肝细胞生长因子对体外培养的人外泌汗腺腺上皮细胞迁移、凋亡及p-Akt表达的影响

Effect of hepatocyte growth factor on the migration and apoptosis of, as well as p-Akt expression in cultured human eccrine sweat gland epithelial cells

目的 研究肝细胞生长因子（HGF）对人外泌汗腺上皮细胞（hESGc）的迁移、凋亡及p-Akt表达的影响.方法 在无血清培养基中分别加入不同浓度HGF（2、20、40μg/L）培养hESGc,采用细胞损伤愈合模型,培养20h后计数进入刮痕区的细胞,观察HGF对hESGc移行的作用.采用Annexin V-FITC及PI双染（膜联蛋白-异硫氰酸荧光素及碘化丙啶双染）后经流式细胞仪检测细胞凋亡,观察HGF对hESGc凋亡的影响,并用Western印迹法检测细胞中p-Akt的变化.结果 2μg/L HGF无促细胞移行作用,20μg/L和40μg/L HGF组进入划痕区的细胞分别为（23.0±6.3）个和（56.7±7.9）个,与对照组（17.3±5.5）个相比,差异均有统计学意义（t值分别为2.653、15.858,P值〈0.05或0.01）,两种浓度HGF促移行能力分别为33.2%和228.2%,差异有统计学意义（P〈0.01）.与对照组凋亡率14.76%相比,2μg/L HGF和20μg/L HGF组细胞凋亡率无明显变化,其凋亡率分别为14.16%和13.50%,而40μg/L HGF组细胞凋亡率为8.87%,显著下降（t=7.852,P〈0.01）.Western印迹法检测显示加入HGF后Akt被磷酸化,p-Akt升高.结论 HGF能促进hESGc移行,抑制hESGc凋亡,并刺激p-Akt表达升高.

Objective To investigate the effect of hepatocyte growth factor （HGF） on migration and apoptosis of,as well as phosphorylated-Akt （p-Akt） expression in cultured human eccrine sweat gland epithehal cells （hESGc）.Methods The first generation of hESGc were cultured in keratinocyte serum free medium （KSFM） and treated with various concentrations （2,20,40μg/L） of HGF for different durations.Then,cell scratch test was performed to detect cell migration,a double staining flow cytometry assay using annexin VFITC/propidium iodide to detect cell apoptosis.and Western blot to measure the expression of p-Akt.Results HGF of 2μg/L had no effect on the migration of hESGc,while that of 20 μg/L and 40μg/L could promote the migration of hESGc by 33.2% and 228.2%.respectively.The average number of cells migrating into the scrach zone was significantly lower in untreated cell group than that in 20 and 40μg/L HGF-treated cell group （17.3±5.5 vs 23.0±6.3 and 56.7±7.9,t=2.653, 15.858,P〈0.05,0.01, respectively）.The apoptosis rate was 14.76% in untreated cells,14.16%,13.5% and 8.87% in cells treated with HGF of 2,20 and 40μ/L, respectively;there was a significant difference between untreated cells and 40μg/L HGF-treated cells （t=7.852,P〈0.01）.HGF could activate the phosphorylation of Akt protein and increase the expression of p-Akt.Conclusion HGF could promote the migration of,inhibit the apoptosis of,and stimulate the p-Akt expression in.hESGc.