骨化三醇对角质形成细胞作用机制的差异蛋白质组学研究

Differential Proteomic Analysis on the Effect of Calcitriol of Keratinocytes

目的研究骨化三醇对角质形成细胞蛋白质表达的影响,寻找骨化三醇作用的靶蛋白。方法常规培养HaCaT细胞、药物诱导,避光培养48h后用反复冻融法提取细胞全蛋白。将提取的蛋白样品采用固相pH梯度双向凝胶电泳进行分离,应用ImageMaster 2D Platinum 5.0软件对图像进行匹配分析,选取各组中有重叠的药物作用前后浓度改变的10个蛋白质点,经基质辅助激光解吸附飞行时间质谱(MALDI-TOF-MS)进行质谱鉴定。结果成功获得HaCaT细胞的蛋白质组电泳图谱,图像重复性好。药物作用后蛋白质表达有明显变化,以抑制为主。共鉴定出四种蛋白质,其中一种为卵清蛋白样丝氨酸蛋白酶抑制剂1(MNE1)。结论成功建立了HaCaT细胞蛋白质二维展示平台。MNE1是骨化三醇的治疗机制中的靶蛋白之一。

Objective To study the effect of calcitriol on HaCaT cells at the protein level to scan for the target proteins of calcitriol. Methods HaCaT cells were euhured with usual method. Two days after the drugs were added,the cells were collected, and the total proteins were extracted by repeating freezing-thawing process. Then the protein samples were seperated by two-dimensional gel electrophoresis. The 2DE profiles were analyzed by ImageMaster 2D Platinum 5.0 software. Ten proteins of the differential expression were identified by MALDI-TOF-MS. Results Reproducible 2-DE profiles of HaCaT cell were constructed. Perfect gel profiles were obtained. The level of proteins was obviously changed after the drugs were administred, and most of them were decreased. Four proteins were identified by MALDI-TOF-MS. One of them was serine proteinase inhibitor, clade B, member 1 （MNE1）. Conclusion The 2-DE profiles of HaCaT cell were established. MNE1 was one of the target proteins of calcitriol on HaCaT cells.