中华皮肤科杂志 ›› 2015, Vol. 48 ›› Issue (8): 578-580.

• 研究报道 • 上一篇    下一篇

重组人色素上皮衍生因子对HaCaT细胞体外增殖和白细胞介素6、8及血管内皮生长因子表达的影响

李小琼1,魏志平2,刘彦群2   

  1. 1. 徐州医学院
    2. 徐州医学院附属医院皮肤科
  • 收稿日期:2014-10-24 修回日期:2015-03-04 出版日期:2015-08-15 发布日期:2015-07-30
  • 通讯作者: 魏志平 E-mail:xzwzp1961@yahoo.com.cn

Effects of recombinant human pigment epithelium derived factor on in vitro proliferation of and expressions of interleukin-6, -8 and vascular endothelial growth factor in cultured human HaCaT keratinocytes

  • Received:2014-10-24 Revised:2015-03-04 Online:2015-08-15 Published:2015-07-30

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摘要：

目的 探讨重组人色素上皮衍生因子（rhPEDF）对HaCaT细胞体外增殖和白细胞介素6（IL-6）、IL-8及血管内皮生长因子（VEGF）表达的影响。 方法 HaCaT细胞分4组进行不同处理，分别为100 μg/L rhPEDF组、50 μg/L rhPEDF组、25 μg/L rhPEDF组及对照组（只加RPMI 1640培养液）。采用CCK8法检测不同浓度rhPEDF作用于HaCaT细胞24、48、72 h后对细胞体外增殖的影响。RT-PCR和Western印迹法检测rhPEDF对HaCaT细胞IL-6、IL-8、VEGF mRNA及其蛋白表达的影响。统计分析方法采用两因素方差分析、单因素方差分析、SNK-q检验以及Pearson相关分析。 结果 25、50、100 μg/L的rhPEDF作用24、48、72 h后对HaCaT细胞体外增殖均有不同程度的抑制作用。随时间延长和rhPEDF浓度的增加，rhPEDF对HaCaT细胞体外增殖的抑制作用均逐渐增强（F = 1115、329.9，均P < 0.001）。与对照组相比，不同浓度（100、50、25 μg/L）rhPEDF作用于HaCaT细胞48 h后，VEGF、IL-6、IL-8 mRNA及蛋白的表达均下降，差异均有统计学意义（P < 0.05）。随rhPEDF浓度的增加，各浓度rhPEDF组VEGF mRNA、IL-6和IL-8蛋白表达均出现下调，与对照组比较，差异均有统计学意义（P < 0.05）；100 μg/L rhPEDF组的IL-6、IL-8 mRNA表达明显低于25 μg/L rhPEDF组（P < 0.05）；100 μg/L rhPEDF组VEGF蛋白表达分别低于50、25 μg/L rhPEDF组（P < 0.05），而50与25 μg/L rhPEDF组间差异无统计学意义（P > 0.05）。 结论 rhPEDF可抑制HaCaT细胞体外增殖，并可在mRNA及蛋白水平下调IL-6、IL-8、VEGF的表达。

Abstract:

Li Xiaoqiong, Wei Zhiping, Liu Yanqun. Department of Dermatology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, Jiangsu, China Corresponding author: Wei Zhiping, Email: xzwzp1961@aliyun.com 【Abstract】 Objective To investigate the effects of recombinant human pigment epithelium derived factor （rhPEDF） on in vitro proliferation of and expressions of interleukin 6 （IL-6）, IL-8 and vascular endothelial growth factor （VEGF） in cultured human HaCaT keratinocytes. Methods Some cultured HaCaT cells were treated with rhPEDF at various concentrations （25, 50, 100 μg/L） for different durations, and some treated with RPMI 1640 medium only served as the control group. Cell counting kit-8 （CCK8） assay was performed to evaluate cell proliferation after 24-, 48- and 72-hour treatment, reverse transcription （RT）-PCR to measure the mRNA expressions of IL-6, IL-8 and VEGF in HaCaT cells after 24-hour treatment, and Western blot to detect the protein expressions of IL-6, IL-8 and VEGF in HaCaT cells after 48-hour treatment. Statistical analysis was carried out by two- and one-way analysis of variance, Student-Newman-Keuls （SNK）-q test and Pearson correlation analysis. Results After treatment with rhPEDF of 25－100 μg/L for 24 － 72 hours, the proliferation of HaCaT cells was significantly inhibited to different extents compared with the control group （all P < 0.05）, and the inhibition rate significantly increased with the increase in treatment duration and concentrations of rhPEDF （F = 1115, 329.9, respectively, both P < 0.001）. Moreover, there was a significant decrease in the expressions of IL-6, IL-8 and VEGF mRNAs （at 24 hours） and proteins （at 48 hours） in HaCaT cells after treatment with rhPEDF of 25 － 100 μg/L compared with the control group （all P < 0.05）. The expression levels of VEGF mRNA as well as IL-6 and IL-8 proteins all significantly decreased with the increase of rhPEDF concentrations （all P < 0.05）. The mRNA expressions of IL-6 and IL-8 were significantly lower in the 100-μg/L rhPEDF group than in the 25-μg/L rhPEDF group （both P < 0.05）, and the protein expression of VEGF was significantly weaker in the 100-μg/L rhPEDF group than in 25- and 50-μg/L rhPEDF groups （both P < 0.05）, but similar between the 25- and 50-μg/L rhPEDF groups （P > 0.05）. Conclusions rhPEDF can inhibit the proliferation of HaCaT cells, and down-regulate the mRNA and protein expressions of IL-6, IL-8 and VEGF.