| Objective: 1.The quality and yield of wool are very important to textile industry,and there are significant differences in wool properties and yield between small-tailed Han sheep and Suffolk sheep.In this study,RNA-seq and proteome sequencing were used to explore the genes that cause wool traits and growth differences,and to understand the related signal pathways that affect hair follicle growth and development.2.Taurine(Tau)is widely distributed in animals in free form and participates in the maintenance and regulation of almost all normal physiological functions.In addition,Tau is added to some shampoos as an effective ingredient to improve hair growth,suggesting that Tau may have an effect on hair follicle development,but its mechanism is not clear.And so far,the effect of Tau on animal hair has been poorly studied.In this paper,sheep skin fibroblasts,mouse animal models and sheep hair follicles cultured in vitro were used as research objects to explore the effect of Tau on the growth of hair follicles.The model of androgenetic alopecia(AGA)in mice was established to explore the resistance effect of Tau on AGA.Methods: 1.The growth rate of hair on the side of Suffolk sheep and small-tailed Han sheep was detected.The body side skin of Suffolk sheep and small-tailed Han sheep were selected as experimental materials,and the skin tissue morphology and structure of Suffolk sheep and small-tailed Han sheep were observed by HE staining.2.The body side skin of Suffolk sheep and small-tailed Han sheep were selected as experimental materials,and the differences of trait related gene and protein expression between the skin tissue of Suffolk sheep and small-tailed Han sheep were compared by RNA-Seq and proteome sequencing.3.We narrowed the screening threshold of RNA-seq,compared the differences of hair growth-related genes expression between Suffolk sheep and small-tailed Han sheep,and verified the most significant signal pathway by immunohistochemistry.4.Sheep skin fibroblasts were cultured in vitro,and the effects of Tau on sheep skin fibroblasts and the expression of genes related to sheep hair follicle development were studied by immunofluorescence,CCK-8 test,cell scratch,flow cytometry,trypan blue staining and RNA-seq.5.The animal model of C57BL/6 female mice was established.The mice were randomly divided into three groups,with 8 mice in each group,which were divided into negative control group(fed with normal drinking water),experimental group(fed with drinking water containing 1% Tau)and positive control group(locally coated with 2% minoxidil).HE staining,Ki67 immunofluorescence and ELISA were used to explore the promoting effect of Tau on hair follicle growth.6.The AGA model of C57BL/6 male mice was established.Mice were randomly divided into five groups: negative control group(intraperitoneal injection of normal saline),model group(intraperitoneal injection of dihydrotestosterone(DHT)1 mg/d/Wt),low-dose group of treatment group(intraperitoneal injection of DHT 1 mg/d/Wt+Tau 2 mg/d/Wt),high-dose group of treatment group(intraperitoneal injection of DHT 1 mg/d/Wt+Tau 10 mg/d/Wt)and positive control group(intraperitoneal injection of DHT 1 mg/d/Wt+ local application of 2% minoxidil).HE staining,ELISA test,Ki67 immunofluorescence staining,Tunel apoptosis test,q RT-PCR and Western blot were used to explore the resistance of Tau to AGA.7.Sheep hair follicles cultured in vitro were divided into two groups: A(Tau group and Con group)and B(negative control group,DHT group,treatment group and positive control group).The effects of Tau on the growth of sheep hair follicles and its resistance to AGA were analyzed respectively.Results:1.In the same growth time,Suffolk sheep’s wool length is longer than that of small-tailed Han sheep,indicating that it grows faster;Morphological observation showed that compared with small-tailed Han sheep,Suffolk sheep had more hair follicles,smaller diameter and higher density.2.The results of RNA-Seq screening of genes related to hair traits showed that 1213 differential genes were screened based on the difference multiple FC≥1.4 or FC≤0.714 and P<0.05,among which 644 genes were up-regulated and 569 genes were down-regulated in Suffolk sheep compared with small-tailed Han sheep.Screening proteins related to hair traits by proteome sequencing showed that 99 differentially expressed proteins were screened based on the difference multiple FC≥1.4 or FC≤0.714 and P<0.05,among which 47 proteins were up-regulated and 52 proteins were down-regulated in Suffolk sheep compared with small-tailed Han sheep.Combined analysis of RNA-Seq and proteome sequencing showed that the intermediate filament was significantly enriched,and the genes involved were KRT35,KRT13 and KAP13-1-like.PPAR signaling pathway was significantly enriched,and the gene involved was FABP4.3.The results of RNA-seq screening of hair growth-related genes showed that 496 differentially expressed genes were screened based on the difference multiple │log2(FC)│≥1 and P<0.05,among which238 genes were up-regulated and 258 genes were down-regulated in Suffolk sheep compared with small-tailed Han sheep.Hair cycle and hair development function were significantly enriched,and the genes involved were KRT16 and Wnt10 a.KEGG enrichment showed that drug metabolism-cytochrome P450,platelet activation,linoleic acid metabolism,taurine and hypotaurine metabolic signal pathways were significantly enriched.The results of immunohistochemical test showed that Tau was mainly located in hair matrix,inner and outer root sheaths,hair shaft,epidermis and sebaceous glands,and the content in hair papilla was relatively small.The results of optical density analysis showed that the content of Tau in the skin tissue of Suffolk sheep was significantly higher than that of small-tailed Han sheep,which was 1.9times that of small-tailed Han sheep,and the difference was extremely significant(P<0.01).4.The results of cytological test showed that the cultured cells were fibroblasts.Tau could enter fibroblasts