摘要：

目的: 探讨直流电刺激(direct current stimulation,DCS)对人口腔黏膜成纤维细胞(human oral mucosa fibroblast,hOMF)增殖的影响及相关机制。方法: 以不同强度(0、10、25、50、100 μA)、时长(0、5、10、30、60 min)、频率(0、1、2、3次/d)的DCS作用于hOMF,应用CCK-8法检测细胞增殖情况。通过酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测DCS组与对照组(接种的细胞贴壁后,随机设置DCS组及对照组)24、48 h细胞培养上清液中表皮细胞生长因子(epidermal growth factor,EGF)和血管内皮生长因子(vascular endothelial growth factor,VEGF)的浓度。通过蛋白质印迹法(Western blotting)检测经DCS后的hOMF中蛋白激酶B(protein kinase B,Akt)及磷酸化蛋白激酶B(phosphorylated protein kinase B,p-Akt)、哺乳动物雷帕霉素靶蛋白(mammalian rapamycin target protein,mTOR)及磷酸化哺乳动物雷帕霉素靶蛋白(phosphorylated mammalian rapamycin target protein,p-mTOR)、磷酸酶和张力蛋白同源物(phosphatase and tensin homology deleted on chromosome ten,PTEN)的表达水平。结果: 通过检测DCS强度梯度、时间梯度、频率梯度的吸光度值可知, 25 μA的电流强度(P<0.01)、10 min的刺激时长(P<0.001)及2次/d的刺激频率(P<0.01)为hOMF的显著增殖刺激条件。相较于对照组,DCS组24 h和48 h细胞培养上清液中VEGF的浓度均有所升高,24 h培养上清液中的VEGF浓度升高更为明显(P<0.01)。在不同时长的DCS下,刺激5、10 min后的p-mTOR和p-Akt的蛋白表达升高(P<0.05),PTEN的蛋白表达降低(P<0.001)。结论: DCS可促进hOMF的增殖,诱导细胞因子VEGF的高表达,可能与PTEN/Akt/mTOR信号通路的激活有关。

关键词: 直流电刺激, 人口腔黏膜成纤维细胞, PTEN/Akt/mTOR信号通路

Abstract:

Objective: To investigate the effect of direct current stimulation(DCS) on the proliferation of human oral mucosal fibroblasts (hOMF) and its related mechanism. Methods: DCS with different intensity (0,10,25,50,100 μA), duration (0, 5, 10, 30, 60 min), and frequency (0, 1, 2, 3 time/day) was acted on hOMF. CCK-8 was used to detect cell proliferation. The concentration of vascular endothelial growth factor (VEGF) in the 24 h and 48 h cell culture supernatant of DCS group and control group (after the cells adhere to the wall, DCS group and control group were randomly set) was detected by enzyme-linked immunosorbent assay (ELISA). The expression levels of protein kinase B(Akt), phosphorylated protein kinase B (p-Akt), mammalian rapamycin target protein(mTOR), phosphorylated mammalian rapamycin target protein (p-mTOR), phosphatase and tensin homology deleted on chromosome ten (PTEN) in hOMF after DCS were detected by Western blotting. Results: By detecting the optical density (OD) values of DCS intensity gradient, time gradient and frequency gradient, 25 μA current intensity (P<0.01), 10 min stimulation duration (P<0.001) and 2 times/day stimulation frequency (P<0.01) were the stimulation conditions for significant prolife ration of hOMF. Compared with the control group, the concentration of VEGF in 24 hand 48 h cell culture supernatant in DCS groups increased, and the concentration of VEGF in 24 h culture supernatant increased more significantly (P<0.01). Under different durations of DCS, the protein expression of p-mTOR and p-Akt increased significantly at 5 min and 10 min(P<0.05), and the protein expression of PTEN decreased significantly (P<0.001). Conclusion: DCS can promote the proliferation of hOMF and induce the high expression of cytokine VEGF, which may be related to the activation of PTEN/Akt/mTOR signal pathway.

Key words: direct current stimulation, human oral mucosa fibroblast, PTEN/Akt/mTOR signal pathway

中图分类号: 

• R782