Effect Of Connexin 40 On The Proliferation Of Dermal Papilla Cells In Albas Cashmere Goats

Cashmere produced by Inner Mongolia Albas Cashmere Goat is a high-grade textile raw material with high economic value,known as"soft gold."Cashmere goat hair follicles(HF)are divided into primary and secondary HF,and primary HF produces wool and secondary HF produces cashmere.The development of secondary HF have an annual cyclic periodicity with three periods every year,which are anagen from August to October,catagen from December to February(the next year),as well as telogen from February to July.Dermal papilla cells(DPCs)accumulate at the bottom of HF and are one of the key cells in the regulation of hair follicle cycling cycle and hair growth.Connexin 40(Cx40)belongs to a member of the gap junction protein family with a molecular weight of 40k Da and can regulate gap junction channels(GJCs),which one of the most important functions is the direct communication of hydrophilic small molecules,atoms,and ions between cells.Among the differentially expressed genes between anagen and telogen in secondary HF obtained by high-throughput transcriptome sequencing,the expression of Cx40 in anagen was much higher than that in telogen,which indicated that this molecule played an important role in the process of hair follicle growth and development.The DPCs are located at the bottom of HF,and their proliferation is closely related to hair follicle growth and development.Therefore,the aim of this experiment was to investigate the effect of Cx40 on dermal papilla cells proliferation in cashmere goats.This study was carried out from three aspects as follows,and the experimental materials were dorsal skin tissue as well as primary dermal papilla cells(PHF-DPCs)and secondary dermal papilla cells(SHF-DPCs)in Albas Cashmere Goats.1.Morphological observation of HF and culture and identification of DPCs in cashmere goatsThe dorsal skin tissue samples of cashmere goats were collected,paraffin sections were prepared and HE staining was performed.The results showed that the number of HF was more than that in telogen,and the hair ball morphology was expanded and full in anagen compared with that in the telogen.The DPCs were cultured with DMEM/F12 culture medium containing 10%FBS,and their status was observed at 24h,48h,and 72h.The results showed that the growth of DPCs was agglutinated,and the growth rate of SHF-DPCs was faster than that of PHF-DPCs.To test the accuracy of the cells used in the experiment,immunofluorescence was performed with the DPCs surface-specific marker proteinsα-SMA and Lam,and the results were positive.2.Effect of positive and negative regulated Cx40 on the proliferation of DPCs in cashmere goatsIn terms of cell model preparation,Cx40 overexpression vector and interference vector were transfected into PHF-DPCs and SHF-DPCs respectively through Lipofectamine~?2000 Reagent,then c DNA and protein samples were extracted,and the m RNA and protein expression of Cx40 were detected by real-time quantitative PCR and Western blot.The results showed that the expression levels of Cx40 m RNA and protein were increased in DPCs transfected with overexpression vector,and decreased in DPCs transfected with interference vector;The interference vector was transfected into DPCs first,and then the overexpression vector was transfected for rescue experiment,and the results showed that the m RNA and protein expression levels of Cx40 were restored,indicating that the overexpression and interference Cx40 DPCs models were successfully constructed.In terms of proliferation ability detection,CCK-8 and Ed U proliferation assays were adopted to study the effect of Cx40 on the cell proliferation of PHF-DPCs and SHF-DPCs.The results presented that the proliferation of DPCs transfected with Cx40 overexpression vector was increased compared with the control,while given that transfected with Cx40 interference vector,the proliferation of DPCs was decreased compared with the control,meanwhile,the proliferation of DPCs in the rescue assay was restored.These results indicated that Cx40 positively regulated the proliferation of DPCs.3.Study on signaling pathway of cashmere goat DPCs regulated by Cx40In this study,three signaling pathways(PI3K/AKT,ERK/MAPK and p38/MAPK)closely related to cell growth,differentiation,proliferation and migration were selected to detect the effect of Cx40 on them.The results suggested that Cx40was available to achieve the proliferation of DPCs by elevating the phosphorylation levels of AKT,ERK and p38 pathways.The results of this study indicated that Cx40 positively regulated the proliferation of PHF-DPCs and SHF-DPCs to promote the development of HF,and the effect was related to the increase of phosphorylation levels of AKT,ERK and p38signaling pathways.