Regulatory Role Of Dlx3 On The Proliferation Of Arbas Cashmere Goat Dermal Papilla Cells

The dermal papilla cells are known as the regulatory center of the hair follicle development cycle.Many signal pathways are involved in the regulation of the proliferation of dermal papilla cells,and thus affect the growth and development of hair follicles.Goat hair follicles have two types,namely primary hair follicles and secondary hair follicles.Primary hair follicles produce wool and secondary hair follicles produce cashmere.In this experiment,the primary dermal papilla cells and secondary dermal papilla cells were used as cell models to construct a Dlx3 gene overexpressed cell line and Dlx3 gene interfering cell line.The purpose of the study was to observe the effect of Dlx3 gene on the proliferation of dermal papilla cells,so that to provide experiment basis for understanding the regulation of related gene expression on hair follicle growth and development in the future.1.Cultivation and identification of primary and secondary dermal papilla cells in Arbas Cashmere GoatThe primary and secondary dermal papilla cells of Arbas cashmere goats were successfully cultured in DMEM medium containing 15%FBS.?-SMA and CD144 markers were used to detect the cultured cells by fluorescence immunochemistry.The results showed that more than 90% of the cells showed positive expression of ?-SMA and CD144 markers,indicating that the cultured cells were dermal papilla cells,which could be used in the study of related experiments.2.Construction and transfection of over expression vector and interference vectorTwo kinds of vectors were successfully constructed by gene fragment cloning,enzyme digestion,connection and transformation,which were Dlx3 gene overexpression vector and Dlx3 gene interference carrier.The transfection method of electrotransfer was used to transfer the overexpressed vector of Dlx3 gene and the plasmid of interference carrier into primary and secondary dermal papilla cells.After successful transfection,the Dlx3 gene overexpressed cell line and Dlx3 gene interfering cell line were established.3.The effect of Dlx3 gene on proliferation and apoptosis of dermal papilla cellsThe expression levels of Dlx3,TP63,Hoxc13 and Lef1 genes related to hair follicle development were detected by q-PCR and Western Blot.CCK8 was used to detect the effect of overexpression and interference of Dlx3 gene on cell proliferation.Apoptosis was detected by the expression of Dlx3 gene and the effect of interference on the apoptosis of dermal papilla cells.Primary and secondary dermal papilla cells without transferring plasmid were used as experimental control group.In primary dermal papilla cells,the Dlx3 gene expression in the Dlx3 gene overexpression experimental group and the DLX3 gene interfering experimental group was 1.53 and 0.18 times of the control group respectively.In the secondary dermal papilla cells,the Dlx3 gene expression in the Dlx3 gene overexpression experimental group and the Dlx3 gene interfering experimental group was 1.12 and 0.10 times of the control group respectively.The results indicated that Dlx3 gene overexpressing cell line and Dlx3 gene interference cell line were successfully constructed in two the dermal papilla cells.The expression of TP63 gene in the Dlx3 gene overexpressed primary and secondary dermal papilla cells was 0.14 and 0.15 times of the control respectively,and the expression in Dlx3 gene interfering primary and secondary cells was 1.19 and 3.11 times of the control respectively.The expression of Hoxc13 gene in the overexpressed primary and secondary dermal papilla cells was 2.66 and 2.04 times of the control respectively,and the expression in the interfered primary and secondary cells was 0.20 and 0.19 times of the control respectively.Over expression and interference of Lef1 gene in primary dermal papilla cells were 3.08 and 0.27 of the control.Over expression and interference of Lef1 gene in secondary dermal papilla cells were 1.81 and 0.14 times of the control.By analyzing the gene expression of Dlx3,Hoxc3,Lef1 and TP63,the expression trend of Dlx3 gene was consistent with the change trend of Hoxc13 and Lef1 gene,so it showed that the expression of Dlx3 gene was positively related to the Hoxc13 and Lef1 gene.The expression of TP63 gene was negatively correlated with Dlx3.By comparing the expression of Hoxc13,Lef1,and TP63 gene in the overexpressed and interfered cell lines of the Dlx3 gene,the expression of Hoxc13 gene,Lef1 gene and TP63 gene in primary dermal papilla cells and secondary dermal papilla cells was different.The results of CCK8 proliferation test showed that in primary dermal papilla cells,the proliferation rates of Dlx3 gene overexpression group in 24 h,48h and 72 h were significantly higher than that of the experimental control group,and the proliferation rate in 48 h was significantly increased,while the proliferation rates of the interference group were significantly lower than that of the experimental control group.In primary dermal papilla cells,the proliferation rate of Dlx3 gene overexpression group was higher than that of the experimental control group,while the proliferation rate of the interference group was lower than that of the experimental control group.In the primary dermal papilla cells,the apoptosis rate of Dlx3 overexpressed cells was 0.45 times of the control,and the apoptosis rate of Dlx3 interfered cells was 12.5 times of the control.In the secondary dermal papilla cells,the apoptosis rate of Dlx3 overexpressing cells was 0.86 times of the control group,and the apoptosis rate of Dlx3 interference cells was 1.61 times of the control group.It could be concluded that over expression of Dlx3 gene could inhibit cell apoptosis and reduce cell apoptosis rate,while Dlx3 gene interference could induce apoptosis rate increasing significantly.The results of this research indicated that Dlx3 gene played a regulatory role in the development of hair follicles by increasing primary and secondary dermal papilla cells to proliferate and inhibit apoptosis.