Influence of pH value on tube formation of human dermal microvascular endothelial cells and its molecular mechanism
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摘要:
目的 探讨pH值对人真皮微血管内皮细胞（HDMEC）成管的影响并研究其分子机制，为促进创面愈合过程中血管新生的研究提供理论依据。 方法 采用实验研究方法。取第4、5代对数生长期HDMEC进行实验，制备pH值分别为6.4、6.6、6.8、7.0、7.2、7.4、7.6、7.8的培养液，用其适应性培养细胞（培养方式下同）24 h后进行后续实验。另培养36 h，采用流式细胞仪测定胞质pH值的相对荧光值并对胞质pH值的相对荧光值与培养液pH值进行相关分析。另培养1.5、2.5、3.5、4.5、5.5 d，采用细胞计数试剂盒8检测细胞增殖活性。采用Oris TM细胞迁移检测试剂盒检测去掉播种塞后0（即刻）、24、48 h细胞迁移剩余面积。进行三维基质胶细胞成管实验，检测另培养48 h细胞成管的管腔直径。另培养48 h，采用蛋白质印迹法检测细胞中蛋白激酶B（Akt）磷酸化位点473、308的蛋白表达。样本数均为3。对数据行Pearson相关性分析、单因素方差分析、析因设计方差分析、重复测量方差分析与Bonferroni校正。 结果 另培养36 h，与pH值6.4培养液相比，pH值6.8~7.8培养液培养细胞胞质pH值的相对荧光值均显著升高（ P<0.05）；与pH值6.6~7.0培养液相比，pH值7.4~7.8培养液培养细胞胞质pH值的相对荧光值均显著升高（ P<0.05），且pH值6.6培养液培养细胞胞质pH值的相对荧光值明显低于pH值7.0、7.2培养液（ P值均<0.05）；pH值7.6、7.8培养液培养细胞胞质pH值的相对荧光值均显著高于pH值7.2、7.4培养液（ P<0.05）。胞质pH值的相对荧光值与培养液pH值呈显著正相关（ r=0.99， P<0.05）。8种pH值培养液另培养1.5 d细胞增殖活性相近（ P>0.05）。另培养2.5 d，与pH值7.6培养液相比，pH值6.4~6.8培养液培养细胞增殖活性均显著下降（ P<0.05）。另培养3.5 d，与pH值6.4~6.8培养液相比，pH值7.0~7.8培养液培养细胞增殖活性均显著升高（ P<0.05）；与pH值7.6培养液相比，pH值7.0~7.4、7.8培养液培养细胞增殖活性均显著下降（ P<0.05）。另培养4.5、5.5 d，与pH值6.4培养液相比，pH值6.8~7.8培养液培养细胞增殖活性均显著升高（ P<0.05）；与pH值6.6、6.8培养液相比，pH值7.0~7.8培养液培养细胞增殖活性均显著升高（ P<0.05）。另培养4.5 d，pH值7.6培养液培养细胞增殖活性显著高于pH值7.0培养液（ P<0.05）。另培养5.5 d，与pH值7.0培养液相比，pH值7.2~7.6培养液培养细胞增殖活性均显著升高（ P<0.05）；与pH值7.2、7.4培养液相比，pH值7.6培养液培养细胞增殖活性显著升高（ P值均<0.05），pH值7.8培养液培养细胞增殖活性显著降低（ P值均<0.05）。去掉播种塞后0 h，8种pH值培养液培养细胞迁移剩余面积相近（ P>0.05）。去掉播种塞后24 h，与pH值6.4培养液相比，pH值6.6~7.8培养液培养细胞迁移剩余面积均显著缩小（ P<0.05）；与pH值6.6、6.8培养液相比，pH值7.0~7.6培养液培养细胞迁移剩余面积均显著缩小（ P<0.05）。去掉播种塞后48 h，与pH值6.4、6.6培养液相比，pH值7.0~7.8培养液培养细胞迁移剩余面积均显著缩小（ P<0.05）；pH值7.2、7.4培养液培养细胞迁移剩余面积均显著小于pH值6.8、7.0、7.8培养液（ P<0.05），均显著大于pH值7.6培养液（ P<0.05）；pH值7.6培养液培养细胞迁移剩余面积显著小于pH值6.8、7.8培养液（ P值均<0.05）。另培养48 h，pH值7.0、7.2、7.4、7.6、7.8培养液培养细胞成管的管腔直径分别为（5.0±0.5）、（7.6±0.9）、（8.5±0.7）、（11.0±0.8）、（5.3±0.8）μm，均显著长于pH值6.4培养液的（2.8±0.8）μm（ P<0.05）；pH值6.6［（4.2±0.3）μm］、6.8［（4.5±0.6）μm］、7.0、7.8培养液培养细胞成管的管腔直径均显著短于pH值7.6培养液（ P<0.05）。另培养48 h，与pH值6.4、6.6培养液相比，pH值6.8~7.8培养液培养细胞中Akt磷酸化位点473、308蛋白表达均明显升高（ P<0.05），且pH值6.6培养液培养细胞中Akt磷酸化位点308蛋白表达明显高于pH值6.4培养液（ P<0.05）；与pH值6.8培养液相比，pH值7.0、7.4~7.8培养液培养细胞中Akt磷酸化位点473蛋白表达均明显升高（ P<0.05）；与pH值7.6培养液相比，pH值7.0~7.4、7.8培养液培养细胞中Akt磷酸化位点473蛋白表达均明显降低（ P<0.05）；与pH值7.8培养液相比，pH值7.0~7.6培养液培养细胞中Akt磷酸化位点308蛋白表达均明显升高（ P<0.05）。 结论 pH值可调控HDMEC形成毛细血管的管腔直径，该调控作用与Akt的激活密切相关；7.2~7.6为构建组织工程毛细血管的适宜pH值。 