Department of Applied Physiology, Faculty of Medicine, University of Miyazaki, Miyazaki 889-1692, Japan.
Biomed Res Int. 2013;2013:963457. doi: 10.1155/2013/963457. Epub 2013 May 2.
In the present investigation, we examined the effect of Hyuganatsu (Citrus tamurana) extract (HE) on skin fibroblast (TIG-119) proliferation and migration during in vitro wound healing. HE selectively inhibited proliferation of TIG-119 cells at higher concentration (>1.0 mg/mL); at lower concentrations (0.1, 0.25, 0.5, and 0.75 mg/mL), it exhibited linear and time-dependent cell proliferation. In vitro scratch wound healing studies showed that the HE also accelerated the migration of cells towards the wounded region. Cytometric analysis demonstrated that HE extract did not alter G1/0 and S phases of cell cycle in any concentration studied; however, G2/M phases of cell cycle were significantly (P < 0.05) accelerated at 0.75 mg/mL dose. RT-PCR and Western blotting analysis indicated that HE markedly overexpressed levels of Rac-1, Rho-A, and Cdc-42 mRNA and the respective proteins. Cyclin-dependent kinases (Cdk-1 and -2) gene expression activity was significantly (P < 0.05) increased, but protein content decreased during treatment with HE. The induction of Cdk-1 and -2 by HE was abolished by inhibitors, transcription (DRB), and translation (CHX), implying transcriptional regulation that required de novo protein synthesis.
在本研究中，我们研究了日向夏柑橘（Citrus tamurana）提取物（HE）对体外伤口愈合过程中皮肤成纤维细胞（TIG-119）增殖和迁移的影响。HE 在较高浓度（>1.0mg/mL）时选择性地抑制 TIG-119 细胞的增殖；在较低浓度（0.1、0.25、0.5 和 0.75mg/mL）时，呈线性和时间依赖性细胞增殖。体外划痕愈合实验表明，HE 还能加速细胞向受伤区域迁移。细胞计量分析表明，HE 提取物在研究的任何浓度下均未改变细胞周期的 G1/0 和 S 期；然而，在 0.75mg/mL 剂量下，细胞周期的 G2/M 期明显（P < 0.05）加速。RT-PCR 和 Western blot 分析表明，HE 显著上调 Rac-1、Rho-A 和 Cdc-42 mRNA 及其相应蛋白的表达水平。细胞周期蛋白依赖性激酶（Cdk-1 和 -2）基因表达活性显著增加（P < 0.05），但在用 HE 处理时蛋白含量降低。HE 诱导的 Cdk-1 和 -2 的表达被抑制剂（DRB）和翻译抑制剂（CHX）阻断，这意味着需要新合成蛋白质的转录调控。
