Effect of P62 on the migration and motility of human epidermal cell line HaCaT in high glucose microenvironment and its mechanism
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摘要:
目的 研究高糖微环境下P62蛋白对人表皮细胞株HaCaT迁移和运动性的影响及其可能的分子机制，以探讨糖尿病足创面难愈合的机制。 方法 采用实验研究方法。取对数生长期HaCaT进行实验。取细胞，按随机数字表法（分组方法下同）分为正常对照组（培养液含终物质的量浓度5.5 mmol/L的葡萄糖）及高糖（培养液含终物质的量浓度30.0 mmol/L的葡萄糖）24 h组、高糖48 h组、高糖72 h组。正常对照组细胞行常规培养72 h，高糖72 h组细胞行高糖培养72 h，高糖48 h组细胞先常规培养24 h再高糖培养48 h，高糖24 h组细胞先常规培养48 h再高糖培养24 h后，采用蛋白质印迹法检测P62蛋白表达。取细胞，分为正常对照组、高糖组，分别同前培养48 h后，采用免疫荧光法检测P62蛋白表达（以绿色荧光表示）。取细胞，分为阴性对照小干扰RNA（siRNA）组、P62-siRNA-1组、P62-siRNA-2组、P62-siRNA-3组，并转染相应试剂，于转染后72 h，采用蛋白质印迹法检测P62蛋白表达。取细胞，分为正常糖+阴性对照siRNA组、正常糖+P62-siRNA组、高糖+阴性对照siRNA组、高糖+P62-siRNA组，并行相应处理，于转染后72 h，采用蛋白质印迹法检测P62蛋白表达；行划痕试验检测并计算划痕后24 h细胞迁移率（样本数为9）；在活细胞工作站下，观察3 h内细胞运动范围并计算运动速度（正常糖+阴性对照siRNA组、正常糖+P62-siRNA组、高糖+阴性对照siRNA组、高糖+P62-siRNA组观察细胞数分别为76、75、80、79个）。取细胞，分为正常糖+磷酸盐缓冲液（PBS）组、高糖+PBS组、高糖+N-乙酰半胱氨酸（NAC）组，行相应处理后，于培养48 h，分别采用蛋白质印迹法及免疫荧光法检测P62蛋白表达。除划痕试验外，其余实验各组样本数均为3。对数据行单因素方差分析、LSD检验。 结果 与正常对照组比较，高糖24 h组、高糖48 h组及高糖72 h组细胞P62蛋白表达量均明显增加（P<0.01）。培养48 h，高糖组细胞中P62的绿色荧光强于正常对照组。转染后72 h，与阴性对照siRNA组比较，P62-siRNA-1组、P62-siRNA-2组和P62-siRNA-3组细胞P62蛋白表达量均明显减少（P<0.01）。转染后72 h，与正常糖+阴性对照siRNA组比较，正常糖+P62-siRNA组细胞P62蛋白表达量明显减少（P<0.01），高糖+阴性对照siRNA组细胞P62蛋白表达量明显增加（P<0.01）；与高糖+阴性对照siRNA组比较，高糖+P62-siRNA组细胞P62蛋白表达量明显减少（P<0.01）。划痕后24 h，与正常糖+阴性对照siRNA组［（55±7）%］比较，正常糖+P62-siRNA组细胞迁移率明显升高［（72±14）%，P<0.01］，高糖+阴性对照siRNA组细胞迁移率明显下降［（37±7）%，P<0.01］；与高糖+阴性对照siRNA组比较，高糖+P62-siRNA组细胞迁移率明显升高［（54±10）%，P<0.01］。观察3 h内，高糖+阴性对照siRNA组细胞运动范围较正常糖+阴性对照siRNA组缩小，正常糖+P62-siRNA组细胞运动范围较正常糖+阴性对照siRNA组增大，高糖+P62-siRNA组细胞运动范围较高糖+阴性对照siRNA组增大。与正常糖+阴性对照siRNA组比较，正常糖+P62-siRNA组细胞运动速度明显增加（P<0.01），高糖+阴性对照siRNA组细胞运动速度明显下降（P<0.01）；与高糖+阴性对照siRNA组比较，高糖+P62-siRNA组细胞运动速度明显增加（P<0.01）。培养48 h，与正常糖+PBS组比较，高糖+PBS组细胞P62蛋白表达量明显增加（P<0.01）；与高糖+PBS组比较，高糖+NAC组细胞P62蛋白表达量明显减少（P<0.01）。培养48 h，高糖+PBS组细胞中P62的绿色荧光强于正常糖+PBS组，而高糖+NAC组细胞中P62的绿色荧光弱于高糖+PBS组。 结论 在HaCaT中，高糖微环境可促进P62蛋白表达；敲减P62蛋白可促进其迁移并增加运动性；高糖微环境下活性氧增加可能是P62表达增加的潜在机制。 Abstract:Objective To investigate the effect of P62 on the migration and motility of human epidermal cell line HaCaT in high glucose microenvironment and its possible molecular mechanism, so as to explore the mechanism of refractory diabetic foot wound healing. Methods The method of experimental research was used. HaCaT cells in logarithmic growth phase was taken for experiment. The cells were collected and divided into normal control group (culture solution containing glucose with final molarity of 5.5 mmol/L) and high glucose (culture solution containing glucose with final molarity of 30.0 mmol/L) 24 h group, high glucose 48 h group, and high glucose 72 h group according to the random number table (the same grouping method below). The cells in normal control group were routinely cultured for 72 h, cells in high glucose 72 h group were cultured with high glucose for 72 h, cells in high glucose 48 h group were routinely cultured for 24 h then cultured with high glucose for 48 h, cells in high glucose 24 h group were routinely cultured for 48 h then cultured with high glucose for 24 h. Then the protein expression of P62 was detected by Western blotting. The cells were collected and divided into normal control group and high glucose group. After being correspondingly cultured for 48 h as before, the protein expression of P62 was detected by immunofluorescence