Abstract:Objective To explore the influence of pH value on tube formation of human dermal microvascular endothelial cells (HDMECs) and study its molecular mechanism, so as to provide theoretical basis for the study of promoting angiogenesis in the process of wound healing. Methods The experimental study methods were applied. HDMECs of 4 or 5 passages in the logarithmic growth phase were collected for experiments. Culture mediums with pH values of 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, and 7.8 were prepared, and the cells were adaptively cultured (the same culture method below) for 24 h before further experiments being carried out. After another 36 h of culture, the relative fluorescence value of cytoplasmic pH value was measured by flow cytometry, and the correlation analysis between the relative fluorescence value of cytoplasmic pH value and the medium pH value was carried out. After another 1.5, 2.5, 3.5, 4.5, and 5.5 days of culture, the cell proliferation activity was detected with cell counting kit 8. Oris TM cell migration detection kit was used to detect the remaining area of cell migration at 0 (immediately), 24, and 48 h after removing the cell seeding stopper. Three-dimensional stromal gel cell tube formation experiment was carried out to detect the lumen diameter of tube formed by cells after another 48 h of culture. The protein expressions of phosphorylation sites 473 and 308 of protein kinase B (Akt) were detected by Western blotting after another 48 h of culture. The sample number was 3. Data were statistically analyzed with Pearson correlation analysis, one-way analysis of variance, analysis of variance for factorial design, analysis of variance for repeated measurement, and Bonferroni correction. Results After another 36 h of culture, the relative fluorescence values of cytoplasmic pH value of cells cultured in pH 6.8-7.8 mediums were significantly higher than the level in pH 6.4 medium ( P<0.05); compared with those in pH 6.6-7.0 mediums, the relative fluorescence values of cytoplasmic pH value of cells cultured in pH 7.4-7.8 mediums were significantly increased ( P<0.05), and the relative fluorescence value of cytoplasmic pH value of cells cultured in pH 6.6 medium was significantly lower than that in pH 7.0 or 7.2 mediun (with P values all <0.05); the relative fluorescence values of cytoplasmic pH value of cells cultured in pH 7.6 and 7.8 mediums were significantly higher than those in pH 7.2 and 7.4 mediums ( P<0.05). The relative fluorescence value of cytoplasmic pH value was significantly positively correlated with the medium pH value ( r=0.99, P<0.05). The proliferation activity was similar among cells cultured in 8 mediums of different pH values for another 1.5 days ( P>0.05). After