Expression Of NGF In Skin Tissue Of Cashmere Goat And Its Effect On Proliferation Of Outer Root Sheath Cells

The growth and development of secondary hair follicles of cashmere goats are closely related to the quantity and quality of hair production.Development of hair follicles in mammals are generally experienced in three cycles(telogen? agaen?catagen),which are the result of a series of signal molecule interactions.In the development of hair follicles,the proliferation of Outer root sheath(ORS)cells is considered to be one of the important power sources of hair follicle growth and development.it is a series of signal molecules interact with each other control results.In this process,the proliferation of Outer root sheath(ORS)cells is considered to be one of the important sources of hair follicle growth and development.Recent studies have found that nerve growth factors in addition to the role of the nervous system,but also in some non-nervous system to play a biological function,Studies have found that nerve growth factor(NGF)and its receptors exist in different stages of mouse hair follicle development,and NGF may affect the morphological changes of hair follicles in mice,suggesting that NGF is a key factor in the development of mouse hair follicles during the development of hair follicles in mice.The following questions are worthy of further study:(1)whether NGF and its receptor tyrosine kinase A(tyrosine kinase A,TrkA)is present in the skin follicles of cashmere goats,and if so,what are their functions?(2)Are cashmere goat ORS cells a NGF-secreting cell or target cell and are involved in the proliferation of ORS cells? In view of the above hypothesis,this paper has carried on the system research,the result is as follows: 1.The expression of NGF and its receptor TrkA in the skin tissue of cashmere hair growth cycleRT-PCR was used to detect the expression of NGF and its receptor TrkA mRNA in skin tissues.The expression of NGF and its receptor TrkA in skin tissue of the three stages of hair follicle were analyzed by real-time fluorescence quantitative PCR and Western blot.The results show: The mRNA of NGF and its receptor TrkA is present in the skin tissues of various stages of follicular growth cycle.The expression level of NGF and its receptor TrkA mRNA in the agaenperiodskin tissue was significantly higher than that in the telogen and catagen(p <0.05).The level of NGF protein in the agaenperiod skin tissue was significantly higher than that in the telogen and catagen(p <0.05),There was no significant difference in the protein level of TrkA during the agaen,but significantly higher than that of the catagen(p <0.05).The morphological changes of hair follicles in three stages of hair follicle growth were observed by HE staining.The results show: Primary hair follicle(PF)and secondary hair follicle(SF)were more than the telogen and catagen,and the follicle structure was complete and clear.The number of PF and SF decreased significantly during the catagen.In the telogen,the number of PF and SF is small,and the ORS and IRS layers of PF are clearly visible,but the ORS and IRS layers of SF are not clear.The proteins of NGF and TrkA were localized by indirect immunofluorescence.The results show: NGF and TrkA proteins are present at all stages of PF and SF follicle growth cycles;During the agaen,high expression of NGF was detected in ORS and IRS cells,and positive for TrkA was detected in ORS cells.The signal intensity is the highest in the agaen,followed by the telogen and the lowest catagen.2.Expression of NGF and its receptor TrkA in secondary hair follicle-derived ORS cells in vitroThe isolation and culture conditions of ORS cells separated from SF in theagaen period.The results showed that the optimized neutral protease digestion method was in accordance with the requirement of in vitro culture from the point of view of isolation.The isolated ORS cells were cultured in medium for 2 to 3 days,and the cells were polygonal and few were oval.The results of the specific protein CK19 showed that the purity of ORS cells was more than 90%.The immunological activity of NGF and TrkA was detected by indirect immunofluorescence.The signal intensity of NGF in the nucleus was higher than that in cytoplasm,and there was no immunological activity of TrkA in the nucleus.The results of ELISA showed that ORS cells could secrete NGF in vitro,but the secretion was decreased significantly with the increase of in vitro culture time.3.Effect of NGF on Proliferation of Secondary Hair Follicle ORS Cells in Cashmere GoatsThe effect of NGF on the proliferation of ORS cells was studied by addition of exogenous NGF recombinant protein and its receptor blocker K252 a.The results show: 20 ng / mL or 100 ng / mL NGF recombinant protein could promote the proliferation of secondary hair follicle ORS cells in cashmere goats,and the promotion of proliferation induced by NGF disappeared when treated with 20 ng / mL K252 a.Indicating that the NGF / TrkA system can promote the proliferation of ORS cells in a dose-dependent manner.In addition,we investigated the presence or absence of cAMP-response element binding protein(CREB)in ORS cells by immunofluorescence.The results show: The immunoreactive signal of CREB was detected in ORS cells,and the signal intensity in the nucleus was stronger than that in the cytoplasm.Exogenous addition of 20 ng / mL or 100 ng / mL NGF recombinant protein can significantly improve the activity of CREB in cells,and NGF could inhance the activity of intracellular CREB throght binding with TrkA.Therefore,The NGF/TrkA system plays an important role in the maintenance and promotion of PCNA expression by studying the mRNA expression of proliferating cell nuclear antigen(PCNA).In summary,the presence of NGF and its receptor TrkA mRNA and protein in cashmere hair follicle tissue was confirmed in this study,and significantly higher in agaen than in other periods.The proteins of NGF and its receptor TrkA can be detected in outer root sheath cells cultured in vitro,and the outer root sheath cells can secrete NGF in vitro.In addition,we also found that the NGF / TrkA pathway affects the activity of transcription factor CREB,by whichNGF modulates the expression of proliferating-related gene PCNA.The results of this study elucidated the expression of NGF in hair follicles and its functuion,which might provided a novol way for further study on the growth mechanism of hair.