Effect of rapamycin on the migration of human epidermal cell line HaCaT and its possible molecular mechanism
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摘要: 目的 探讨雷帕霉素对人表皮细胞株HaCaT迁移的影响,并初步分析其分子机制。 方法 采用含体积分数10%FBS的RPMI 1640培养液(简称培养液)常规培养HaCaT细胞。(1)取对数生长期细胞,按照随机数字表法将其分为对照组及1、5、50、100、200 nmol/L雷帕霉素组,每组6孔。5个雷帕霉素组细胞分别加入含相应质量浓度雷帕霉素的培养液,对照组细胞加入含二甲基亚砜(DMSO)的培养液。常规培养4 h后,采用酶标仪检测细胞增殖活性(以吸光度值表示)。(2)取对数生长期细胞,同实验(1)分组培养,每组1孔。常规培养4 h后,在活细胞工作站下观察各组细胞3 h内运动范围,计算细胞曲线运动速度,筛选雷帕霉素适宜实验浓度。(3)取对数生长期细胞,按随机数字表法将其分为雷帕霉素组和对照组,每组1孔。雷帕霉素组细胞加入含50 nmol/L雷帕霉素的培养液,对照组细胞加入含DMSO的培养液。常规培养4 h后行划痕实验,观察划痕后0(即刻)、5、10、15 h的划痕面积并计算后3个时相点的细胞迁移率。(4)取对数生长期细胞,同实验(3)分组培养,每组1孔。用蛋白质印迹法检测细胞黏着斑激酶(FAK)活性(以磷酸化FAK与FAK灰度值比值表示)。上述实验重复3次或5次。对数据行单因素方差分析、LSD检验、
t 检验。 结果 (1)对照组及1、5、50、100、200 nmol/L雷帕霉素组细胞增殖活性分别为1.22±0.28、1.29±0.38、1.12±0.27、1.20±0.29、1.15±0.30、1.39±0.40,组间比较差异无统计学意义(F =2.112,P =0.068)。(2)1、5 nmol/L雷帕霉素组细胞的运动范围与对照组接近,其余3个雷帕霉素组细胞的运动范围明显小于对照组。组间细胞曲线运动速度总体比较,差异有统计学意义(F =3.525,P =0.004)。1、5 nmol/L雷帕霉素组细胞曲线运动速度分别为(0.8±0.4)、(0.8±0.8)μm/min,与对照组[(0.9±0.5)μm/min]相近(P 值均大于0.05);50、100、200 nmol/L雷帕霉素组细胞曲线运动速度分别为(0.7±0.5)、(0.7±0.4)、(0.7±0.4)μm/min,明显慢于对照组(P 值均小于0.01)。选择50 nmol/L雷帕霉素用于后续实验。(3)划痕后0、5 h,雷帕霉素组细胞划痕面积与对照组相近;划痕后10、15 h,雷帕霉素组细胞划痕面积较对照组大。划痕后5 h,对照组及雷帕霉素组的细胞迁移率分别为(17.5±2.6)%、(15.8±3.5)%,组间比较差异无统计学意义(t =1.951,P >0.05)。雷帕霉素组划痕后10、15 h的细胞迁移率分别为(42.5±4.0)%、(71.3±9.2)%,明显低于对照组的(46.9±6.7)%、(88.0±7.7)%,t 值分别为2.732、6.746,P 值均小于0.01。(4)雷帕霉素组细胞FAK活性为0.16±0.08,明显低于对照组的0.46±0.14,t =4.967,P <0.01。 结论 在HaCaT细胞中,FAK信号通路对雷帕霉素敏感,FAK活性下降可能参与介导雷帕霉素对HaCaT细胞迁移的抑制效应。Abstract: Objective To explore the effects of rapamycin on the migration of human epidermal cell line HaCaT, and to analyze its molecular mechanism. Methods HaCaT cells were conventionally cultured with RPMI 1640 culture medium containing 10% fetal calf serum (hereinafter referred to as culture medium). (1) According to the random number table, HaCaT cells in logarithmic phase were divided into control group and 1, 5, 50, 100, 200 nmol/L rapamycin groups, with 6 wells in each group. The cells in rapamycin groups were cultured with culture medium containing rapamycin in corresponding mass concentration, and the cells in control group were cultured with culture medium containing dimethyl sulfoxide (DMSO) instead. After being conventionally cultured for 4 hours, proliferative activity of cells was determined with microplate reader (denoted as absorbance value). (2) HaCaT cells in logarithmic phase were grouped and cultured as that in experiment (1), with 1 well in each group. After being conventionally cultured for 4 hours, range of movement of cells in 3 hours was observed under live cell imaging workstation, and their curvilinear movement speeds were calculated. Then the suitable concentration of rapamycin was selected for experiments (3) and (4). (3) HaCaT cells in logarithmic phase were divided into control group and rapamycin group according to the random number table, with 1 well in each group. The cells in rapamycin group were cultured with culture medium containing 50 nmol/L rapamycin, and the cells in control