another 2.5 days of culture, the proliferation activity of cells cultured in pH 6.4-6.8 mediums was significantly decreased compared with that in pH 7.6 medium ( P<0.05). After another 3.5 days of culture, the proliferation activity of cells cultured in pH 7.0-7.8 mediums was significantly higher than that in pH 6.4-6.8 mediums ( P<0.05); compared with that in pH 7.6 medium, the proliferation activity of cells cultured in pH 7.0-7.4 and 7.8 mediums was significantly decreased ( P<0.05). After another 4.5 or 5.5 days of culture, the proliferation activity of cells cultured in pH 6.8-7.8 mediums was significantly higher than that in pH 6.4 medium ( P<0.05); compared with that in pH 6.6 and 6.8 mediums, the proliferation activity of cells cultured in pH 7.0-7.8 mediums was significantly increased ( P<0.05). After another 4.5 days of culture, the proliferation activity of cells cultured in pH 7.6 medium was significantly higher than that in pH 7.0 medium ( P<0.05). After another 5.5 days of culture, the proliferation activity of cells cultured in pH 7.2-7.6 mediums was significantly increased compared with that in pH 7.0 medium ( P<0.05); the proliferation activity of cells cultured in pH 7.2 and 7.4 mediums was significantly lower than that in pH 7.6 medium (with P values all <0.05) but significantly higher than that in pH 7.6 medium (with P values all <0.05). Immediately after removing the cell seeding stopper, the remaining migration areas were similar among cells cultured in 8 mediums of different pH values ( P>0.05). At 24 h after removing the cell seeding stopper, the remaining migration areas of cells cultured in pH 6.6-7.8 mediums were significantly smaller than the area in pH 6.4 medium ( P<0.05); compared with those in pH 6.6 and 6.8 mediums, the remaining migration areas of cells cultured in pH 7.0 to 7.6 mediums were significantly reduced ( P<0.05). At 48 h after removing the cell seeding stopper, compared with those in pH 6.4 and 6.6 mediums, the remaining migration areas of cells cultured in pH 7.0-7.8 mediums were significantly reduced ( P<0.05); the remaining migration areas of cells cultured in pH 7.2 and 7.4 mediums were significantly smaller than those in pH 6.8, 7.0, and 7.8 mediums ( P<0.05) but significantly larger than the area in pH 7.6 medium ( P<0.05); the remaining migration area of cells cultured in pH 7.6 medium was significantly smaller than that in pH 6.8 or 7.8 medium (with P values all <0.05). After another 48 h of culture, the lumen diameters of tubes formed by cells cultured in pH 7.0, 7.2, 7.4, 7.6, and 