基质细胞衍生因子-1促进毛囊角质细胞增殖的研究

Proliferation of keratinocytes in hair follicles induced by stromal cell derived factor-1

目的观察基质细胞衍生因子-1(SDF-1)对体外培养的大鼠毛囊角质细胞增殖的影响。方法 2019年6月至2020年1月,采用免疫荧光检测大鼠毛囊角质细胞(购自中国赛百慷生物有限公司)标志物角蛋白14(K14)的表达量。细胞计数试剂盒(CCK-8)检测0、5、10、25、50 μg/L浓度SDF-1作用大鼠毛囊角质细胞24 h后,10、25 μg/L浓度SDF-1作用大鼠毛囊角质细胞48 h后,及10、25 μg/L浓度SDF-1加入50 nmol/L趋化因子受体-4(CXCR4)拮抗剂普乐沙福(AMD3100)作用大鼠毛囊角质细胞24 h后毛囊角质细胞增殖;5-乙炔基-2’-脱氧尿苷(EdU)标记法分别检测对照组(0 μg/L SDF-1),25 μg/L SDF-1组,25 μg/L SDF-1+50 nmol/L AMD3100组作用大鼠毛囊角质细胞24 h后毛囊角质细胞增殖。组间比较采用t检验。结果细胞免疫荧光显示95%以上细胞K14染色呈阳性。CCK-8检测结果显示与0 μg/L浓度SDF-1(103.04±0.99)比较5 μg/L浓度SDF-1对细胞增殖活性无明显影响(100.67±4.59,t=0.905,P>0.05),差异无统计学意义,10、25、50 μg/L浓度SDF-1促进毛囊角质细胞增殖(111.44±4.95、124.59±2.08、125.11±0.48,t10=3.221,P10<0.05;t25=8.624,P25<0.01;t50=8.464,P50<0.01),差异有统计学意义。与浓度为25 μg/L比较,SDF-1浓度为10 μg/L促增殖作用减弱(t=5.043,P<0.01),差异有统计学意义,浓度为50 μg/L促增殖作用无明显变化(t=0.199,P>0.05),差异无统计学意义。10、25 μg/L浓度的SDF-1作用48 h(98.26±5.24、112.39±2.30)与作用24 h(111.44±4.95、124.59±2.08)比较其促增殖作用减弱(t10=3.167,P10<0.05;t25=6.816,P25<0.01),差异有统计学意义。10、25 μg/L浓度SDF-1加入50 nmol/L AMD3100作用毛囊角质细胞24 h后,毛囊角质细胞增殖明显受到抑制(84.20±1.06、98.51±0.92,t10=9.325,P10<0.01;t25=19.900,P25<0.01),差异有统计学意义。EdU检测结果进一步验证SDF-1具有促进毛囊角质细胞增殖作用(SDF-1组:38.59±0.33,对照组:22.45±2.59,t=10.700,P<0.01),AMD3100可抑制SDF-1促增殖作用(22.12±1.96,t=10.700,P<0.01),差异有统计学意义。结论 SDF-1在10~50 μg/L浓度范围内对体外培养的大鼠毛囊角质细胞有促进增殖作用,且该增殖作用能被AMD3100抑制。

Objective To observe the effect of stromal cell-derived factor-1(SDF-1)on the proliferation of rat hair follicle keratinocytes cultured in vitro.Methods From June 2019 to January 2020,immunofluorescence was used to detect the expression of keratin 14(K14),a marker of rat hair follicle keratinocytes purchased from iCell Bioscience Inc.Cell count kit-8(CCK-8)assay was used to detect the proliferation of hair follicle keratinocytes treated with 0,5,10,25,50μg/L SDF-1 for 24 h,the proliferation of hair follicle keratinocytes treated with 10,25μg/L SDF-1 for 48 h,and the proliferation of hair follicle keratinocytes treated with 10,25μg/L SDF-1 combined with 50 nmol/L CXCR4 antagonist plerixafor(AMD3100)for 24 h.5-Ethynyl-2’-deoxyuridine(EdU)detected the proliferation of hair follicle keratinocytes in control group(0μg/L SDF-1),25μg/L SDF-1 group and 25μg/L SDF-1 adding 50 nmol/L AMD3100 group for 24 h.T test was used to analyze the results.Results Cell immunofluorescence showed that more than 95%of the cells were positive for K14 staining.CCK-8 experiment showed that compared with SDF-1 at concentration of 0μg/L(103.04±0.99),5μg/L of SDF-1 had no effect on the proliferation activity of hair follicle keratinocytes(100.67±4.59,t=0.905,P>0.05).SDF-1 at concentrations of 10,25,50μg/L promoted the proliferation of hair follicle keratinocytes(111.44±4.95,124.59±2.08,125.11±0.48,t10=3.221,P10<0.05;t25=8.624,P25<0.01;t50=8.464,P50<0.01).Compared with SDF-1 at concentration of 25μg/L,the proliferation capacity of hair follicle keratinocytes is reduced at concentration of 10μg/L(t=5.043,P<0.01),and has no significant change at concentration of 50μg/L(t=0.199,P>0.05).The proliferative effect of SDF-1 at the concentrations of 10μg/L and 25μg/L for 48 h(98.26±5.24,112.39±2.30)was reduced compared with the effect of 24 h(111.44±4.95,124.59±2.08,t10=3.167,P10<0.05;t25=6.816,P25<0.01).After 10μg/L and 25μg/L of SDF-1 were added to 50 nmol/L AMD3100,the proliferation of hair follicle keratinocytes was obviously inhibited(84.20±1.06,98.51±0.92,t10=9.325,P10<0.01;t25=19.900,P25<0.01).The results of EdU further verified that SDF-1 can promote the proliferation of hair follicle keratinocytes(SDF-1 group:38.59±0.33,control group:22.45±2.59,t=10.700,P<0.01),and AMD3100 can restrain the proliferative effect of SDF-1(22.12±1.96,t=10.700,P<0.01).Conclusion SDF-1 in a certain concentration range can promote the proliferation of hair follicle keratinocytes,and this proliferative effect can be suppressed by AMD3100.