钙泊三醇诱导的HaCaT细胞Wnt/β-Catenin信号通路差异表达基因的筛选

Screening for Differentially Expressed Genes of Human Wnt Signaling Pathway in Calcipotriol-induced HaCaT Cell Line

目的研究钙泊三醇(calcipotriol)对HaCaT细胞Wnt/β-Catenin信号通路的影响,探讨其治疗银屑病的机制。方法分别用1.0×(10-7～10-11)mol/L的钙泊三醇刺激HaCaT细胞,CKK-8法检测其对HaCaT细胞增殖的影响。再用1.0×10-7mol/L的钙泊三醇处理细胞后,抽提实验组和对照组细胞的总RNA,用紫外吸收测定法和变性琼脂糖凝胶电泳法检测RNA产量和质量;合成cDNA后与含有89条基因的Wnt信号通路PCR芯片进行杂交。结果当钙泊三醇的浓度为1.0×(10-7～10-8)mol/L时,作用HaCaT细胞48h后,钙泊三醇组的吸光值较空白对照组显著降低,该两组有25条基因表达差异达2倍以上,其中21条显著上调,4条显著下调。这些差异表达基因涉及细胞增殖与分化、胚胎形成、细胞内信号传导和多组织器官发育等。结论钙泊三醇通过影响Wnt信号通路中CTNNB1,LRP5,TCF7L1,WNT7B和RPL13A等相关基因表达,对角质形成细胞的增殖分化发挥调节作用。

Objective To investigate the roles of Calcipotriol on Wnt signaling pathway in HaCaT cells via PCR array technology, and explore its mechanisms for psoriasis treatment. Methods Cultured HaCaT cells were trea- ted with various concentrations of calcipotriol and the proliferation of HaCaT cells were observed by CKK-8 assay. HaCaT cells were cultured in vitro. Total RNA was extracted from cells of Calcipotriol-treated and control group. RNA yield and quality were assessed by UV absorbance and denaturing agarose gel electro- phoresis assay. First strand cDNA was synthesized and hybridized to a human Wnt Signaling RT2 ProfilerTM PCR Genome Array containing 89 genes. Subsequently, the signal images were scanned by a laser scanner and acquired images were analyzed by software. Results Calcipotriol caused a dose-dependent decrease in the number of HaCaT cells. The proliferation of 48h cultured HaCdF cells was inhibited by Calcipotriol with concentrations from 1.0 ×（10-7 ～ 10-8） moL/L in vitro. A total of 25 genes were identified to be differential- ly expressed between Calcipotriol-treated group and control group. Of these genes, 21 were up-regulated and 4 were down-regulated. These differentially expressed genes were associated with cell proliferation and differ- entiation, the embryo formation, cell signal transduction growth and development of tissue and organ, and so on. Conclusion Calcipotriol may affect the expression of Wnt signaling-related genes such as CTNNB1, LRP5, TCFTL1, WNTTB and RPL13A ,which regulate the proliferation and differentiation of keratinocytes.