Effect Of Versican Gene On Agglutinative Growth Of Dermal Papilla Cells

Background and objective:Dermal papilla can induce formation of the hair follicle even when the in vitro cultured dermal papilla cells are transplanted into normal skin. Every hair follicle has its own inherent growth rhythm but asynchronous growth cycle, so periodical growth of single hair follicle is regulated by signal of self cells of many hair follicles. Currently, little has known about expression of all kinds of genes in growth cycle of hair follicle and deeper study is needed in molecular biological study of the hair follicles. However, there is no report on successful in vitro construction of the hair follicle, for many researches only simulated taction environment for follicular epithelium and mesenchyma but could not provide in vivo environment containing cytokine and extracellular matrix, which lacked basic condition for formation of the hair follicle. It is clear that cytokine and extracellular matrix play important role in development of the hair follicle. Expression of Versican gene is positively correlated with agglutinative growth of dermal papilla cells, while Wnt/β-catenin signal pathway controlled expression of Versican gene. We presumed that Versican gene is key gene controlling agglutinative growth of dermal papilla cells. In the study, by means of enzyme digestion, we effectively and conveniently isolated dermal papilla cells and cultured them in vitro to observe their biological characters. Meanwhile, Western blot, Wnt-3a adenovirus infection and siRNA method were used to explore possible role of Versican gene in agglutinative growth pattern of the dermal papilla cells so as to provide basis for further studying biological characters of the hair follicle and probing influencing factors of hair follicle formation, and give experimental evidence and theoretical support for strategy in regulating agglutinative growth of dermal papilla cells with Versican as target.Methods:By using enzyme digestion, fibroblasts including dermal papilla cells and dermal sheath cells as well as epidermal keratinocytes were in vitro cultured and passaged to investigate changes of agglutinative growth of different generation dermal papilla cells. Adenovirus plasmid pAdEasy-1/Wnt-3a was recombined and amplified. Western blot was employed to detect expression changes ofβ-catenin and Versican of different generation dermal papilla cells. At the same time, expression changes ofβ-catenin and Versican were observed in low generation and high generation dermal papilla cells, high generation dermal papilla cells induced by Wnt-3a in conditioned media with Wnt-3a and malpighian cells as well as in control group of malpighian cells, fibroblasts and dermal sheath cells. Recombinant adenovirus plasmid pAdEasy-1/Wnt-3a was used to transfect dermal papilla cells to observe the effect of exogenously overexpressing Wnt-3a on growth pattern of dermal papilla cells. mRNA expressions of Wnt-3a,β-catenin and different subtypes of Versican were detected by RT-PCR and the expression ofβ-catenin and Versican in different groups by laser confocal microscopy. Moreover, adenovirus Wnt-3a was used to infect high generation dermal papilla cells to comparatively investigate expression changes ofβ-catenin and Versican in high generation dermal papilla cells and observe growth changes of high generation dermal papilla cells after infection with Wnt-3a adenovirus. siRNA synthesis fragments were used to transfect low generation dermal papilla cells to investigate the effect of siRNA decreasing expression of Versican gene on agglutinative growth of low generation dermal papilla cells.Results:Culture results showed that dermal papilla cells were characterized by agglutinative growth, which was in accordance with previous reports. Agglutination may weaken with increased generation of cells. Western blot was employed to observe protein level changes ofβ-catenin and Versican of different generation of dermal papilla cells and showed that protein expression ofβ-catenin and Versican decreased gradually especially Versican protein with maturation of dermal papilla cells from low generation to high generation. Expressions of two genes of low generation dermal papilla cells showed obvious difference compared with fibroblasts, dermal sheath cells and malpighian cells in control group, while high generation dermal papilla cells showed very significant difference. Results of agglutinative growth mode of different generation of dermal papilla cells showed that agglutinative growth gradually weakened with increase of generation, which was roughly identical to time and amplitude of Versican gene. We successfully constructed Wnt-3a expression adenovirus. mRNA and protein expression ofβ-catenin and Versican genes after Wnt-3a infected high generation dermal papilla cells: posterior to infection with adenovirus Wnt-3a, expressions ofβ-catenin and Versican gene were up-regulated and up-regulation of Versican gene reached basically same level of low generation dermal papilla cells; posterior to infection with Wnt-3a, high generation dermal papilla cells grew at agglutinative pattern, which was similar to agglutinative growth of low generation dermal papilla cells. siRNA could depress expression of Versican gene in low generation of dermal papilla cells at a time- and dose-related fashion either at mRNA level or protein level. However, siRNA exerted no effect on expression ofβ-catenin. Versican siRNA could change growth mode of low generation of dermal papilla cells and make previous agglutinative growth mode disappear, which was similar to growth mode of high generation of dermal papilla cells.Conclusions:(1)Versican protein level is closely correlated with agglutinative growth of DPC. We proved, for the first time, the relation between Versican and agglutinative growth of DPC by studying expressions of Versican andβ-catenin in different generation of DPC. With increase of generation, the agglutinative growth of DPC tended to be slowed and expressions of DPCβ-catenin and Versican gradually decreased, especially Versican. While expression of Versican is positively related with characteristic change of agglutinative growth of DPC.(2) Signal regulation of Wnt participates in regulation of characteristic agglutinative growth of DPC. Over expression of Wnt-3a can markedly induce agglutinative growth of DPC, which is possibly related to regulating expressions of downstream-related genesβ-catenin and Versican at certain threshold value. Conditioned medium of KC can partially induce agglutinative growth of DPC, which is due to the reason that KC contains some cytokines.(3) Wnt-3a has definite regulative effect on DPC expressingβ-catenin and Versican. Over expression of Wnt-3a can induce up-regulation of expressions ofβ-catenin and Versican, especially that of Versican, in human DPC. In the meantime, gene and protein levels ofβ-catenin and Versican change dissymmetrically, which can preliminarily make clear that the course of Wnt-3a regulating Versican protein level is mainly regulating transcription and affected by multiple factors. (4) Specific knock-down of gene expression of DPC Versican by means of RNA interference technique,results in decrease of expression of low generation of DPC Versican protein, at a time- and dose-dependent fashion. Subtype V0/V1 of Versican exerts remarkable effect on agglutinative growth of DPC, while subtypes V2 and V3 may be excluded from such regulation course. When expression of low generation DPC Versican is knocked down, the agglutinative growth of DPC gradually disappears, which further proves critical role of Versican gene in agglutinative growth of DPC.