through cell membrane,and could promote the proliferation and migration of sheep skin fibroblasts and inhibit apoptosis.Adding too much Tau to fibroblasts would lead to a large number of cell deaths.After adding proper amount of Tau to the cells,The results of RNA-seq showed that 81 differential genes were screened according to the differential multiple │log2(FC)│≥1 and P<0.05,among which 50 genes were up-regulated and 31 genes were down-regulated in the experimental group compared with the control group.The proliferation item of fibroblasts was significantly enriched.KEGG enrichment showed that linoleic acid metabolism,caffeine metabolism,steroid hormone biosynthesis,arachidonic acid metabolism and insulin secretion were significantly enriched.The results of RNA-seq after excessive Tau was added to the cells showed that 5499 differential genes were screened according to the differential multiple │log2(FC)│≥1 and P<0.05,among which 2088 genes were up-regulated and 3411 genes were down-regulated in the experimental group compared with the control group.Cell cycle-related signal pathways were significantly enriched.5.The results of mouse zoology experiments showed that the hair growth rate of the experimental group and the positive control group were obviously faster than that of the negative control group.The results of HE staining showed that the number of hair follicles and dermal thickness in the experimental group and the positive control group were extremely significantly higher than those in the negative control group(P<0.01).Ki67 immunofluorescence staining showed that the fluorescence intensity and the number of positive cells in the skin of mice in the experimental group and the positive control group were significant(P<0.05)and extremely significant(P<0.01)higher than those in the negative control group.The results of ELISA showed that the contents of linoleic acid,caffeine,arachidonic acid and insulin in the skin of the experimental group were significantly(P<0.05)and extremely significant(P<0.01)higher than those in the negative control group.The content of steroid hormones was lower than that of negative control group,and the difference was significant(P<0.05).The contents of serum linoleic acid and insulin in the experimental group were higher than those in the negative control group,with no significant difference(P>0.05).The contents of arachidonic acid and caffeine were significantly(P<0.05)and extremely significant(P<0.01)higher than those in the negative control group.The content of steroid hormones was lower than that of negative control group,and the difference was significant(P<0.05).6.The research results of androgen alopecia mouse model show that the hair growth rate of negative control group,treatment group and positive control group is faster than that of model group.The results of HE staining showed that the dermal thickness,the number of hair follicles and the depth of hair roots in the negative control group,the treatment group and the positive control group were all higher than those in the model group,and the difference was extremely significant(P<0.01).The results of Ki67 immunofluorescence showed that the number of skin staining positive cells in negative control group,treatment group and positive control group was higher than that in model group,and the difference was extremely significant(P<0.01).Tunel results showed that the number of positive cells in the skin of mice in negative control group,treatment group and positive control group was extremely significantly lower than that in model group(P<0.01).The results of q RT-PCR and Western blot showed that the expressions of AR,TGF-β1,TGF-β2 and DKK1 in the skin of mice in negative control group,treatment group and positive control group were significantly(P<0.05)and extremely significant(P<0.01)lower than those in model group.7.The research results of sheep hair follicles in vitro showed that the growth rate and survival rate of hair follicles in Tau group were extremely significantly higher than those in Con group(P<0.01);The growth rate of hair follicles in negative control group,treatment group and positive control group was significantly higher than that in DHT group,and the difference was extremely significant(P<0.01).Conclusion: 1.Compared with small-tailed Han sheep,Suffolk sheep may have faster wool growth,more wool yield and better wool quality.It shows that there are differences in wool development between Suffolk sheep and small-tailed Han sheep.2.The results of RNA-seq and proteomic sequencing showed that there were significant differences in key genes affecting wool traits(Bending,diameter,hardness and toughness),such as KRT35,KRT13,KAP13-1-like and FABP4.3.RNA-seq analysis showed that the genes that caused the hair growth difference between Suffolk sheep and small-tailed Han sheep might be Wnt10 a and KRT16;Signal pathways may be drug metabolism-cytochrome P450,platelet activation,linoleic acid metabolism,Taurine and hypotaurine metabolism signal pathways.4.Tau can promote hair follicle growth.It may promote the growth of sheep hair follicles by promoting the proliferation,migration and inhibiting the apoptosis of sheep skin fibroblasts.In addition,Tau may promote the proliferation of mouse skin hair follicle matrix keratinocytes.And increase the content of linoleic acid,caffeine,arachidonic acid and insulin in skin and serum,and reduce the content of steroid hormones to promote hair follicle growth.5.Tau can resist AGA,possibly through two pathways,one of which may be by reducing the expression of AR induced by DHT.Another way may be to reduce the expression of TGF-β1,TGF-β2 and DKK1 induced by DHT,which counteracts the inhibition of keratinocyte growth and the induction of apoptosis.6.Tau can promote the growth of sheep hair follicles in vitro and improve the survival rate of hair,and at the same time,it has certain resistance to the growth inhibition of sheep hair follicles caused by DHT. |
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