| Inner Mongolia Arbas Cashmere Goat is famous for its high cashmere yield and good quality.At present,the research on the mechanism of cashmere growth has attracted more and more attention.This research not only can deeply understand the regulation mechanism of villi,but also has important significance in the breeding work of cashmere goats and related scientific research.In this study,primary hair follicle dermal papilla cells(PHF-DPC)and secondary hair follicle-dermal papilla cells(SHFDPC)which are closely related to the growth and development of cashmere were selected as the cell model,the hair follicle development related genes SPP1(Secreted phosphoprotein 1)and HGF(hepatocyte growth factor)were selected to constructe SPP1 and HGF gene overexpression and interference vectors,then transfected PHFDPC and SHF-DPC to observed the effects of over expression and low expression of target genes on the proliferation of dermal papilla cells,so that to provide experimental basis for further research on the regulation of hair follicle growth and development by related genes.1.Cultivation and identification of PHF-DPC and SHF-DPC of Inner Mongolia Albas Cashmere GoatsPHF-DPC and SHF-DPC were cultured in DMEM medium containing 10% FBS,and the two kinds of cells were identified by cell immunofluorescence assay.The results showed that both cells displayed ?-SMA and CD144 marker protein positive expression,demonstrating that the cultured cells were indeed PHF-DPC and SHF-DPC,which could be used for further experimental studies.2.Construction of overexpression vector and interference vectorThe target genes were cloned,and the overexpression vector and interference vector of SPP1 gene and HGF gene were successfully constructed by restriction enzyme digestion and ligation experiments.3.Transfection and detection of SPP1 gene overexpression and interference vector in the cellsThe SPP1 gene overexpression vector and interference vector were successfully transferred into PHF-DPC and SHF-DPC by liposome transfection.The expression of SPP1 gene was detected by q-PCR and Western Blot in cells successfully transfected with both vectors.Results displayed that the expression levels of SPP1 overexpression and interference group in PHF-DPC were 14.03 and 0.89 times than that of the experimental control group,respectively.In SHF-DPC,the expression levels of the SPP1 overexpression and interference groups were 8.19 and 0.37 times than that of the control group,respectively.The results showed that the cell lines with overexpression and interference of SPP1 gene were successfully established.4.Regulation of SPP1 gene on proliferation of DPCThe proliferation detection of CCK8 showed that in PHF-DPC,the proliferation rate of SPP1 gene overexpression group was significantly higher than that of the experimental control group at the first day,the second day and the third day,and the proliferation rate was particularly accelerated at the second day,while that of the interference group was significantly lower than that of the experimental control group.In SHF-DPC,the proliferation rate of the SPP1 gene overexpression group was also 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.The effect of SPP1 gene on the proliferation of PHF-DPC and SHF-DPC was further detected by EdU assay,and PHF-DPC and SHF-DPC without any plasmid transfection were used as the control group.The results showed that the number of EdU positive cells in the DPC transfected with SPP1 overexpression vector was significantly higher than that in the control group,and the number of EdU positive cells transfected with SPP1 interference vector was significantly lower than that in the control group.The results showed that the overexpression of SPP1 gene promoted the proliferation of DPC,while the proliferation of DPC decreased after the interference of SPP1 gene.The expression levels of CD44,IL-6 and ITGB1 genes associated with DPC proliferation were detected by q-PCR and Western Blot,and cells not transfected with any plasmid were used as experimental control groups.The results showed that in the DPC transfected with SPP1 gene overexpression vector,the expression levels of CD44,IL-6 and ITGB1 increased,while in the DPC transfected with SPP1 gene interference vector,the expression levels of CD44,IL-6 and ITGB1 decreased.The results showed that the changes of SPP1 gene expression in the two DPCS were consistent with the changes of CD44,IL-6 and ITGB1 gene expression.It was proved that SPP1 gene was positively correlated with the expression of CD44,IL-6 and ITGB1 genes in the two DPCS.The results further indicated that SPP1 gene had a positive regulatory effect on DPC proliferation.5.Transfection and detection of HGF gene overexpression vector and interference vector in the cellsHGF gene overexpression vector and interference vector were successfully transferred into PHF-DPC and SHF-DPC by liposome transfection.Changes in HGF gene expression were detected by q-PCR and Western Blot in cells successfully transfected with the two vectors.PHF-DPC and SHF-DPC without any vector transfection were used as the control group.It was found that the expression levels of HGF overexpression and interference groups in PHF-DPC were 2.98 and 0.46 times as high as those in the control group,while in SHF-DPC,the expression levels of HGF overexpression and interference groups were 18.83 and 0.62 times as high as those in the control group.The results showed that HGF gene overexpression and interference cell lines were successfully established.6.Regulation of HGF gene on proliferation of DPCThe proliferation detection of CCK8 showed that in SHF-DPC,the proliferation rate of the overexpression group of HGF gene was significantly higher than that of the experimental control group,and the proliferation rate was significantly accelerated at the second day,while the proliferation rate of the interference group was significantly lower than that of the experimental control group.In PHF-DPC,the proliferation rate of the HGF gene overexpression group was also 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.The effect of HGF gene on the proliferation of PHFDPC and SHF-DPC was further detected by EdU assay.PHF-DPC and SHF-DPC without any plasmid transfection were used as the control group.The results displayed that according to the observation of cell growth,the number of EdU positive cells in the DPC transfected with HGF overexpression vector was significantly higher than that in the control group,and the number of EdU positive cells in the DPC transfected with HGF interference vector was significantly lower than that in the control group.The results showed that the overexpression of HGF gene promoted the proliferation of DPC,while the proliferation of DPC decreased after the interference of HGF gene.The expression levels of TGF-? and HGF gene-specific receptor C-met genes associated with DPC proliferation were detected by q-PCR and Western Blot.The results showed that the expression of C-met gene was increased and the expression of TGF-? gene was decreased in DPC transfected with HGF gene overexpression vector.The expression of C-met gene in DPC transfected with HGF gene interference vector was decreased,and TGF-? gene expression was increased.The results showed that the expression of HGF gene in both DPCs was positively correlated with the expression of C-met gene and negatively correlated with TGF-?.The above experimental results show that both SPP1 and HGF genes and their related genes play an important role in the proliferation of DPC,and SPP1 and HGF genes play a positive role in the proliferation of DPC.In the dermal papilla cells,SPP1 gene is positively correlated with CD44,IL-6 and ITGB1 genes.HGF gene is positively correlated with C-met gene and negatively correlated with TGF-? gene.The experimental results further indicated that the SPP1 and HGF genes had a positive regulatory effect on the proliferation of DPC. |
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