摘要
构建Wnt-3a的真核表达载体并稳定转染大鼠成纤维细胞Rat-1,建立Wnt信号通路持续激活的细胞模型,以探讨Wnt信号通路激活对该细胞的生长以及某些表型特征的影响。结果表明:Wnt信号通路持续激活时,Rat-1细胞形态表现为细胞长度的增加,具折光性以及呈绳索状的成束密集排布;MTT以及流式细胞仪检测表明稳定转染后细胞增殖率明显高于正常对照组,进入G2期的细胞增多,细胞增殖分裂能力增强;Transwell小室迁移实验证实转染组的细胞迁移率略高于对照组,但无显著性差异;体外划痕实验表明,稳定转染后的Rat-1细胞在划痕后伤口愈合时间显著缩短。结果提示:体外Wnt信号通路的激活能够引起成纤维细胞某些表型改变,并促进细胞增殖,加速体外伤口的修复。
The Wnt signaling pathway is thought to be functionally conserved in vertebrates and invertebrates and plays an important role during the embryonic and postembryonic development. Recent studies indicated that this pathway may be also involved in the controlled proliferation and migration of some kinds of fibroblasts during the wound healing process. To verify this assumption in vitro,we chose Rat- 1, a kind of rat fibroblasts to investigate the regulation of Wnt signaling pathway to the growth and changes of several phenotypes of this kind of cells. Full length Wnt-3a cDNA was inserted in pcDNA 3.1 vector to construct the Wnt-3a mammalian expression vector, which was stably transfected into Rat- 1 cells, and then to establish a cell model in which Wnt signaling pathway was constantly activated. When Wnt signaling pathway was activated constantly, Rat-1 cells exhibited morphological changes： grew more densely as a monolayer, adopted an elongated and refractile appearance, forming cord-like bundles lined up in a uniform direction. The results of MTT assay and FCM analysis indicated that more Rat-1/Wnt-3a cells entered into G2 phase and the proliferation rate of the Rat- 1/Wnt-3a cells increased significantly compared to the non-transfected cells. Though the migration of Rat- l/Wnt-3a cells increased slightly by the method of Transwell migration assay, there was no statistic significance compared to the non-transfected cells. The result of in vitro scrape wound healing assay showed that for Rat- 1/Wnt-3a cells the time course of wound healing decreased significantly. It is therefore concluded that the activation of Wnt signaling pathway can regulate some of the phenotypes of Rat-I cells, facilitate cell proliferation and promote the scrape wound healing in vitro.
出处
《生理学报》
CAS
CSCD
北大核心
2005年第4期505-510,共6页
Acta Physiologica Sinica
基金
ThisworkwassupportedbytheExcellentPh.DDissertationCultivatingFund
ShanghaiMedicalcollege
FudanUniversity(No.021101013).