7.8 mediums were (5.0±0.5), (7.6±0.9), (8.5±0.7), (11.0±0.8), and (5.3±0.8) μm, respectively, which were significantly longer than (2.8±0.8) μm in pH 6.4 medium ( P<0.05); the lumen diameters of tubes formed by cells cultured in pH 6.6 ((4.2±0.3) μm), 6.8 ((4.5±0.6) μm), 7.0, and 7.8 mediums were significantly shorter than the diameter in pH 7.6 medium ( P<0.05). After another 48 h of culture, compared with those in pH 6.4 and 6.6 mediums, the protein expressions of Akt phosphorylation sites 473 and 308 of cells cultured in pH 6.8 to 7.8 mediums were significantly increased ( P<0.05). Moreover, the protein expression of Akt phosphorylation site 308 of cells cultured in pH 6.6 medium was significantly higher than that in pH 6.4 medium ( P<0.05); compared with the expression in pH 6.8 medium, the protein expressions of Akt phosphorylation site 473 of cells cultured in pH 7.0 and 7.4-7.8 mediums were significantly increased ( P<0.05); compared with the expression in pH 7.6 medium, the protein expressions of Akt phosphorylation site 473 of cells cultured in pH 7.0-7.4 and 7.8 mediums were significantly decreased ( P<0.05); compared with the expression in pH 7.8 medium, the protein expressions of Akt phosphorylation site 308 of cells cultured in pH 7.0 to 7.6 mediums were significantly increased ( P<0.05). Conclusions pH value can regulate the lumen diameter of HDMEC-formed capillaries, which is closely related to the activation of Akt. 7.2-7.6 is the appropriate pH value for constructing tissue engineered capillaries. -
参考文献
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1 8种pH值培养液另培养36 h人真皮微血管内皮细胞胞质pH值的相对荧光值比较（样本数为3，
注：先于相应pH值培养液中进行24 h适应性培养，再进行后续培养检测；与pH值6.4培养液相比，aP<0.05；与pH值6.6培养液相比，bP<0.05；与pH值6.8培养液相比，cP<0.05；与pH值7.0培养液相比，dP<0.05；与pH值7.2培养液相比，eP<0.05；与pH值7.4培养液相比，fP<0.05
2 8种pH值培养液培养人真皮微血管内皮细胞去掉播种塞后各时间点细胞迁移剩余面积（黄色虚线标记区域） 5-氯甲基荧光素二乙酸酯×10。2A、2B、2C、2D、2E、2F、2G、2H与2I、2J、2K、2L、2M、2N、2O、2P和2Q、2R、2S、2T、2U、2V、2W、2X.分别为pH值6.4、6.6、6.8、7.0、7.2、7.4、7.6、7.8培养液培养细胞去掉播种塞后0（即刻）、24、48 h，图2A~2H细胞迁移剩余面积相近，图2M、2N、2O细胞迁移剩余面积明显较图2I~2L及2P小，图2U、2V、2W细胞迁移剩余面积明显较图2Q~2T及2X小，其中图2W细胞迁移剩余面积最小
注：先于相应pH值培养液中进行24 h适应性培养，再进行后续培养检测
3 8种pH值培养液另培养48 h人真皮微血管内皮细胞在Matrigel基质胶内成管（黄色箭头指示）情况 倒置相差显微镜×100。3A、3B、3C、3D、3E、3F、3G、3H.分别为pH值6.4、6.6、6.8、7.0、7.2、7.4、7.6、7.8培养液培养细胞，图3A、3B、3C、3D管腔直径较短，图3E、3F、3G、3H管腔直径较长，其中图3G管腔直径最长
注：先于相应pH值培养液中进行24 h适应性培养，再进行后续培养检测
4 8种pH值培养液另培养48 h人真皮微血管内皮细胞成管的管腔直径比较（样本数为3，
注：先于相应pH值培养液中进行24 h适应性培养，再进行后续培养检测；与pH值6.4培养液相比，aP<0.05；与pH值6.6培养液相比，bP<0.05；与pH值6.8培养液相比，cP<0.05；与pH值7.0培养液相比，dP<0.05；与pH值7.6培养液相比，eP<0.05
5 蛋白质印迹法检测的8种pH值培养液另培养48 h人真皮微血管内皮细胞Akt磷酸化位点蛋白表达
