋¹,刘瑶²,刘航航²,胡建¹,赵爽³,杨世茂³()

1.武汉大学口腔医学院,湖北省口腔基础医学重点实验室-省部共建国家重点实验室培育基地,口腔生物医学教育部重点实验室（武汉大学）,武汉 430072
2.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口腔颌面外科,成都 610041
3.济南市口腔医院口腔颌面外科,济南 250001

收稿日期:2018-07-12 修回日期:2018-12-19 出版日期:2019-04-01 发布日期:2019-04-28
通讯作者: 杨世茂 E-mail:yangshimao1986@163.com
作者简介:廖锋,主治医师,博士,E-mail： wb003230@whu.edu.cn
基金资助:
国家自然科学基金(81621062);四川省科技创新团队项目(2017TD0016)

Effect of Angelica sinensis polysaccharide on the osteogenic differentiation of bone marrow mesenchymal stem cells of rats with high glucose levels

Feng Liao¹,Yao Liu²,Hanghang Liu²,Jian Hu¹,Shuang Zhao³,Shimao Yang³()

1.The State Key Laboratory Breeding Base of Basic Science of Stomatology, Hubei Province & Key Laboratory of Oral Biomedicine (Wuhan University), Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan 430072, China
2.State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Dept. of Oral and Maxillo-facial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
3.Dept. of Oral and Maxillofacial Surgery, Jinan Stomatology Hospital, Jinan 250001, China

Received:2018-07-12 Revised:2018-12-19 Online:2019-04-01 Published:2019-04-28
Contact: Shimao Yang E-mail:yangshimao1986@163.com
Supported by:
The National Natural Science Foundation of China(81621062);Technology Innovation Team Project of Sichuan Province(2017TD0016)

摘要/Abstract

摘要：

目的探讨当归多糖（ASP）对高糖状态下大鼠骨髓间充质干细胞（BMSCs）成骨向分化的影响。方法培养并收集第3代BMSCs进行成骨成脂分化诱导鉴定。将BMSCs分为3组进行培养：正常对照组（葡萄糖浓度5.5 mmol·L ^-1）、高糖组（葡萄糖浓度25.5 mmol·L ^-1）、ASP+高糖组（葡萄糖浓度25.5 mmol·L ^-1+40 mg·L ^-1 ASP）。CCK8检测各组BMSCs的增殖活性,茜素红染色和碱性磷酸酶活性检测成骨活性。实时荧光定量聚合酶链反应检测成骨标记基因Runt相关转录因子2（Runx-2）、锌指结构转录因子（Osx）、骨钙蛋白（OCN）、Ⅰ型胶原酶（COL-Ⅰ）mRNA及Wnt/β-catenin信号通路关键因子CyclinD1及β-catenin的mRNA表达。建立2型糖尿病大鼠模型,将大鼠分为3组：正常对照组（正常大鼠）、糖尿病组（糖尿病大鼠）、糖尿病+ASP组（糖尿病大鼠,ASP喂养）,制备大鼠胫骨骨缺损,进行组织学检测,观察骨缺损修复情况。结果高糖组、ASP+高糖组的BMSCs增殖高于正常对照组（P<0.05）,高糖组和ASP+高糖组二者之间无统计学差异（P>0.05）。高糖组中BMSCs钙结节数量、碱性磷酸酶活性及Runx-2、OCN、Osx、COL-Ⅰ、CyclinD1、β-catenin的mRNA表达均低于正常对照组和ASP+高糖组（P<0.05）,正常对照组和ASP+高糖组之间无统计学差异（P<0.05）。组织学检测结果显示,糖尿病组骨小梁数量少于正常对照组和糖尿病+ASP组（P<0.05）,而正常对照组和糖尿病+ASP组二者之间无统计学差异（P>0.05）。结论 ASP可促进高糖状态下大鼠BMSCs的成骨分化及2型糖尿病大鼠的骨缺损修复,这种促进作用可能与Wnt/β-catenin信号通路的激活有关。

关键词: 当归多糖, 骨髓间充质干细胞, 成骨分化, 2型糖尿病

Abstract:

