| T cell factor 4 (TCF-4) is a member of TCF gene family, which is the key factor of the Wnt signaling pathway, and plays molecule "switch" dual role (transcriptional activation or repression) in the pathway regulating transcription process.Currently multiple target genes regulated TCF4 have been identified, the function of them are involved in cell cycle, multiple aspects of cell proliferation, apoptosis, and cell adhesion, etc.. In this study, express of TCF-4 gene in was up and down-regulated in vitro in cashmere goat primary and secondary dermal papilla cells, so that to explore the function and influence of TCf-4 gene in cashmere goat primary and secondary dermal papilla cells.1. Construction of TCF-4 overexpression and interference vectorsFirstly,sequences of Arbas cashmere goats TCF-4 cDNA was cloned correctly, then TCF-4 gene overexpression and interfering carrier vectors were designed and constructed.The TCF-4 overexpression vector was composed of CAG promoter, TCF-4, polyA, CMV promoter and DsRed2.The CAG promoter could be efficiently start the espression of target gene, and CMV-DsRed2 expressed red fluorescence protein, to detect the expression of TCF-4 gene in transgenic cell.Accroding to cashmere goat TCF-4 gene cDNA sequenceing, we designed and constructed three TCF-4 interference carrier vectors and a nonsense sequence control vector for TCF-4 gene down-regulation.2. Effects of TCF-4 overexpression on development-related genes expression in primary and secondary DPCs.In this study, two types of TCF-4 overexpressed hair follicle-ralated cells(pHFDPCs and sHFDPCs) were established using liposome-mediated method. The results showed that TCF-4 was overexpressed, the expression of PI3K and β-catenin was up-regalated and the expression of AKT and GSK-3β had no significant changes in pHFDPCs, and the expression of PI3K, β-catenin and AKT was up-regulated and GSK-3β was down-regulated in sHFDPCs.3. Effects of the TCF-4 down-regulation on development-related genes expression in primary and secondary DPCs.Two types of hair follicle-ralated cells (pHFDPCs and sHFDPCs) transfected with TCF-4 interference vectors were established using liposome-mediated method. Total RNA of the two DPCs was abstracted, then transfection efficiency of those three vectors was compared by realtime PCR, and the highest efficiency vectors of shRNA-1 in this reserch. The results showed that when TCF-4 was down-regulated, the expression of PI3K and β-catenin was down-regulate, GSK-3β was up-regulated and the expression of AKT had no significant change in pHFDPCs, and the expression of PI3K, β-catenin and AKT was down-regualated and GSK-3β was up-regulated in sHFDPCs.The results of this research provided experimental bases to understand the biological effects and development and growth during cyclical regulatory mechanism of TCF-4 on hair follicle cells. |
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