中华皮肤科杂志 ›› 2013, Vol. 46 ›› Issue (6): 397-400.

• 论著 • 上一篇    下一篇

神经纤毛蛋白-1及血管内皮生长因子促HaCaT细胞增殖的研究

杨晓红1,曹毅2,解凡3,李园园1,翁鹤3,罗宏宾1   

  1. 1. 浙江省中医院
    2. 浙江中医药大学第一附属医院
    3. 浙江中医药大学附属第一医院
  • 收稿日期:2012-07-09 修回日期:2013-02-08 出版日期:2013-06-15 发布日期:2013-06-01
  • 通讯作者: 曹毅 E-mail:caoyi1965@163.com
  • 基金资助:
    载NRP-1基因壳聚糖缓释微球治疗糖尿病足研究

Promotive effect of neuropilin-1 and vascular endothelial growth factor on the proliferation of HaCaT cells

  • Received:2012-07-09 Revised:2013-02-08 Online:2013-06-15 Published:2013-06-01

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摘要： 【摘要】 目的 探讨神经纤毛蛋白-1（neuropilin-1，NRP-1）和细胞外调节蛋白激酶（ERK）在血管内皮生长因子（vascular endothelial growth factor ,VEGF）促HaCaT 细胞增殖中的可能机制。方法 培养HaCaT细胞，转染NRP-1质粒EX-O0008-M02，采用逆转录（RT）-PCR法检测转染后NRP-1 mRNA表达，蛋白免疫印迹法检测NRP-1蛋白质表达。将部分培养的HaCaT细胞分为脂质体对照组、对照质粒组以及目的质粒组分别予以脂质体、对照质粒pReceiver-M02及目的质粒EX-O0008-M02转染，各组分别加入PBS、MEK1/2抑制剂（U0126）、VEGF或U0126联合VEGF进行处理后，噻唑蓝（MTT）法检测HaCaT细胞增殖的改变，蛋白免疫印迹法观察ERK1/2的磷酸化情况以及增殖细胞核抗原（PCNA）的表达情况。采用One-way ANOVA 检验组间差异，LSD法进行多重比较和校正。结果 RT-PCR和蛋白免疫印迹实验结果提示EX-O0008-M02质粒转染可有效促进HaCaT细胞NRP-1 mRNA及其蛋白质的表达。与脂质体对照组（A570值0.63 ± 0.07）及对照质粒组（A570值0.62 ± 0.13）比较，目的质粒组HaCaT细胞增殖活性（A570值0.88 ± 0.14）明显增强，F = 8.755，P < 0.05。与对照质粒VEGF刺激组（A570值0.88 ± 0.10）比较，目的质粒VEGF刺激组HaCaT细胞的增殖活性（A570值1.14 ± 0.18）亦明显增强，F = 4.591，P < 0.05。与目的质粒VEGF刺激组比较，U0126预处理可以明显抑制VEGF对HaCaT细胞的促增殖作用（A570值0.50 ± 0.13，F = 42.106，P < 0.01）。蛋白免疫印迹结果提示与对照质粒转染细胞比较，目的质粒转染后VEGF对HaCaT细胞ERK1/2的促磷酸化及促PCNA表达得到明显提高，然而此作用可以被U0126有效抑制。结论 ERK1/2信号通路的激活在NRP-1蛋白介导的VEGF促HaCaT细胞增殖作用中可能发挥关键性作用。 【关键词】 神经纤毛蛋白质1； 角蛋白细胞； 血管内皮生长因子类； 糖尿病足

关键词: 角蛋白细胞, 神经纤毛蛋白质1, 血管内皮生长因子类, 糖尿病足

Abstract: YANG Xiao-hong, CAO Yi, XIE Fan, LI Yuan-yuan, WENG He, LUO Hong-bin. Department of Dermatology, First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou 310006, China Corresponding author: CAO Yi, Email: caoyi1965@163.com 【Abstract】 Objective To explore the possible role of neuropilin-1 （NRP-1） and extracellular regulated protein kinases （ERK） in the mechanisms underlying the promotive effect of vascular endothelial growth factor （VEGF） on the proliferation of HaCaT cells. Methods HaCaT cells were cultured in vitro and transfected with a NRP-1 expression plasmid EX-O0008-M02. Reverse transcription （RT-PCR and Western blot were performed to detect the mRNA and protein expression of NRP-1 in HaCaT cells respectively before and after the transfection. Some HaCaT cells were divided into three groups to be transfected with liposome （liposome control group）, control plasmid pReceiver-M02 （plasmid control group）, and objective plasmid EX-O0008-M02 （objective plasmid group）, respectively, and each of the three groups was classified into several subgroups to be treated with phosphate buffer solution （PBS）, U0126 （MEK1/2 inhibitor） and VEGF alone or in combination. After additional culture, methyl thiazolyl tetrazolium （MTT） assay was performed to determine the proliferative activity of HaCaT cells, Western blot to quantify the expression of total and phosphorylated ERK1/2 as well as proliferating cell nuclear antigen （PCNA） protein in HaCaT cells. Intergroup differences were assessed by one-way analysis of variance （ANOVA）, and multiple comparisons and correction were done by using the least significant difference （LSD） test. Results RT-PCR and Western blot analysis confirmed that the transfection with NRP-1 effectively promoted the mRNA and protein expression of NRP-1 in HaCaT cells. A significant increase was observed in cellular proliferative activity （absorbence value at 570 nm） in the objective plasmid group compared with the liposome control group and plasmid control group （0.88 ± 0.14 vs. 0.63 ± 0.07 and 0.62 ± 0.13, F = 8.755, P < 0.05）, also in the VEGF-stimulated objective plasmid group compared with the VEGF-stimulated plasmid control group （1.14 ± 0.18 vs. 0.88 ± 0.10, F = 4.591, P < 0.05）. The U0126 pretreatment markedly suppressed the VEGF-induced proliferation of A375 cells in the objective plasmid group （0.50 ± 0.13 vs. 1.14 ± 0.18, F = 42.106, P < 0.01）. As Western blot analysis suggested, the objective plasmid significantly enhanced the VEGF-induced increase in ERK1/2 phosphorylation degree and PCNA expression intensity in HaCaT cells compared with the control plasmid, but the enhancing effect of objective plasmid was effectively inhibited by U0126. Conclusion The activation of ERK1/2 signaling pathway may play a key role in the NRP-1 protein-mediated promotive effect of VEGF on the proliferation of HaCaT cells. 【Key words】 Neuropilin-1; Keratinocytes; Vascular endothelial growth factors; Diabetic foot

Key words: neuropilin-1, Diabetic foot