骨化三醇通过上调中性粒细胞弹性蛋白酶抑制剂表达抑制角质形成细胞增殖研究.docVIP

骨化三醇通过上调中性粒细胞弹性蛋白酶抑制剂表达抑制角质形成细胞增殖研究 　　摘 要 目的：探讨骨化三醇和中性粒细胞弹性蛋白酶抑制剂serpin B1对人角质形成细胞株HaCaT细胞增殖的影响。方法：骨化三醇组在常规培养的HaCaT细胞中加入骨化三醇，对照组加入等量的无血清培养基，避光培养48 h后采用反复冻融法提取细胞全蛋白，采用固相pH梯度双向凝胶电泳法进行分离，并用ImageMaster 2D Platinum 5.0软件分析蛋白表达的差异，同时采用基质辅助激光解吸飞行时间质谱仪进行蛋白鉴定。serpin B1的表达上调通过反转录聚合酶链式反应法和Western-blotting法予以验证。通过RNA干扰实验、噻唑蓝实验和流式细胞法分析serpin B1对HaCaT细胞增殖的影响。结果：骨化三醇组的serpin B1表达上调。骨化三醇在1×10-9 ～ 1×10-6 mol/L浓度时能抑制HaCaT细胞增殖。干扰serpin B1可促进HaCaT细胞增殖，且使骨化三醇的抑制作用消失。结论：骨化三醇可上调HaCaT细胞中的serpin B1表达，这可能在骨化三醇抑制HaCaT细胞增殖的过程中起着重要作用。 　　关键词 骨化三醇 角质形成细胞 中性粒细胞弹性蛋白酶抑制剂 serpin B1 银屑病 　　中图分类号：R977.24； R758.63 文献标识码：A 文章编号：1006-1533（2016）09-0028-07 　　Calcitriol inhibits keratinocyte proliferation by upregulating the expression of leukocyte elastase inhibitor serpin B1 　　LI Hui1，2*， GUO Zhili2， GU Jun2** 　　（1. Department of Dermatology， General Hospital of Urumuqi Military Region， Urumuqi 830000， China； 　　2. Department of Dermatology， Changhai Hospital， Second Military Medical University， Shanghai 200433， China） 　　ABSTRACT Objective： To evaluate the role of calcitriol and serpin B1 in human keratinocyte cell line HaCaT proliferation. Methods： Calcitriol was added at 1×10-8 mol/L into the culture of HaCaT cell in a calcitriol group while only serum-free Dulbecco’s modified Eagle’s medium instead in a control group. Whole proteins were extracted by freeze-thawing method after cells were incubated in the dark for 48 h and were separated by two-dimensional differential gel electrophoresis. The differences for protein expression were analyzed by ImageMaster 2D Platinum 5.0 and the identification of proteins was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry. The overexpression of serpin B1 was confirmed by reverse transcription polymerase chain reaction and Western-blotting assay. The effect of serpin B1 on HaCaT proliferation was analyzed by RNA interference experiments， methyl thiazole tetrazolium assay and flow cytometry. Results： The expression of ser