肝细胞生长因子对人外泌汗腺上皮细胞促增殖作用的研究

EFFECT OF HEPATOCYTE GROWTH FACTOR ON PROLIFERATION OF CULTURED HUMAN ECCRINE SWEAT GLAND EPITHELIAL CELLS

目的研究肝细胞生长因子(hepatocyte growth factor,HGF)对培养的人外泌汗腺上皮细胞(human eccrinesweat gland epithelial cells,hESGc)的促增殖作用,以及细胞内磷酸化细胞外信号调节激酶(phosphorylated extracellular signal-regulated kinase1/2,p-ERK1/2)蛋白的表达。方法在无血清角质形成细胞培养基(keratinocyte serum free medium,KSFM)中培养hESGc,取第1代细胞进行实验。免疫组织化学染色检测C-met表达。MTT法检测HGF对hESGc的促增殖作用,实验分为3组:空白组、对照组和实验组。空白组仅含200μL KSFM;对照组于96孔板以2×103个/孔接种hESGc,并加入200μLKSFM;实验组于96孔板以2×103个/孔接种hESGc并加入200μLKSFM后,再分别加入不同浓度(2、20、40、80ng/mL)HGF。实验组于加入各浓度HGF前以及加入后2、4d,观察细胞增殖情况。实验组于加入40ng/mL HGF后即刻,5、30、90和120min,采用Westernblot方法检测细胞内p-ERK1/2表达规律。结果hESGc抗-C-met免疫组织化学染色胞浆可见阳性染色,细胞核染色为蓝色。MTT法检测示40、80ng/mL HGF对hESGc具有显著促增殖作用(P<0.05)。在含40ng/mL HGF的KSFM中培养2d,吸光度(A)值为0.2393±0.0709,增殖率为74.2%,对照组A值为0.1374±0.0290;培养4d,40ng/mL HGF实验组A值为0.2878±0.0743,增殖率为74.8%,对照组A值为0.1646±0.0350,两组比较差异均有统计学意义(P<0.05)。在含80ng/mL HGF的KSFM中培养2d,A值为0.2123±0.0592,增殖率为54.5%;培养4d,80ng/mL HGF实验组A值为0.2310±0.0567,增殖率为40.3%;与对照组比较差异均有统计学意义(P<0.05)。p-ERK1/2在40ng/mL HGF刺激5min后表达达高峰,相对积分吸光度值(relative integral absorbance,RIA)为0.5932±0.1922,较刺激后即刻增加8.1倍(P<0.01);刺激30、90及120min后RIA与刺激后即刻比较,差异无统计学意义(P>0.05)结论HGF可促进hESGc增殖,并能引起ERK1/2蛋白磷酸化。

Objective To investigate the effect of hepatocyte growth factor （HGF） on proliferation of cultured human eccrine sweat gland epithelial cells （hESGc） and the involvement of phosphorylation of ERK1/2. Methods hESGc were cultured in keratinocyte serum free medium （KSFM） and the first generation of hESGc was harvested. The expression of C-met was detected by immunocytochemistry. MTT assay was used to detect the effect of HGF on the proliferation of hESGc. The cells were divided into blank group, control group and experimental group. The culture density was 2 × 10^3 cells/hole in control group and experimental group. Two hundred μL KSFM with HGF in different levels was added to every hole. hESGc were cultured in KSFM with HGF at different levels （2, 20, 40 and 80 ng/mL） in experimental group, in KSFM without HGF in control group, and in KSFM without HGF and no hESGc in blank group. The cell proliferation was observed in experimental group 2 and 4 days later. Western blot was used to detect the expression of phosphorylated ERK1/2 at 40 ng/mL HGF after 0, 5, 30, 90 and 120 minutes. Results The results were positive for anti-C-met staining in the cytoplasm. HGF （40 ng/mL and 80 ng/mL） significantly improved the proliferation of hESGc （P 〈 0.05）. When cultured in the KSFM with 40 ng/mL HGF, the cell proliferation rate and the absorbance were 74.2%, 0.239 3 ± 0.070 9 at 2 days and 74.8%, 0.287 8 ± 0.0743 at 4 days; showing significant differences when compared with control group （P 〈 0.05）. When cultured in KSFM with 80 ng/mL HGF, the cell proliferation rate and the absorbance were 54.5%, 0.212 3 ± 0.059 2 at 2 days and 40.3%, 0.2310 ± 0.056 7 at 4 days; showing significant differences when compared with control group （P 〈 0.05）. The expression of p-ERK1/2 reached to the maximum after stimulation of 40 ng/mL HGF for 5 minutes, and relative integral absorbance （RIA） was 0.593 2 ± 0.192 2, increased 8.1 times compared with instant stimulation （P 〈 0.01）. Conclusion HGF could induce the proliferation of hESGc and activate the phosphorylation of ERK1/2 protein.