method (indicated as green fluorescence). The cells were collected and divided into negative control small interfering RNA (siRNA) group, P62-siRNA-1 group, P62-siRNA-2 group, and P62-siRNA-3 group, and transfected with the corresponding reagents. At post transfection hour (PTH) 72, the protein expression of P62 was detected by Western blotting. The cells were collected and divided into normal glucose+negative control siRNA group, normal glucose+P62-siRNA group, high glucose+negative control siRNA group, and high glucose+P62-siRNA group. After the corresponding treatment, the protein expression of P62 was detected by Western blotting at PTH 72 h, the cell migration rate was detected and calculated at 24 h after scratching by scratch test, with the number of samples being 9; and the range of cell movement was observed and the trajectory velocity was calculated within 3 h under the living cell workstation, with the number of samples being 76, 75, 80, and 79 in normal glucose+negative control siRNA group, normal glucose+P62-siRNA group, high glucose+negative control siRNA group, and high glucose+P62-siRNA group, respectively. The cells were collected and divided into normal glucose+phosphate buffered solution (PBS) group, high glucose+PBS group, and high glucose+N-acetylcysteine (NAC) group. After the corresponding treatment, the protein expression of P62 at 48 h of culture was detected by Western blotting and immunofluorescence method, respectively. Except for scratch test and cell motility experiment, the number of samples was all 3 in the rest experiments. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results Compared with the protein expression in normal control group, the protein expressions of P62 of cells in high glucose 24 h group, high glucose 48 h group, and high glucose 72 h group were significantly increased (P<0.01). At 48 h of culture, the green fluorescence of P62 of cells in high glucose group was stronger than that in normal control group. At PTH 72, compared with the protein expression in negative control siRNA group, the protein expressions of P62 of cells in P62-siRNA-1 group, P62-siRNA-2 group, and P62-siRNA-3 group were significantly decreased (P<0.01). At PTH 72, compared with the protein expression in normal glucose+negative control siRNA group, the protein expression of P62 of cells in normal glucose+P62-siRNA group was significantly decreased (P<0.01), while the protein expression of P62 of cells in high glucose+negative control siRNA group was significantly increased (P<0.01); compared with the protein expression in high glucose+negative control siRNA group, the protein expression of P62 of cells in high glucose+P62-siRNA group was significantly decreased (P<0.01). At 24 h after scratching, compared with (55±7)% in normal glucose+negative control siRNA group, the cell migration rate in normal glucose+P62-siRNA group was significantly increased ((72±14)%, P<0.01), while the cell migration rate in high glucose+negative control siRNA group was significantly decreased ((37±7)%, P<0.01); compared with that in high glucose+negative control siRNA group, the cell migration rate in high glucose+P62-siRNA group was significantly increased ((54±10)%, P<0.01). Within 3 h of observation, the cell movement range in high glucose+negative control siRNA group was smaller than that in normal