group were cultured with culture medium containing DMSO. After being conventionally cultured for 4 hours, cells were collected for scratch assay. Wound area was observed at post scratching hour (PSH) 0, 5, 10, and 15, and the migration rates of cells at PSH 5, 10, and 15 were calculated respectively. (4) HaCaT cells in logarithmic phase were grouped and cultured as that in experiment (3), with 1 well in each group. Activity of focal adhesion kinase (FAK) was determined with Western blotting (denoted as the ratio of gray value of phosphorylated FAK to that of FAK). Above-mentioned experiments were independently repeated for three or five times. Data were processed with one-way analysis of variance, LSD test, andt test. Results (1) Proliferative activity of cells in control group and 1, 5, 50, 100, 200 nmol/L rapamycin groups was respectively 1.22±0.28, 1.29±0.38, 1.12±0.27, 1.20±0.29, 1.15±0.30, 1.39±0.40, without statistically significant differences among these groups (F =2.112,P =0.068). (2) The ranges of movement of cells in 1, 5 nmol/L rapamycin groups were similar to the range of movement of cells in control group, while those of cells in 50, 100, 200 nmol/L rapamycin groups were obviously smaller than the range of movement of cells in control group. There were statistically significant differences in cell curvilinear movement speeds among the 6 groups (F =3.525,P =0.004). The curvilinear movement speeds of cells in 1, 5 nmol/L rapamycin groups were respectively (0.8±0.4) and (0.8±0.8) μm/min, and they were similar to the curvilinear movement speed of cells in control group [(0.9±0.5) μm/min, withP values above 0.05]. The curvilinear movement speeds of cells in 50, 100, 200 nmol/L rapamycin groups were respectively (0.7±0.5), (0.7±0.4), (0.7±0.4) μm/min, and they were significantly lower than the curvilinear movement speed of cells in control group (withP values below 0.01). Thus, 50 nmol/L rapamycin was selected for experiments (3) and (4). (3) Compared with those of control group, wound areas of rapamycin group showed no obvious change at PSH 0 and 5, while they were obviously increased at PSH 10 and 15. At PSH 5, migration rate of cells in control group [(17.5±2.6)%] was similar to that in rapamycin group [(15.8±3.5)%,t =1.951,P >0.05]. Migration rates of cells of rapamycin group at PSH 10 and 15 [(42.5±4.0)% and (71.3±9.2)%, respectively] were obviously decreased as compared with those of control group [(46.9±6.7)% and (88.0±7.7)%, witht values respectively 2.732 and 6.746,P values below 0.01]. (4) Compared with that in control group (0.46±0.14), FAK activity of cells in rapamycin group (0.16±0.08) was significantly down-regulated (t =4.967,P <0.01). Conclusions FAK signal pathway is sensitive to rapamycin in HaCaT cells. Inhibition effects of rapamycin on migration of HaCaT cells may be mediated by down-regulated activity of FAK.-
Key words:
- Sirolimus /
- Cell proliferation /
- Cell migration assays /
- Cell movement /
- Focal adhesion kinase /
- HaCaT cells
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