1,25-二羟基维生素D3通过微管动力学调控表皮细胞迁移的研究

1α,25-dihydroxyvitamin D3 regulates epidermal cell migration via mechanism of microtubule dynamics

目的:本研究通过HaCaT细胞体外研究观察不同浓度(包括生理浓度)维生素D3(VD3)对表皮细胞迁移的影响并初步探索其迁移调控机制。方法:选用不同浓度VD3(0 nmol/L、10 2 nmol/L、10 nmol/L、10 -1 nmol/L、10 -3 nmol/L及10 -5 nmol/L)的处理HaCaT细胞(上海中国科学院细胞研究所)后,通过划痕实验、活细胞工作站实验、免疫荧光染色实验和免疫印迹实验观察HaCaT细胞迁移运动和检测α-微管蛋白(α-tubulin)的表达并通过 t检验评估组间差异,研究其迁移调控机制。 结果:与对照组比较(VD3浓度0 nmol/L),高浓度VD3组(10 2、10 nmol/L)HaCaT细胞呈现出更小的迁移范围和速率,α-tubulin表达增高(53.650±5.414和65.370±4.512比76.110±4.386, t=3.224、2.357, P<0.05;29.140±0.678比32.780±1.311, t=2.467, P<0.05;18.070±0.792和16.830±1.028比14.830±0.741, t=3.254、2.336, P<0.05)。生理浓度VD3组(10 -1 nmol/L)HaCaT细胞表现为更大的迁移范围和速率,α-tubulin表达降低(87.010±1.936比76.110±4.386, t=2.274, P<0.05;41.210±2.044比32.780±1.311, t=3.472, P<0.05;11.350±0.691比14.830±0.741, t=3.425, P<0.05)。低浓度VD3组(10 -3、10 -5 nmol/L)HaCaT细胞迁移的范围、速率和α-tubulin表达与对照组比较差异无统计学意义(79.360±6.036和72.370±2.990比76.110±4.386, t=0.298、0.687, P>0.05;34.150±1.588和33.240±1.055比32.780±1.311, t=0.666、0.289, P>0.05;14.310±1.115比14.830±0.741, t=0.403, P>0.05)。 结论:VD3通过微管动力学来调控表皮细胞的迁移,其作用具有双相性和浓度选择性。该研究为进一步揭示局部皮肤VD3应用浓度对伤口愈合作用的细胞和分子机制提供基础。

Objective To investigate the effects of different concentrations(including physiological concentrations)of 1α,25-dihydroxyvitamin D3(VD3)on the migration of epidermal cells and explore the mechanism of migration regulation.Methods HaCaT cells were cultured in medium containing different concentrations of VD3(0,102,10,10-1,10-3 and 10-5 nmol/L),then cell scratch test,live cell workstation experiment,immunofluorescence staining experiment and immunoblotting experiment were utilized to observe the changes in cell migration and detect the expression ofα-tubulin,and the differences between groups were assessed by t test.Results Compared with the control group,the cell migration ability decreased and the expression of polymerizedα-tubulin increased after high concentration VD3(102 and 10 nmol/L)treatment(53.650±5.414 and 65.370±4.512 vs.76.110±4.386,t=3.224,2.357,P<0.05;29.140±0.678 vs.32.780±1.311,t=2.467,P<0.05;18.070±0.792 and 16.830±1.028 vs.14.830±0.741,t=3.254,2.336,P<0.05).Under the physiological concentration,the cells showed obvious migration ability and low expression trend of polymerized tubulin(87.010±1.936 vs.76.110±4.386,t=2.274,P<0.05;41.210±2.044 vs.32.780±1.311,t=3.472,P<0.05;11.350±0.691 vs.14.830±0.741,t=3.425,P<0.05).The migration ability andα-tubulin expression of HaCaT cells cultured in low concentration of VD3(10-3and 10-5 nmol/L)were not significantly different from those in the control group(79.360±6.036 and 72.370±2.990 vs.76.110±4.386,t=0.298,0.687,P>0.05;34.150±1.588 and 33.240±1.055 vs.32.780±1.311,t=0.666,0.289,P>0.05;14.310±1.115 vs.14.830±0.741,t=0.403,P>0.05).Conclusion VD3 regulates the migration of epidermal cells via microtubule dynamics,of which effect is biphasic and concentration selective.This study provides the basis for further revealing the cellular and molecular mechanism of the effect of local skin VD3 on wound healing.