Studies On Isolaiton, Culture And Identification Of Hair Follicle Cells Of Goat And Reconstitution Of Hair Follicle In Vitro

Taking Jining Grey goats as animal models，clarifies the growth regular of hair follicle,impact factors and the interaction mechanism of hair follicle dermal cells and epidermal cellsfrom isolation and culture of the hair follicle cells and reconstitution of hair follicle in vitro.Not only to lay the foundation for improving the quality of the breeding improved ofCashmere quality, but also to offer reference for improving the wool quality, quantity, qualityof fur pattern and human hair research in medical area. Improvement technique of isolatingand culturing the hair follicle cells, constructing reconstitution model of hair follicles in vitrocan lay the foundation for further study in hair follicle differentiation and development,mechanism of cell factors on hair follicle cell injury and skin cell repair, mechanism of thecell factors and hormone on follicle development, wool quality and melanin formation andregulation. The present study based on the successful construction of the platform oninsolation, culture and identification of a variety of skin cells and hair follicle cells from goatin vitro, screening the optimum culture methods and system and building cell line impactfactors and involved many physiological phenomena, such as skin and hair follicles celldifferentiation, interaction between dermal and epidermal cell, mechanism on skin and hairfollicle damage and cleaning.I. Improvement of outer root sheath cells culture system from goat hair follicle in vitro.Adopting enzyme digestion in conjunction with mechanical methods sterile isolatesentire hair follicle, the purified ORS cells were cultured in serum-free keratinocyte mediumcontaining epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) andhydrocortisone. The cells were incubated in a5%CO2atmosphere at37℃in the start ofprimary culture. The ORS cells were split to subcultures when the primary cells formed goodmonolayer. ORS cells were subcultured to8th to10th passages, the medium was replacedwith DMEM/F12medium containing EGF, IGF-I, hydrocortisone and2%FBS for the longterm culture. The cells showed gradually stabilized growth after changing the medium. Detected and identified the biological characteristic of ORS cells. The results show that thecultured cells population doubling time was51.9h, chromosome number is still dominated by2n=60. The results of immunocytochemical staining showed that the cytokeratin19expression was positive, proved that the cultured cells is outer root sheath cells from hairfollicle stem cell differentiation.II. Isolation and identification of dermal papilla cells from goat.The dispase combined with collagenase digestion and laying of adherent culture matrix,separation and purification of DP cells, cultured in DMEM/F12medium containing epidermalgrowth factor (EGF), basic fibroblast growth factor (bFGF) and10%FBS in primary culture.When the primary cells were completely confluent subcultured, the medium was replacedwith MEM and DMEM/F12with EGF, bFGF and2%FBS (1:1) composite medium tocontinue to subculture. The cells showed gradually stabilized growth after changing themedium. The DP cells at7th passage were selected to study on biological characteristics andidentification. The results show that the average cloning efficiency of cell is60%, suggest thecells proliferation vitality were luxuriant. The results of chromosomal analysis indicated thatmajority of the cells were2n=60. The results of immune-fluorescence showed that theexpression of α-SMA and Vimentin were positive in DPC and fully confirmed the subculturedcells. The results proved that the dermal papilla cells were hair follicle dermal source.III. Culture of dermal fibroblasts from the goat and establishment of the dermalfibroblast cell line.The method of4℃dispase cold digestion was used to separate the skin epidermis anddermis, in order to culture the purification of dermal fibroblasts. Then, adopting the primaryexplant combined with enzyme digestion separated and purified DFB. At first dispase wasused to separate epidermis and dermis from skin, and the sterile dermal part was gentlyminced into small pieces. The small tissue pieces were enzyme by trypsin, and then dermalexplants were seeded on the bottom of6-well plates. DMEM/F12medium with containingbasic fibroblast growth factor (bFGF), penicillin, streptomycin, tylosin and10%FBS wasadded to each well of the6-well plates after the pieces attached firmly. The cells wereincubated in a5%CO2atmosphere at37℃in the start of primary culture. When a largenumber of cells were migrated out from cultured dermal explant (about5~6days), the medium was replaced with DMEM medium containing bFGF, penicillin, streptomycin,tylosin and5%FBS to subculture, the medium was replace every3days. The cells werereplaced to the25cm2flasks at the first passage time when the primary dermal fibroblastswere grown to confluency, in favor of dermal fibroblasts cultured long term subculture andcryopreservation in vitro. The stable DFB cell line was established in this research hassubcultured to the56passages, and the growth and proliferation of cells were in goodcondition, genetic stability.IV. Establish and maintain the reconstitution model of hair follicle in vitro.In the sterile ice bath, the rat tail collagen, hyaluronic acid and chondroitin sulfate A weremixed proportion to MEM and DMEM/F12(1:1) composite medium to make of gel, and thenadded to24-well plates after regulated pH of the gel. The purified dermal papilla cells anddermal fibroblasts that were isolated and identificated the early stage were harvest, the cellswere suspended by MEM and DMEM/F12containing5%FBS (1:1) composite medium.Sterile cell suspension was added in24-well plates, formed the dermal cell collagen gel. Thecell collagen gel was incubated in37℃and5%CO2humidity environmental conditions. Thedaily growth and proliferation of DPC and DFB were observed under microscope. When thehair follicle dermal cells reached completely confluence, prepared ORS cell suspension addedthe surface, cultured for3~5days. Observed ORS cells in the hair follicle dermal cell collagengel to form a complete monolayer, removed the old medium and added a small amountK-SFM (just covered the surface of ORS cells) containing IGF-I, EGF at37℃,5%CO2saturated humidity of gas-liquid interface culture, replaced the surface medium every twodays. Through microscopic observation, micrometer measurement and real-time fluorescencequantitative PCR detection, the results show that dermal papilla cells present agglutinativegrowth trend in the gas-liquid interface culture early, but the hair follicle outer root sheathcells and scattered colonies in the dermal fibroblast layer. With the gas-liquid interface culture,dermal papilla cells further agglutinative growth, part of the outer root sheath cells colony bygel dermal fibroblast monolayer migration gathered around the dermal papilla cells, andconstantly surrounded, differentiate into ball kind of type of hair ball structure attached to thedermal fibroblast cell layer. Spherical structure formed by several hair follicle cells graduallyto peripheral tension expansion after10days of culture in vitro, dermal papilla cells to end internal migration growth, and ultimately by the hair bulb structure extensional extend intothe class structure of hair follicle, and hairy fibers from the radical minister, wool fiber colorpart of the students to grow as thick black, with black pigment. The testing data shows, invitro reconstruction of hair follicle structure and normal hair follicles are both in theorganizational structure, wool growth rate, the key gene and hair follicle developmentregulation of expression, there is no significant differences.This study established and optimized conditions and culture system of hair follicle cellsin vitro from goat. Studied the effects of impact factors on hair follicle dermal cells andepidermal cells, successfully established a reconstruction induction system of hair follicle invitro from goat. This provide animal models and lay the foundation for people to reveal theand regulatory mechanism of differentiation and development of goat hair follicle and woolgrowth; provides a reference for medical research and discovery cause of hair disorders, themechanism of such drug treatment. Related factors involved in this study and the cultureconditions reflect the most common interactions among these factors in the experimentalconditions, provide reference for human hair regeneration and canities research.