注：先于相应pH值培养液中进行24 h适应性培养，再进行后续培养检测；Akt为蛋白激酶B，p-Akt-473、p-Akt-308分别为Akt磷酸化位点473、308，GAPDH为3-磷酸甘油醛脱氢酶；条带上方1、2、3、4、5、6、7、8分别指示pH值6.4、6.6、6.8、7.0、7.2、7.4、7.6、7.8培养液
表1 8种pH值培养液另培养各时间点人真皮微血管内皮细胞增殖活性比较（
pH值 样本数 1.5 d 2.5 d 3.5 d 4.5 d 5.5 d 6.4 3 0.489±0.006 0.506±0.016 0.528±0.021 0.542±0.012 0.561±0.016 6.6 3 0.492±0.008 0.510±0.018 0.541±0.019 0.568±0.012 0.573±0.014 6.8 3 0.497±0.005 0.518±0.014 0.557±0.012 0.583±0.014 a 0.627±0.020 a 7.0 3 0.489±0.004 0.527±0.016 0.601±0.014 abc 0.652±0.016 abc 0.678±0.019 abc 7.2 3 0.495±0.004 0.543±0.020 0.638±0.012 abc 0.692±0.018 abc 0.768±0.014 abcd 7.4 3 0.496±0.008 0.556±0.016 0.655±0.016 abc 0.709±0.010 abc 0.811±0.013 abcd 7.6 3 0.486±0.003 0.562±0.014 abc 0.682±0.016 abcdef 0.725±0.019 abcd 0.832±0.016 abcdef 7.8 3 0.496±0.009 0.532±0.018 0.626±0.016 abcg 0.683±0.014 abc 0.707±0.020 abcef 注：先于相应pH值培养液中适应性培养24 h，再进行后续培养检测；处理因素主效应， F=152.35， P<0.001；时间因素主效应， F=683.95， P<0.001；两者交互作用， F=21.82， P<0.001；与pH值6.4培养液相比， a P<0.05；与pH值6.6培养液相比， b P<0.05；与pH值6.8培养液相比， c P<0.05；与pH值7.0培养液相比， d P<0.05；与pH值7.2培养液相比， e P<0.05；与pH值7.4培养液相比， f P<0.05；与pH值7.6培养液相比， g P<0.05 
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表2 8种pH值培养液培养人真皮微血管内皮细胞去掉播种塞后各时间点细胞迁移剩余面积比较（mm 2，
pH值 样本数 0 h（即刻） 24 h 48 h 6.4 3 2.636±0.023 2.240±0.064 1.491±0.052 6.6 3 2.608±0.028 1.900±0.073 a 1.491±0.062 6.8 3 2.608±0.030 1.756±0.068 a 1.246±0.058 7.0 3 2.608±0.027 1.131±0.072 abc 0.814±0.042 ab 7.2 3 2.637±0.025 0.970±0.068 abc 0.262±0.041 abcd 7.4 3 2.608±0.028 0.894±0.063 abc 0.188±0.040 abcd 7.6 3 2.608±0.030 0.684±0.050 abc 0.045±0.011 abcef 7.8 3 2.637±0.030 1.305±0.054 a 0.821±0.023 abefg 注：先于相应pH值培养液中进行24 h适应性培养，再进行后续培养检测；处理因素主效应， F=74.60， P<0.001；时间因素主效应， F=2 019.01， P<0.001；两者交互作用， F=24.28， P<0.001；与pH值6.4培养液相比， a P<0.05；与pH值6.6培养液相比， b P<0.05；与pH值6.8培养液相比， c P<0.05；与pH值7.0培养液相比， d P<0.05；与pH值7.2培养液相比， e P<0.05；与pH值7.4培养液相比， f P<0.05；与pH值7.6培养液相比， g P<0.05
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表3 8种pH值培养液另培养48 h人真皮微血管内皮细胞蛋白激酶B磷酸化位点蛋白表达比较（
pH值 样本数 位点473 位点308 6.4 3 0.433±0.042 0.297±0.055 6.6 3 0.498±0.033 0.467±0.018 a 6.8 3 0.752±0.059 ab 0.826±0.047 ab 7.0 3 0.914±0.036 abc 0.938±0.033 ab 7.2 3 0.826±0.048 ab 0.902±0.028 ab 7.4 3 0.890±0.035 abc 0.935±0.021 ab 7.6 3 1.041±0.029 abcdef 0.901±0.037 ab 7.8 3 0.860±0.033 abcg 0.760±0.032 abdefg 注：先于相应pH值培养液中进行24 h适应性培养，再进行后续培养检测；与pH值6.4培养液相比， a P<0.05；与pH值6.6培养液相比， b P<0.05；与pH值6.8培养液相比， c P<0.05；与pH值7.0培养液相比， d P<0.05；与pH值7.2培养液相比， e P<0.05；与pH值7.4培养液相比， f P<0.05；与pH值7.6培养液相比， g P<0.05
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