低氧诱导因子-1α对骨髓间充质干细胞成骨分化与血管生成相关因子的影响

doi:10.12016/j.issn.2096-1456.2021.07.003

口腔疾病防治 ›› 2021, Vol. 29 ›› Issue (7): 449-455.DOI: 10.12016/j.issn.2096-1456.2021.07.003

低氧诱导因子-1α对骨髓间充质干细胞成骨分化与血管生成相关因子的影响

左新慧¹(),李君¹,韩祥祯¹,刘小元¹,何惠宇^1,²()

1.新疆医科大学第一附属医院（附属口腔医院）口腔修复科,新疆维吾尔自治区乌鲁木齐（830054）
2.新疆维吾尔自治区口腔医学研究所,新疆维吾尔自治区乌鲁木齐（830011）

收稿日期:2020-08-18 修回日期:2021-01-05 出版日期:2021-07-20 发布日期:2021-04-19
通讯作者: 何惠宇
作者简介:左新慧,住院医师,硕士研究生,Email： 13838371011@163.com
基金资助:
新疆维吾尔自治区科技支疆项目(2018E02060);研究生创新创业项目(XJ2020G216)

Effects of hypoxia inducible factor-1α on osteogenic differentiation and angiogenesis related factors of bone marrow mesenchymal stem cells

ZUO Xinhui¹(),LI Jun¹,HAN Xiangzhen¹,LIU Xiaoyuan¹,HE Huiyu^1,²()

1. Department of Prosthodontics, the First Affiliated Hospital of Xinjiang Medical University (Affiliated Stomatological Hospital), Urumqi 830054, China
2. Xinjiang Uygur Autonomous Region Institute of Stomatology, Urumqi 830011, China

Received:2020-08-18 Revised:2021-01-05 Online:2021-07-20 Published:2021-04-19
Contact: Huiyu HE
Supported by:
Xinjiang Uygur Autonomous Region Science and Technology Support Project(2018E02060);Postgraduate Innovation and Entrepreneurship Project(XJ2020G216)

摘要/Abstract

摘要：

目的探讨低氧诱导因子-1α（hypoxia-inducible factor-1α,HIF-1α）在SD大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）中对成骨分化和血管生成相关细胞因子的影响。方法分离并培养BMSCs,采用流式细胞术进行鉴定;分别构建上调和下调HIF-1α基因的质粒载体及对照组质粒,使用Lipofectamine^?LTX转染试剂将各组质粒分别转染至BMSCs,将细胞分为过表达实验组、过表达对照组、低表达实验组和低表达对照组。各组成骨诱导3、7 d后分别进行茜素红染色;RT-PCR检测目的基因HIF-1α,成骨分化特异标志物Runt相关转录因子2（Runt-related transcription factor 2,Runx2）,以及血管生成特异标志物血小板衍生生长因子-BB（platelet derived growth factor-BB,PDGF-BB）、转化生长因子-β（transforming growth factor-β,TGF-β）的mRNA表达水平;用Western blot检测目的蛋白HIF-1α及Runx2、PDGF-BB的蛋白表达量。结果流式鉴定BMSCs表面标记物CD29、CD45阳性表达率分别为98.2%和4.2%;RT-PCR结果显示：过表达实验组BMSCs中HIF-1α、Runx2、TGF-β和PDGF-BB的mRNA表达显著升高（P < 0.001）;低表达组中HIF-1α、Runx2、TGF-β 和PDGF-BB的mRNA表达显著降低（P < 0.001）;Western blot结果显示：过表达实验组BMSCs中HIF-1α、Runx2和PDGF-BB的蛋白表达量均增高（P < 0.001）,低表达实验组中HIF-1α、Runx2和PDGF-BB蛋白表达量降低（P < 0.001）。茜素红染色结果显示,低表达实验组钙结节面积小于其对照组,过表达实验组红色钙结节面积显著大于其对照组,随着成骨诱导时间的增加,各组钙化面积也增大。结论上调和下调HIF-1α后可调控BMSCs的成骨分化及血管生成相关因子表达。

关键词: 低氧诱导因子-1α, 骨髓间充质干细胞, 成骨分化, 血管生成, Runt相关转录因子2, 血小板衍生生长因子-BB, 转化生长因子-β, 磷脂酰肌醇3-激酶, 蛋白激酶B, 骨组织工程

Abstract:

Objective To investigate the level of hypoxia inducible factor-1α (HIF-1α) on osteoblasts and angiogenesis-associated cytokines in bone marrow mesenchymal stem cells (BMSCs) from SD rats.Methods BMSCs were isolated and cultured and identified by flow cytometry. Plasmid vectors containing upregulated and downregulated HIF-1α gene and a control vector were constructed. The plasmids were transfected into BMSCs by Lipofectamine^?LTX transfection reagent, and the cells were divided into an overexpression experimental group, an overexpression control group, a low expression experimental group and a low expression control group. All components were stained with a lizarin red 3 d and 7 d after osteogenesis induction. The mRNA expression levels of the target gene HIF-1α, osteogenic differentiation-specific markers, including Runt-related transcription factor 2 (Runx2) and angiogenic markers, including platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β (TGF-β), were detected by RT-PCR. Western blot was used to detect the protein expression of the target proteins HIF-1α, Runx2, and PDGF-BB.Results The CD29- and CD45-positivity rates of BMSC surface markers identified by flow cytometry were 98.2% and 4.2%, respectively. RT-PCR results showed that the mRNA expression of HIF-1α, Runx2, TGF-β and PDGF-BB was observably increased (P < 0.001). The mRNA expression levels of HIF-1α, Runx2, TGF-β and PDGF-BB in BMSCs from the low expression experimental group were significantly reduced (P < 0.001). Western blot results showed that the expression levels of HIF-1α, Runx2 and PDGF-BB in BMSCs from the overexpression experimental group were all increased (P < 0.001). The expression levels of HIF-1α, Runx2 and PDGF-BB in BMSCs from the low expression experimental group were reduced (P < 0.001). Alizarin red staining results showed that the area of calcium nodules in the low expression experimental group was smaller than that in low expression control group, the area of red calcium nodules in the over expression experimental group was larger than that in over expression control group, and with the increase of osteogenic induction time, the calcification area of each group also increased.Conclusion Upregulation and downregulation of HIF-1α can regulate the osteogenic differentiation and the expression of angiogenesis related factors of BMSCs.

