Introducation Of Mouse Dermal Fibroblasts Into Dermal Papilla-like Cells By Versican Over-expression

The dermal papilla(DP)are a specialized group of fibroblasts located at the base of the hair follicle and are important in guiding hair development,morphogenesis and follicle regeneration.DP cells are difficult to obtain and gradually lose their ability to induce hair regeneration during in vitro two-dimensional culture.Versican is an important marker for identifying the location,shape and size of DPs.The expression of Versican is closely related to the hair induction ability of epidermal hair papilla cells during the anagen phase.Studies have shown that Versican expression in DP cells(DPCs)decreases with increasing generations of in vitro culture and is restored by three-dimensional culture.This thesis aims to investigate the possibility of overexpression of Versican in fibroblasts and the transformation of fibroblasts into DP cells under three-dimensional culture conditions,in order to obtain a large number of hair papilla-like cells with hair-inducing ability.The main studies are as follows:1)Fibroblasts were obtained from the skin of C57 neonatal mice by enzymatic digestion and identified using immunofluorescence staining;Versican V3 overexpression vector was constructed and transfected into fibroblasts and validated by Q-PCR.2)The overexpression vector-transfected DPC spheres were grown on low-adhesion plates coated with soft agarose membranes in inclined culture to form three-dimensional spheres,and whether the overexpression vector-transfected fibroblasts transformed to hair papilla cells after sphere-forming culture was investigated by Q-PCR and cellular immunofluorescence at the gene level and protein level.3)The over-expression vector-transfected fibroblast spheroids and mouse epidermal cells obtained in the above experiments were mixed and injected into the subcutis of nude mice for three weeks to directly observe their hair growth,and to observe the hair follicle structure at the injection site by sectioning and H&E staining.In this thesis,it is verified that the expression of versican in DPCs decreases rapidly over passages.A versican overexpression vector was constructed and the overexpression vector-transfected fibroblasts formed cell spheres in tilted culture,in which DPC-associated marker genes are upregulated,and particularly the expression pattern of WNT signaling pathway gene is similar to that of DPCs.In nude mice melanin precipitation was noticed in the versican overexpression group and H&E staining revealed hair follicle structures at the injection site,which demonstrates the possibility of inducing fibroblasts into DPC-like cells by overexpression of versican.