缺氧对人牙周膜细胞增殖、迁移能力及IGF-1表达的影响  

Influence of hypoxia on proliferation,migration,and insulin-like growth factor-1 expression of human periodontal ligament cells

作　　者：谢佳颖 尹东青[1] Xie Jiaying;Yin Dongqing(Department of Prosth odontics ,the Affiliated Stomatological Hospital of Chongqing Medical University,Key Laboratory of Oral Diseases and Biomedical Chongqing)

机构地区：[1]重庆医科大学附属口腔医院修复科、口腔疾病与生物医学重庆市重点实验室

出　　处：《重庆医科大学学报》2019年第5期561-567,共7页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:81700982);重庆市卫生局高端人才资助项目(编号:2015HBRC010);重庆市医学科研计划资助项目(编号:2013-2-071);重庆市渝北区科技计划资助项目(编号:2016(社)18号)

摘　　要：目的:探讨缺氧对人牙周膜细胞(human periodontal ligament cells,hPDLCs)生物学活性的影响。方法:选择第 5 代hPDLCs,在严重缺氧(1% O2)、轻度缺氧(5% O2)和常氧(21% O2)3 种环境下培养,用 MTT 法评价细胞增殖率,用倒置显微镜观察细胞形态变化,细胞划痕试验评价细胞迁移能力,用免疫荧光染色染色研究细胞中低氧诱导因子-1(hypoxia inducible factor-1,HIF-1)的表达及分布情况,用 RT-PCR 及 Western blot 检测 HIF 和 IGF-1 在不同氧浓度环境下的 mRNA 及蛋白表达水平。结果:①与正常 hPDLCs 相比,在 1% O2 浓度下,hPDLCs 的数量在一个显微镜视野下明显减少,排列稀疏,结构发生改变。②hPDLCs 的增长速率随着氧浓度的下降而减少,氧气浓度越低,增殖速率越慢。③在划痕处理后 72 h,在 3 组不同氧浓度下,hPDLCs 均向划痕区域迁移,但严重缺氧组的移动速率明显小于轻度缺氧和常氧组(划痕 72 h:1% O2 组 0.917±0.023,5% O2 组0.742±0.046,21% O2 组 0.453±0.039,F=530.237,P=0.002)。④HIF/DAPI 染色结果显示在常氧条件下,HIF-1 主要表达在细胞质中,随着氧气浓度降低,HIF 逐渐转移至细胞核内。结论:缺氧能够抑制人牙周膜细胞的增殖和迁移能力,改变 hPDLCs 的形态。在缺氧条件下,HIF 从细胞质转移到细胞核中,HIF 和 IGF-1 的表达增加,提示缺氧微环境对人牙周膜细胞的生物学活性有一定影响。Objective:To investigate the influence of hypoxia on the bioactivities of human periodontal ligament fibroblasts (hPDLCs). Methods:The fifth-generation hPDLCs were selected and cultured under the condition of severe hypoxia (1% O2),mild hypoxia(5% O2),or normoxia(21% O2). MTT assay was used to measure the proliferation rate of hPDLCs. An inverted microscope was used to observe cell morphological changes. Wound healing assay was used to observe the migration ability of hPDLCs. Immunofluorescence staining was used to measure the expression and distribution of hypoxia inducible factor-1 (HIF-1) in hPDLCs,and RT-PCR and Western blot were used to measure the mRNA and protein expression of HIF-1 and insulin-like growth factor-1 (IGF-1) under different levels of O2. Results:Compared with normal hPDLCs,the hPDLCs cultured in 1% O2 had a significant reduction in cell number in a microscopic field,with sparse arrangement and a change in structure. The growth rate of hPDLCs decreased with the reduction in oxygen concentration;the lower the oxygen concentration,the slower the proliferation rate. At 72 hours after scratch in the wound healing assay,hPDLCs migrated to the scratched area in all three groups,but the severe hypoxia group had a significantly lower migration rate than the mild hypoxia group and the normoxia group(0.917±0.023 vs. 0.742±0.046/0.453±0.039,F=530.237,P=0.002). HIF/DAPI staining showed that under the condition of normal oxygen,HIF-1 was mainly expressed in the cytoplasm,and with the gradual reduction in oxygen concentration,HIF-1 gradually migrated to the nucleus. Conclusion:Hypoxia can inhibit the proliferation and migration of hPDLCs and change their morphology. Under the hypoxic condition,HIF-1 gradually migrates from the cytoplasm to the nucleus,and there are increases in the expression of HIF-1 and IGF-1,suggesting that hypoxia has certain influence on the bioactivity of hPDLCs.

关 键 词：缺氧 人牙周膜细胞 细胞增殖 细胞迁移 低氧诱导因子-1 

分 类 号：R783.6[医药卫生—口腔医学] R783.3[医药卫生—临床医学]