The Study On Directional Differentiation Of Human Amniotic Fluid Stem Cells Into Sweat Gland-like Cells

Skin is the largest organ of the human body,mainly responsible for protecting the body,sweating,feeling hot and cold and pressure functions.The number of deaths caused by accidental injuries every year ranks second only to traffic accidents.Every year,about 20 million people in China suffer from various degrees of burns,of which about 5%of burn patients need hospitalization,which is a big burn country.At present,the medical level has reached a high survival rate for the treatment of patients with extensive deep burn.However,the wounds healed by patients usually lose many functions of normal skin due to scar formation.The scar tissue does not contain sweat glands and can not sweat,which greatly affects the quality of life of patients.Sweat gland formation is a very complex process.Sweat gland cells are somatic cells and belong to terminal differentiation cells.It is difficult to carry out large-scale culture and expansion in vitro.For large area burn patients,it is impossible to extract sweat gland cells from large area skin of patients,resulting in very few sweat gland cells.However,patients need a lot of sweat gland cells,which leads to slow clinical application of sweat gland regeneration and functional skin repair.Therefore,how to improve the quality of life of survivors of extensive deep burn has become a new challenge for clinical burn treatment,and the study of sweat gland regeneration has become one of the important topics.In recent years,with the development of stem cell research,stem cells play a certain role in the repair of skin injury,including sweat gland reconstruction.Bone marrow mesenchymal cells(BMSCs)are used in the treatment of severe burns.Although researchers have made some progress in the study of stem cells differentiating into sweat gland cells,the specific mechanism of sweat gland differentiation and development is not clear enough.Therefore,it is of great significance to find the mechanism of sweat gland differentiation and development from stem cells to sweat gland cells in order to provide a basis for the research of sweat gland reconstruction.The reconstruction of functional sweat glands in skin tissue engineering is a hot and difficult problem in medical research nowadays.The difficulty of proliferation and passage of sweat gland cells in vitro makes the reconstruction of sweat glands lack of seed cells.The research on differentiation of stem cells sweat gland cells has made some progress,but there are still differences in culture system and differentiation mechanism.In view of this,the method of isolation,culture and identification of human amniotic fluid stem cells in vitro was established.The method of isolation,culture,purification and identification of human exocrine sweat gland cells in vitro was established.Human amniotic fluid stem cells were differentiated into sweat gland-like cells in conditioned medium.At the same time,this topic proved that the differentiated sweat gland-like cells are similar to human amniotic fluid stem cells and have low immunogenicity,which provides seed cells for functional sweat gland reconstruction in vitro,and has innovative research and important clinical significance.At the same time,the key molecule was identified in the process of differentiation,which provided a theoretical basis for other types of stem cells to differentiate into sweat gland-like cells,made the functional reconstruction of sweat glands possible,and improved the quality of life of patients with extensive burns after treatment.PART I Differentiation of human amniotic fluid stem cells into sweat gland-like cells and identification of their biological characteristicsObjective:To establish a method for isolation,culture and identification of human amniotic fluid stem cells in vitro,and establish a method for isolation,culture,purification and identification of human exocrine sweat gland cells in vitro.Conditional medium was used to differentiate human amniotic fluid stem cells into sweat gland-like cells,providing sufficient seed cells for functional reconstruction of sweat gland.Methods:(1)In vitro culture and identification of human amniotic fluid stem cells:The collected amniotic fluid samples were centrifuged to obtain cells,cultured adherently,digested by trypsinase,and separated by magnetic bead cell sorting(MACS)from amniotic fluid cells.The AFS cells with positive expression of CD 117 were isolated and cultured.The expression of surface specific antigen of AFS was determined by flow cytometry.(2)Isolation,culture and identification of sweat gland cells:Babies and young children with multiple finger samples were removed from subcutaneous fat,soaked in PBS containing bivalent antibodies,then rinsed with PBS,cut the skin into small pieces of tissue less than 1 cm*1 cm with ophthalmic scissors,digested by Dispase II enzyme at 4?C for 18 hours,and separated the epidermis and dermis with tweezers.Collagenase IV was used to digest the epidermal dermal tissue in incubator for 1 hour.The sweat gland tissue was free.The whole sweat gland tissue(including ducts and glands)was picked up under inverted microscope.Some ducts hovering around the gland were pulled out with insulin needles,and the junction between ducts and glands was cut off.The intermediate connection between ducts and glands was removed to separate the mechanically separated glands.Body parts were cultured in sweat gland medium.The morphology of human exocrine sweat gland cells was observed under inverted microscope,gene expression of sweat gland cells was analyzed by PCR,and the expression of CEA and other keratin K14,K8 and K18 were detected by immunofluorescence staining.