低氧对人牙囊细胞生物学特性的影响

doi:10.7518/hxkq.2017.03.004

摘要/Abstract

摘要：

目的研究低氧对人牙囊细胞（hDFCs）生物学特性的影响。方法利用组织块酶消化法从年轻恒牙中分离培养hDFCs;采用免疫荧光技术检测细胞表面标志物,多向诱导实验检测细胞多向分化潜能;模拟体外低氧微环境,将细胞分为常氧组（20%O₂）和低氧组（2%O₂）,分别对两组细胞行Transwell小室试验检测低氧对细胞迁移的影响,采用CCK-8法检测低氧对细胞增殖的影响。通过实时定量聚合酶链反应（qRT-PCR）和Western blot分别从基因和蛋白水平检测hDFCs多能性相关标志物于不同氧体积分数下的表达;分别对两组细胞进行成骨诱导,qRT-PCR检测成骨相关基因,茜素红染色评估矿化结节的形成。结果 hDFCs具有较强的干细胞特征,具有成骨、成脂及成神经多向分化能力,符合间充质干细胞基本标准,能够满足牙组织工程构建对种子细胞的需求。低氧有利于hDFCs多能性的保持,同时促进了hDFCs的迁移和增殖。hDFCs于低氧中进行诱导时,其成骨分化能力得到增强。结论低氧微环境对维持hDFCs多能性,促进hDFCs增殖、迁移和分化有重要作用。

关键词: 低氧, 人牙囊细胞, 增殖, 迁移, 分化

Abstract:

Objective This study aimed to investigate the effects of hypoxia on the characteristics of human dental follicle cells (hDFCs). Methods The tissue explant collagenase method was used to isolate hDFCs from young permanent teeth. The immunofluorescence technique was used to detect cell surface markers, and the multi-differentiation potential was detected by multilineage differentiation induction assay. Then, the hypoxic microenvironment was physically mimicked, and the cells were divided into the normoxia group (20%O₂) and the hypoxia group (2%O₂). The effects of hypoxia on cell migration and proliferation were examined by Transwell chamber test and CCK-8 assay, respectively. The gene and protein expression levels of stemness-related markers at both oxygen concentrations were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. After osteogenic induction of both groups, qRT-PCR was performed to evaluate the osteogenesis-related gene, and alizarin red staining was used to assess the formation of mineralized nodules. Results With the multi-differentiation capacity of osteogenic cells, adipogenic cells, and nerves, hDFCs demonstrate strong stem cell characteristics and possess the criteria of mesenchymal stem cells, which can meet the requirements of seed cells in dental tissue engineering. Hypoxia was conducive to the maintenance of hDFC stemness. Hypoxia promoted the migration and proliferation of hDFCs. The hDFCs were induced to osteogenic differentiation under hypoxic conditions, thereby enhancing osteogenesis. Conclusion Hypoxic microenvi-ronment plays an important role in maintaining the stemness and promoting the proliferation, migration, and differentiation of hDFCs. Thus, this microenvironment could also serve several important functions in future clinical applications.

Key words: hypoxia, human dental follicle cells, proliferation, migration, differentiation

中图分类号:

R 780.2

梁熙, 陈国庆, 田卫东. 低氧对人牙囊细胞生物学特性的影响[J]. 华西口腔医学杂志, 2017, 35(3): 245-252.

Xi Liang, Guoqing Chen, Wei-dong Tian. Effect of hypoxia on the biological characteristics of human dental follicle cells[J]. West China Journal of Stomatology, 2017, 35(3): 245-252.

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基因	引物序列（5’—3’）
β-actin	F：AGCCTCGCCTTTGCCG
	R：CTCGTCGCCCACATAGGAAT
Nanog	F：TGGACACTGGCTGAATCCTTC
	R：CGCTGATTAGGCTCCAACCAT
Sox-2	F：CAGCTCGCAGACCTACATGAAC
	R：CTGGAGTGGGAGGAAGAGGTAAC
Oct-4	F：TCCCATGCATTCAAACTGAGG
	R：CCAAAAACCCTGGCACAAACT
Runx2	F：CTTTACTTACACCCCGCCAGTC
	R：AGAGATATGGAGTGCTGCTGGTC
OSX	F：TCAACAACTCTGGGCAAAGCA
	R：TTAGCATAGCCTGAGGTGGGTG
BSP	F：CGAACAAGGCATAAACGGCACCAG
	R：TTCTCCATTGTCTCCTCCGCTGCT
OPN	F：CAGTTGTCCCCACAGTAGACAC
	R：GTGATGTCCTCGTCTGTAGCATC