Objective: To explore the effects of hypoxia inducible factor-1α (HIF-1α) on P311 and its influence on the migration of murine epidermal stem cells (ESCs) under hypoxia in vitro. Methods: Two kinds of murine ESCs were isolated and obtained from 15 neonatal wild-type C57BL/6J mice and 5 congeneric source P311 gene knock-out mice, respectively. The first passage of cells were used in the following experiments after morphologic observation and detection of expression of cell surface markers CD71 and CD49f with flow cytometer. (1) After cell scratch assay, according to the random number table (the same dividing method below), ESCs of P311 gene knock-out mice were divided into normoxia group (cells were cultured with complete medium in normoxic carbon dioxide incubator, and the subsequent normoxic treatments were the same) and hypoxia group (cells were cultured in hypoxic carbon dioxide incubator containing 1% oxygen, and the subsequent hypoxic treatments were the same), with 12 inserts in each group. ESCs of wild-type mice were divided into normoxia group, pure hypoxia group, dimethyl sulfoxide (DMSO) control group (2 μL DMSO solvent was added for 1 h of normoxia treatment before hypoxia treatment), HIF-1α inhibitor group (cells were treated with 11 μmol/L HIF-1 inhibitor of 2 μL under normoxia condition for 1 h before hypoxia treatment), HIF-1α stabilizer group (the cells were treated with 2 μmol/L FG-4592 of 2 μL under normoxia condition for 1 h before hypoxia treatment), with 12 inserts in each group. Three inserts of each time point in each group were adopted respectively to measure the residual width of scratch under inverted phase contrast microscope at post scratch hour (PSH) 0 (immediately), 12, 24, and 48. (2) After hypoxia treatment, the protein level of HIF-1α in ESCs of wild-type mice was detected by Western blotting at post hypoxia hour (PHH) 0, 12, 24, and 48. (3) ESCs of wild-type mice were divided into pure hypoxia group, DMSO control group, HIF-1α inhibitor group, and HIF-1α stabilizer group as that of experiment (1) with the same treatment. The mRNA expression of P311 and expression of P311 in ESCs were determined by real-time fluorescent quantitative reverse transcription polymerase chain reaction and immunocytochemical staining, respectively, at PHH 0 (immediately), 12, 24, and 48 (with sample numbers of 12). (4) The second passage of human embryonic kidney 293 (HEK-293) cells were divided into empty vector hypoxia group (cells were cultured under hypoxia condition after being transfected with empty vector plasmid), P311 normoxia group (cells were cultured under normoxia condition after being transfected with P311 reporter gene plasmid), P311 hypoxia group (cells were cultured under hypoxia condition after being transfected with P311 reporter gene plasmid), P311 hypoxia+ HIF-1α inhibitor group (cells which were incubated with HIF-1α inhibitor were cultured under hypoxia condition after being transfected with P311 reporter gene plasmid). The luciferase activity was detected at post culture hour (PCH) 0 and 12, respectively, and then the P311 transcriptional