(3)Human AFS cells differentiate into sweat gland-like cells:Conditioned sweat gland cell medium(CM)was collected and used to induce differentiation of human AFS using sweat gland differentiation medium(CM=1:1,supplemented with human epidermal growth factor of 50ng/ml)and replaced every day.The differentiated sweat gland-like cells were detected by RT-PCR,immunofluorescence staining,flow cytometry and transmission electron microscopy.Result:(1)Human AFS expressed embryonic stem cell markers,some expressed mesenchymal stem cell markers,but not hematopoietic stem cell markers.Its differentiation potential was between ESCs and adult stem cells.(2)Human exocrine sweat glands were observed under optical inversion microscopy.The free sweat gland tissues could be observed in globular and straight ducts.Human sweat gland cells cultured in vitro were typical epithelial cells,arranged closely,similar to paving roads,with clear cell boundaries,clear nuclei and certain proliferative capacity.The results of PCR showed that human sweat gland cells cultured in vitro had certain proliferative capacity.Oct-4,Sox-2 and Nanog,Klf-4 and c-Myc,CEA and K14,K8,K18 and K19 were not expressed in ES cells.Immunofluorescence staining showed that keratin K14,K8,K18 and CEA were expressed in human sweat gland cells cultured in vitro.(3)Human AFS was successfully induced to differentiate into sweat gland-like cells using sweat gland differentiation medium(SGDM).The cell morphology changed from mesenchymal cell-like to epithelial cell-like.RT-PCR results showed that sweat gland development-related genes EDA and EDAR increased significantly on the 14th day of differentiation,then decreased to normal sweat gland cell level on the 28th day of differentiation.The expression of K8 and CEA increased significantly and approached the level of normal sweat gland cells after 28 days of differentiation.Immunofluorescence staining showed that the differentiated sweat gland-like cells expressed EDA,EDAR,CEA,and K8,which were similar to normal sweat gland cells.The efficiency of flow cytometry in detecting the differentiation of human AFS into sweat gland-like cells was about 30%.Transmission electron microscopy showed that the differentiated sweat gland-like cells expressed EDA,EDAR,CEA and K8?There are microvilli on the cell membrane,a special cell structure of sweat gland cells.Conclusions:In this study,the isolation,culture and identification of human amniotic fluid stem cells in vitro were comprehensively analyzed,and the isolation,extraction,culture,purification and passage of human sweat gland cells in vitro were analyzed.The identification of sweat gland cells was completed,which provided a theoretical basis for further study of sweat gland cells.On the basis of obtaining good initial cells and target cells,we designed a new induction and differentiation medium SGDM,which can induce human AFS to differentiate into sweat gland-like cells successfully.The sweat gland-like cells obtained by SGDM are similar to normal sweat gland cells,which lays a foundation for other types of stem cells to differentiate into sweat gland-like cells,and also for sweat gland differentiation and development.The research provides a new way.PART I I Identification of biological functions of sweat gland-like cells derived from human amniotic fluid stem cellsObjective:To establish a method of differentiation of human AFS into sweat gland-like cells mediated by SGDM,and to identify its biological characteristics.Meanwhile,in the process of differentiation,the related molecules of sweat gland differentiation were searched.Methods:After removing the undifferentiated human AFS,the differentiated sweat gland-like cells were added into three culture gels in vitro,cultured for 21 days,sliced and observed the structure of the gel;sweat gland-like cells were taken out and replaced with sweat gland cell culture medium supplemented with CaCl2,stained with Fluo 3/AM,and the fluorescence signal intensity of Flou 3 was observed under confocal microscope with different concentrations of acetylcholine.Flow cytometry was used to detect the fluorescence intensity.Sweat adenoid cells and human amniotic fluid stem cells were co-cultured with activated T lymphocytes to observe the potential effects on T cell proliferation.The role of unknown factors(UFCM)in CM and additional EGF in SDGM was analyzed by designing several different differentiation media.Result:(1)Immunohistochemical results showed that differentiated sweat gland-like cells could form sweat gland-like structures similar to those of normal human sweat glands in gel,and the number of sweat gland-like structures formed in gel was about 24.Immunofluorescence staining showed that the sweat gland secretory cells expressed EDA,EDAR,CEA,K8,K18,EMA,CD133 and Na-K ATPase;(2)Acetylcholine increased the concentration of free Ca2+in the cells by reflecting the fluorescence intensity.The results showed that the differentiated sweat gland-like cells had partial sweat gland function;(3)co-culture with activated T cells.The results showed that sweat gland-like cells were similar to human amniotic fluid stem cells and had low immunogenicity;(4)RT-PCR results showed that UFCM played an important role in SDGM after differentiation in several different differentiation media,while EGF could improve the differentiation efficiency of SGDM,and further detection showed that Shh might be included in CM and play an important role in the formation of sweat gland-like structure.Conclusions:In the process of human AFS differentiating into sweat gland-like cells mediated by sweat gland induction and differentiation medium SGDM,the functional types of sweat gland-like cells were identified,including the potential of sweat secretion,the ability of three-dimensional culture in vitro to form sweat gland-like structure,the ability of inhibiting T cell activation,and the molecule related to sweat gland-like structure formation in CM,Shh,was found in the process of differentiation.This provides a new method for the study of stem cells differentiating into sweat gland cells,lays a foundation for the study of sweat gland differentiation and development,and makes the functional reconstruction of sweat gland possible.