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

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。

Abstract:

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 ob－serve 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 dif－ferent 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 mi－gration 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 mi－grated to the nucleus. Conclusion：Hypoxia can inhibit the pro－liferation and migration of hPDLCs and change their morpholo－gy. Under the hypoxic condition，HIF-1 gradually migrates from the cytoplasm to the nucleus，and there are increases in the ex－pression of HIF-1 and IGF-1，suggesting that hypoxia has cer－tain influence on the bioactivity of hPDLCs.