regulatory binding site of HIF-1α and the promoter sequence of P311 were predicted and searched by bioinformatics methods. Data were processed with factorial design variance analysis, one-way analysis of variance, LSD test and Bonferroni correction. Results: (1) The results of ESCs. The cells showed cobblestone-like pattern and different clonal morphology due to the different cell proliferation potential. The proportion of CD71(-)CD49f(+) cells accounted for about 85%. The identification results indicated that the cells showed strong stem cell properties and high purity. Compared with those in cells of normoxia group of P311 gene knock-out mice, the residual widths of scratch of cells in pure hypoxia group were smaller at PSH 12 and 24 (with P values below 0.05), and those in hypoxia group, normoxia group of wild-type mice, DMSO control group, HIF-1α inhibitor group, and HIF-1α stabilizer group were smaller at PSH 12 (with P values below 0.05). Compared with those in cells of normoxia group of wild-type mice, the residual widths of scratch of cells in hypoxia group, pure hypoxia group, and DMSO control group were smaller at PSH 12 and 24 (with P values below 0.05), and the residual width of scratch of cells in HIF-1α stabilizer group was smaller at PSH 12 (P<0.05). Compared with those of cells in pure hypoxia group, the residual widths of scratch of cells in hypoxia group were wider at PSH 12 and 24 (with P values below 0.05), and the residual width of scratch of cells in HIF-1α inhibitor group was wider at PSH 12 (P<0.05), and those of cells in HIF-1α stabilizer group were smaller at PSH 12 and 24 (with P values below 0.05). There was no obvious difference in the width of scratch in cells among the 7 groups (F=19.02, P>0.05). The protein levels of HIF-1α in ESCs of wild-type mice at PHH 0, 12, 24, and 48 were respectively 1.02±0.05, 2.56±0.09, 1.60±0.17, and 1.17±0.03. Compared with that at PHH 0, the protein level of HIF-1α at PHH 12 was significantly enhanced (P<0.01). It began to decline at PHH 24 but was still higher than that at PHH 0 (P<0.05), and the protein level of HIF-1α at PHH 48 was close to the normoxia level (P>0.05). Compared with those of cells in pure hypoxia group, the mRNA expressions of P311 of cells in HIF-1α inhibitor group were significantly decreased at each time point (with P values below 0.05), and those in HIF-1α stabilizer group were significantly increased at PHH 12 and 24 (with P values below 0.05). Compared with those of cells in HIF-1α inhibitor group, the mRNA expressions of P311 of cells in DMSO control group and HIF-1α stabilizer group were significantly increased at PHH 0, 12, and 24 (with P values below 0.05). Compared with those of cells in pure hypoxia group, the expressions of P311 of cells in HIF-1α inhibitor group were significantly decreased at each time point (with P values below 0.05), while those in HIF-1α stabilizer group were significantly increased at PHH 12 and 24 (with P values below 0.05). Compared with those of cells in HIF-1α inhibitor group, the expressions of P311 of cells in DMSO control group and HIF-1α