Optimization Of A Conditioned Medium For Hair Follice Stem Cell Of Albas Cashmere Goat And Effects Of Small Compound On Its Growth Characteristics

Inner Mongolia Albas cashmere goat is one of the precious genetic resources in China.It is concentrated in the western part of Inner Mongolia Autonomous Region and is famous for its excellent cashmere quality.Cashmere are fibers grown from skin hair follicles,and their growth,repair and self-renewal are closely related to the periodic proliferation,differentiation and migration of hair follicles stem cells.It is of great significance to understand the mechanism of hair follicle stem cells in regulating the growth of cashmere to improve the yield and quality of cashmere.In this study,Albas cashmere goat hair follicle stem cells(gHFSCs)were used as the research objec to optimize their conditional culture system and explore the effects of small molecular compounds on their growth characteristics,in order to lay a foundation for the study of cashmere growth mechanism of Albas cashmere goat,to provide theoretical basis for breeding work.In this study,the purification system of hair follicle stem cells of Albas cashmere goats was established,and the cells were identified from the aspects of cell morphology and molecular markers.By observing the effects of primary hair follicle stem cells(PHFSCs)and secondary hair follicle stem cells(SHFSCs)cultured in different media,the most suitable medium for the growth of hair follicle stem cells of Albas cashmere goats was found,and the largest number of hair follicle stem cells with high purity could be obtained in the shortest time Furthermore,small molecular compounds were added into the primary hair follicle stem cell culture medium,and the changes of PHFSCs were detected by observing the morphological characteristics of cells and detecting the changes of hair follicle stem cell markers genes so as to explore the influence of a certain concentration of Y-27632 on the growth characteristics of primary hair follicle stem cells of Albas cashmere goats1.Purification and identification of gHFSCsPHFSCs and SHFSCs with high purity and high activity were obtained by differential trypsin digestion and rapid adherent method of type IV collagen.The cells were identified by observing the morphological characteristics of cells and detecting the expression of marker genes at different levels by real-time quantitative PCR and Western blot.The results showed that the purified cells were consistent with the morphological characteristics of hair follicle stem cells,and the hair follicle stem cell marker genes of p63,CD34,Itgβ1,Lgr5,K19 and Sox9 were highly expressed at the transcription level and protein level.The cells were identified as Arbas cashmere goat hair follicle stem cells.2.Screening of optimal serum concentration of gHFSCs medium and establishment of serum reducing culture systemIn this study,we observed the morphological characteristics of PHFSCs and SHFSCs cultured in different media,and detected the proliferation activity and clone formation rate.The results showed that the growth morphology of PHFSCS and SHFSCs in DMEM/12+6% FBS medium was the best,and the proliferation activity and clone formation rate were significantly higher than those in DMEM/F12+2%FBS medium and DMEM/F12+10% FBS medium.FBS concentration in 6% was selected as the optimal serum supplemental concentration for gHFSCs in basic medium.And the morphology of PHFSCs and SHFSCs in KSR+2% FBS medium was good,and the proliferation activity and clone formation rate were not significantly different from those in KSR+6% FBS and DMEM/F12+6% FBS medium.It was proved that PHFSCs and SHFSCs could grow well in serum reduced medium.3.Study on the effect of rock inhibitor Y27632 on PHFSCsThe effects of different concentrations of Y-27632 on the growth characteristics of PHFSCs were further explored by observing the morphology of PHFSCs,detecting the proliferation activity,colony formation rate,cryopreservation recovery rate and 24-hour adherent rate.The results showed that PHFSCs with the addition of 10 μM Y-27632 in the medium had good morphology,and the morphology of hair follicle stem cells with the expansion to the 11 th generation still maintained good morphological characteristics,The proliferation rate and clone formation rate of PHFSCs were significantly higher than those of the control group and PHFSCs treated with 20 μM Y-27632.The number of gHFSCs treated with 20 μM Y-27632 showed a negative growth and few clones were formed.When 10 μM Y-27632 was added during cryopreservation,the resuscitation rate and 24-hour adherence rate of PHFSCs were significantly higher than those of control group and 20μM Y-27632 treatment group.Real-time quantitative PCR and Western Blot were used to detect the expression of hair follicle stem cell markers at different levels.The results showed that the gHFSCs treated with 10 μM Y-27632 overexpressed the marker genes p63,CD34,Itgβ1,Lgr5,K19,Sox9.It was proved that 10 μM Y-27632 could promote the growth and proliferation of PHFSCs,maintain the stem cell characteristics and protect the cells during cryopreservation.The growth of PHFSCs and the formation of clones were inhibited by 20 μM Y27632.Furthermore,the proliferation activity of cells was detected by CCK-8,and it was found that the growth rate of PHFSCs treated with 10 μM Y-27632 and 10μM ERK signaling pathway inhibitor U0126 was significantly lower than that of PHFSCs treated with 10 μM Y-27632,and the growth rate of PHFSCs treated with 10μM Y-27632 and 10 μM U0126 was consistent with that of control group.It was also found that the growth promoting effect of Y27632 could be reversed by adding an inhibitor of ERK signaling pathway(U0126)and it was preliminarily speculated that ERK signaling pathway was related to this growth promoting mechanism of Y27632.