Key words: hypoxia inducible factor-1α, bone marrow mesenchymal stem cells, osteogenic differentiation, angiogenesis, Runt-related transcription factor 2, platelet derived growth factor-BB, transforming growth factor-β, phosphatidylinositol 3-kinase, protein kinase B, bone tissue engineering

中图分类号:

左新慧,李君,韩祥祯,刘小元,何惠宇. 低氧诱导因子-1α对骨髓间充质干细胞成骨分化与血管生成相关因子的影响[J]. 口腔疾病防治, 2021, 29(7): 449-455.

ZUO Xinhui,LI Jun,HAN Xiangzhen,LIU Xiaoyuan,HE Huiyu. Effects of hypoxia inducible factor-1α on osteogenic differentiation and angiogenesis related factors of bone marrow mesenchymal stem cells[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2021, 29(7): 449-455.

图/表 6

表1 RT-PCR引物序列

Table 1 Synthetic primers used for RT-PCR

Primers	Sequence
GAPDH	F:5’-GGCACAGTCAAGGCTGAGAATG-3’ R:5’-ATGGTGGTGAAGACGCCAGTA-3’
HIF-1α	F:5’-CACCGCAACTGCCACCACTG-3’ R:5’-TGAGGCTGTCCGACTGTGAGTAC-3’
Runx2	F:5’-CCATAACGGTCTTCACAAATCCT-3’ R:5’-TCTGTCTGTGCCTTCTTGGTTC-3’
PDGF-BB	F:5’-TCTCTGCTGCTACCTGCGTCTG-3’ R:5’-AAGGAGCGGATGGAGTGGTCAC-3’
TGF-β	F:5’-GGCACCATCCATGACATGAACCG-3’ R:5’-GCCGTACACAGCAGTTCTTCTCTG-3’

图1 BMSCs的培养（× 50）

Figure 1 Cell culture of BMSCs（× 50） a: the primary cells were round; b: P3 cells, the cells adhered to the wall and grew in long fusiform or whirlpool shape;BMSCs： bone marrow mesenchymal stem cells

图2 质粒转染BMSCs 48 h后（× 100）

Figure 2 BMSC transfected with plasmid after 48 h （× 100） BMSCs that were successfully transfected with DNA plasmid showed green fluorescence expression, and the cell growth density was approximately 75%. The expression level of the low expression experimetal group was approximately 76%, and that of the control group was approximately 70%. The fluorescence level of the overexpression experimetal group was 73%, and that of the control group was approximately 68%. BMSCs： bone marrow mesenchymal stem cells

图3 各组BMSCs茜素红染色结果（×50）

Figure 3 BMSC alizarin red staining results for each group（×50） The area of calcium nodules in the low expression group was smaller than that in the control group and smaller than the area of nodules in the overexpression experimental group. The area of red calcium nodules in the overexpression experimental group was significantly larger than that in the control group, and the calcification area in each group increased significantly with increasing osteogenic induction time. BMSCs： bone marrow mesenchymal stem cells

表2 各组成骨分化与血管生成相关因子mRNA表达

Table 2 mRNA expression of osteogenic differentiation and angiogenesis related factors $\bar{x}\pm s$

Gene names	Low expression experimental group	Low expression control group	t	P	Over expression experimental group	Over expression control group	t	P
HIF-1α	0.373 ± 0.020	1.023 ± 0.231	22.522	<0.001	1.563 ± 0.128	1.013 ± 0.171	0.896	<0.001
Runx2	0.618 ± 0.012	1.025 ± 0.229	32.776	<0.001	25.462 ± 0.406	1.023 ± 0.204	6.278	<0.001
PDGF-BB	0.097 ± 0.011	1.009 ± 0.155	16.531	<0.001	3.322 ± 0.111	1.009 ± 0.147	1.705	<0.001
TGF-β	0.447 ± 0.013	1.023 ± 0.222	9.203	<0.001	1.734 ± 0.016	1.006 ± 0.112	17.389	<0.001

图4 各组成骨分化与血管生成相关因子表达

Figure 4 Protein expression of osteogenic differentiation and angiogenesis related factors 1: low expression experimental group; 2: low expression control group; 3: over expression experimental group; 4: over expression control group; HIF-1α: hypoxia inducible factor-1α; Runx2: runt-related transcription factor 2; PDGF-BB: platelet derived growth factor-BB; TGF-β: transforming growth factor-β; ***P<0.001

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低氧诱导因子-1α对骨髓间充质干细胞成骨分化与血管生成相关因子的影响

Effects of hypoxia inducible factor-1α on osteogenic differentiation and angiogenesis related factors of bone marrow mesenchymal stem cells

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