stabilizer group were significantly increased at PHH 12 and 24 (with P values below 0.05). (2) The results of HEK-293 cells. At PCH 0, there was no significant difference in the luciferase activity among cells of empty vector hypoxia group, P311 normoxia group, P311 hypoxia group, and P311 hypoxia+ HIF-1α inhibitor group (F=13.33, P>0.05). At PCH 12, the luciferase activity of cells in P311 hypoxia group was higher than that in empty vector hypoxia group (P<0.01). The luciferase activity of cells in hypoxia group was higher than that in P311 normoxia group (P<0.05), while that of cells in P311 hypoxia+ HIF-1α inhibitor group was lower than that in P311 hypoxia group (P<0.01). Conclusions: HIF-1α may increase the migration of murine ESCs through inducing the expression of P311 at the early stage of hypoxia.
目的： 体外条件下模拟缺氧环境，探讨P311和缺氧诱导因子1α(HIF-1α)相互作用及对小鼠表皮干细胞(ESC)迁移影响。 方法： 取15只C57BL/6J野生型新生小鼠，5只同属来源P311基因敲除新生小鼠，分离培养2种小鼠ESC，取第1代细胞行形态学观察，采用流式细胞仪检测细胞表面标志物CD71、CD49f表达后行以下实验。(1)取2种小鼠ESC划痕处理后，立即按随机数字表法(下同)将P311基因敲除小鼠ESC分为常氧组(将细胞加入完全培养基置于常氧二氧化碳培养箱培养，常氧处理下同)和缺氧组(将细胞置于含体积分数1%氧气的缺氧二氧化碳培养箱培养，缺氧处理下同)，每组12个插件；将野生型小鼠ESC分为常氧组、单纯缺氧组、二甲基亚砜(DMSO)对照组(用2 μL DMSO溶剂常氧处理1 h后行缺氧培养)、HIF-1α抑制剂组(加入11 μmol/L HIF-1抑制剂2 μL常氧培养1 h后行缺氧培养)、HIF-1α稳定剂组(加入2 μmol/L FG-4592 2 μL常氧处理1 h后行缺氧培养)，每组12个插件。每组各时相点取3个插件分别于划痕后0(即刻)、12 、24、48 h在100倍倒置相差显微镜下测量划痕剩余宽度。(2)取野生型小鼠ESC行缺氧培养，蛋白质印迹法检测缺氧0(即刻)、12、24、48 h HIF-1α蛋白表达。(3)取野生型小鼠ESC同实验(1)分为单纯缺氧组、DMSO对照组、HIF-1α抑制剂组、HIF-1α稳定剂组并行相应处理，分别在缺氧0、12、24、48 h采用实时荧光定量RT-PCR法检测P311 mRNA表达，免疫细胞化学染色法观测P311表达(样本数均为12)。(4)取第2代人胚胎肾293(HEK-293)细胞分为空载缺氧组(转染空载质粒后进行缺氧培养)、P311常氧组(转染P311报告基因质粒后进行常氧培养)、P311缺氧组(转染P311报告基因质粒后进行缺氧培养)、P311缺氧＋HIF-1α抑制剂组(HIF-1抑制剂处理后转染P311报告基因质粒然后进行缺氧培养)，分别于培养0、12 h检测荧光素酶活性，采用生物信息学方法预测HIF-1α对P311转录调节的结合位点并查找P311启动子序列。对数据行析因设计方差分析、单因素方差分析、LSD检验以及Bonferroni校正。 结果： (1)ESC结果。细胞呈现铺路石样改变，细胞由于增殖潜能不同而呈现不同克隆状态。CD71(－)CD49f(＋)细胞占85%左右，说明ESC的干性较强、纯度较高。与P311基因敲除小鼠常氧组比较，单纯缺氧组细胞划痕后12、24 h划痕剩余宽度较小(P值均小于0.05)，缺氧组、野生型小鼠常氧组、DMSO对照组、HIF-1α抑制组、HIF-1α稳定剂组细胞划痕后12 h划痕剩余宽度较小(P值均小于0.05)。与野生型小鼠常氧组比较，缺氧组、单纯缺氧组、DMSO对照组细胞划痕后12、24 h划痕剩余宽度较小(P值均小于0.05)，HIF-1α稳定剂组细胞划痕后12 h划痕剩余宽度较小(P<0.05)。与单纯缺氧组比较，缺氧组细胞划痕后12、24 h划痕剩余宽度较大(P值均小于0.05)，HIF-1α抑制剂组细胞划痕后12 h划痕剩余宽度较大(P<0.05)，HIF-1α稳定剂组细胞划痕后12、24 h划痕剩余宽度较小(P值均小于0.05)。7组小鼠细胞间划痕后48 h划痕剩余宽度总体比较差异不明显(F＝19.02，P>0.05)。野生型小鼠ESC缺氧0、12、24、48 h的HIF-1α蛋白表达量分别为1.02±0.05、2.56±0.09、1.60±0.17、1.17±0.03。与缺氧0 h比较，HIF-1α蛋白表达量缺氧12 h明显增加(P<0.01)，缺氧24 h开始下降但仍高于缺氧0 h(P<0.05)，缺氧48 h后降至常氧水平(P>0.05)。与单纯缺氧组比较，HIF-1α抑制剂组细胞缺氧各时相点P311 mRNA表达量均明显下降(P值均小于0.05)，HIF-1α稳定剂组细胞P311 mRNA表达量则在缺氧12、24 h明显升高(P值均小于0.05)。与HIF-1α抑制剂组比较，DMSO对照组和HIF-1α稳定剂组细胞在缺氧0、12、24 h P311 mRNA表达量均明显升高(P值均小于0.05)。与单纯缺氧组比较，HIF-1α抑制剂组细胞缺氧各时相点P311表达量均明显下降(P值均小于0.05)，而HIF-1α稳定剂组细胞P311表达量则在缺氧12、24 h明显升高(P值均小于0.05)。与HIF-1α抑制剂组比较，DMSO对照组和HIF-1α稳定剂组细胞P311表达量在缺氧12、24 h明显升高(P值均小于0.05)。(2)HEK-293细胞结果。培养0 h，空载缺氧组、P311常氧组、P311缺氧组、P311缺氧＋HIF-1α抑制剂组细胞荧光素酶活性差异无统计学意义(F＝13.33，P>0.05)。培养12 h，P311缺氧组细胞荧光素酶活性较空载缺氧组高(P<0.01)；P311缺氧组细胞荧光素酶活性较P311常氧组高(P<0.05)；P311缺氧＋HIF-1α抑制剂组细胞荧光素酶活性较P311缺氧组低(P<0.01)。 结论： 缺氧早期，HIF-1α可能通过诱导P311表达促进小鼠ESC迁移。.
Keywords: Cell migration assays; Epidermal stem cells; Hypoxia-inducible factor 1; P311; Wound healing.