Objective This study aims to evaluate the effect of Angelica sinensis polysaccharide (ASP) on the osteogenic differentiation of the bone marrow mesenchymal stem cells (BMSCs) of rats with high glucose levels. Methods Rat BMSCs were isolated and identified by osteogenic and adipo-genic differentiation. Then, the BMSCs were divided into three groups as follows: normal control group (5.5 mmol·L ^-1glucose), high glucose group (25.5 mmol·L ^-1glucose), and ASP+high glucose group (25.5 mmol·L ^-1 glucose +40 mg·L ^-1 ASP). The proliferation activities of the BMSCs were detected by CCK8. Alizarin red staining, and alkaline phosphatase activity were used in the examination of osteogenic activity. Quantitative real time-polymerase chain reaction was used to detect the expression levels of the osteogenic genes (Runx2, Osx, OCN, Col-Ⅰ) and the key factors of Wnt/β-catenin signal pathway (CyclinD1, β-catenin). In vivo, a type 2 diabetes rat model was established. The rats were divided into three groups, namely, the normal control group (normal rats), diabetes group (diabetic rats), diabetes+ASP group (diabetic rats, ASP feeding). Then, the tibia bone defect was established. The repair of bone defects in each group was observed through histological examination. Results The proliferation of BMSCs was higher in the high glucose group and ASP+high glucose group than in the normal control group (P<0.05). No significant difference was observed between the high glucose group and ASP+high glucose group (P>0.05). The number of calcium nodules of BMSCs; alkaline phosphatase activity; and the mRNA expression of Runx2, OCN, Osx, Col-Ⅰ, CyclinD1, β-catenin in the high glucose group were lower than those in the normal control and ASP+high glucose groups (P<0.05). No significant difference was observed between the normal control and ASP+high glucose groups (P>0.05). The bone mass was significantly lower in the bone defect of the diabetes group than in the bone defect of the normal control or diabetes+ASP group (P<0.05). No statistical difference was found between the normal control and diabetes+ASP groups (P>0.05). Conclusion ASP can promote the osteogenic differentiation of rat BMSCs under high glucose culture and induce bone regeneration in rats with type 2 diabetes. These features may be related to the activation of the Wnt/β-catenin signaling pathway.

Key words: Angelica sinensis polysaccharide, bone marrow mesenchymal stem cells, osteogenic differentiation, type 2 diabetes mellitus

中图分类号:

Q254

廖锋,刘瑶,刘航航,胡建,赵爽,杨世茂. 当归多糖对高糖状态下大鼠骨髓间充质干细胞成骨向分化的影响[J]. 华西口腔医学杂志, 2019, 37(2): 193-199.

Feng Liao,Yao Liu,Hanghang Liu,Jian Hu,Shuang Zhao,Shimao Yang. Effect of Angelica sinensis polysaccharide on the osteogenic differentiation of bone marrow mesenchymal stem cells of rats with high glucose levels[J]. West China Journal of Stomatology, 2019, 37(2): 193-199.

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基因	引物序列（5’-3’）
Runx2	F：GCGGACGAGGCAAGAGTT
	R：TTGGTGCTGAGTTCAGGGAG
Osx	F：CTGTGAAACCTCAAGTCCTATGGA
	R：GCTCTGCAGTCAAGGGAGATG
OCN	F：CAGGTGCAAAGCCCAGCGACT
	R：AGGGGATCTGGGTAGGGGGCT
Col-Ⅰ	F：CCACCTGCCTCTGGCTTCT
	R：AGCTGTGGAGGAGGGTTTCA
β-catenin	F：TGGCAACCAAGAAAGCAAG
	R：CTGAACAAGAGTCCCAAGGAG
CyclinD1	F：CCCTCGGTGTCCTACTTCA
	R：GTTTGTTCTCCTCCGCCTCT
GAPDH	F：AGACCTTCAACACCCCAG
	R：CACGATTTCCCTCTCAGC

当归多糖对高糖状态下大鼠骨髓间充质干细胞成骨向分化的影响

Effect of Angelica sinensis polysaccharide on the osteogenic differentiation of bone marrow mesenchymal stem cells of rats with high glucose levels

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