glucose+negative control siRNA group, while the cell movement range in normal glucose+P62-siRNA group was larger than that in normal glucose+negative control siRNA group, and the cell movement range in high glucose+P62-siRNA group was larger than that in high glucose+negative control siRNA group. Compared with that in normal glucose+negative control siRNA group, the cell trajectory speed in normal glucose+P62-siRNA group was significantly increased (P<0.01), while the cell trajectory speed in high glucose+negative control siRNA group was significantly decreased (P<0.01); compared with that in high glucose+negative control siRNA group, the cell trajectory speed in high glucose+P62-siRNA group was significantly increased (P<0.01). At 48 h of culture, compared with that in normal glucose+PBS group, the protein expression of P62 of cells in high glucose+PBS group was significantly increased (P<0.01); compared with that in high glucose+PBS group, the protein expression of P62 of cells in high glucose+NAC group was significantly decreased (P<0.01). At 48 h of culture, the green fluorescence of P62 of cells in high glucose+PBS group was stronger than that in normal glucose+PBS group, while the green fluorescence of P62 of cells in high glucose+NAC group was weaker than that in high glucose+PBS group. Conclusions In HaCaT cells, high glucose microenvironment can promote the protein expression of P62; knockdown of P62 protein can promote the migration and increase the mobility of HaCaT cells; and the increase of reactive oxygen species in high glucose microenvironment may be the underlying mechanism for the increase of P62 expression. -
Key words:
- Diabetic foot /
- Reactive oxygen species /
- Cell migration assays /
- P62 /
- High glucose /
- Wound repair
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1 蛋白质印迹法检测正常对照组和高糖培养3个时间点组HaCaT中P62蛋白表达
注：GAPDH为3-磷酸甘油醛脱氢酶；条带上方1、2、3、4分别指示正常对照组、高糖24 h组、高糖48 h组、高糖72 h组
2 正常对照组和高糖组HaCaT培养48 h的P62蛋白表达 异硫氰酸荧光素-4'，6-二脒基-2-苯基吲哚×630。2A、2B、2C.分别为正常对照组P62染色、细胞核染色、P62与细胞核染色重叠图片，细胞核完整，P62蛋白表达较少，主要分布于细胞质；2D、2E、2F.分别为高糖组P62染色、细胞核染色、P62与细胞核染色重叠图片，细胞核完整，P62蛋白表达较图2A多
注：P62阳性染色为绿色，细胞核阳性染色为蓝色
3 蛋白质印迹法检测转染siRNA后4组HaCaT中P62蛋白表达
注：GAPDH为3-磷酸甘油醛脱氢酶，siRNA为小干扰RNA；条带上方1、2、3、4分别指示阴性对照siRNA组、P62-siRNA-1组、P62-siRNA-2组、P62-siRNA-3组
4 蛋白质印迹法检测高糖微环境下siRNA对4组HaCaT转染后72 h的P62蛋白表达的作用
注：GAPDH为3-磷酸甘油醛脱氢酶，siRNA为小干扰RNA；条带上方1、2、3、4分别指示正常糖+阴性对照siRNA组、正常糖+P62-siRNA组、高糖+阴性对照siRNA组、高糖+P62-siRNA组
5 4组HaCaT处理完成后划痕后各时间点细胞划痕面积情况 倒置光学显微镜×100。5A、5B、5C、5D.分别为正常糖+阴性对照小干扰RNA（siRNA）组、正常糖+P62-siRNA组、高糖+阴性对照siRNA组、高糖+P62-siRNA组划痕后0 h（即刻），划痕情况相近；5E、5F、5G、5H.分别为正常糖+阴性对照siRNA组、正常糖+P62-siRNA组、高糖+阴性对照siRNA组、高糖+P62-siRNA组划痕后24 h，图5F划痕面积明显小于图5E，图5G划痕面积明显大于图5E，图5H划痕面积明显小于图5G
6 活细胞工作站观察4组HaCaT 3 h内的运动范围。6A、6B、6C、6D.分别为正常糖+阴性对照siRNA组、正常糖+P62-siRNA组、高糖+阴性对照siRNA组、高糖+P62-siRNA组，其中图6B、6D运动范围分别较图6A、6C明显增大
注：siRNA为小干扰RNA；细胞运动起点均为坐标（0，0），运动终点为位于4个象限中的圆点，连接两者之间的曲线为细胞运动轨迹
7 蛋白质印迹法检测高糖微环境下NAC对3组HaCaT培养48 h的P62蛋白表达的影响
注：GAPDH为3-磷酸甘油醛脱氢酶，PBS为磷酸缓冲盐溶液，NAC为N-乙酰半胱氨酸；条带上方1、2、3分别指示正常糖+PBS组、高糖+PBS组、高糖+NAC组
8 3组HaCaT培养48 h的P62蛋白表达 异硫氰酸荧光素-4'，6-二脒基-2-苯基吲哚×630。8A、8B、8C.分别为正常糖+PBS组P62染色、细胞核染色、P62与细胞核染色重叠图片，细胞核完整，P62蛋白表达较少，主要分布于细胞质；8D、8E、8F.分别为高糖+PBS组P62染色、细胞核染色、P62与细胞核染色重叠图片，细胞核完整，P62蛋白表达较图8A多；8G、8H、8I.分别为高糖+NAC组P62染色、细胞核染色、P62与细胞核染色重叠图片，细胞核完整，P62蛋白表达较图8D少
注：P62阳性染色为绿色，细胞核阳性染色为蓝色；PBS为磷酸缓冲盐溶液，NAC为N-乙酰半胱氨酸
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