Study On The Reconstruction Of Hair Follicle-like Organs By Co-culture Of Human Hair Follicle Stem Cells And Hair Papillary Cells

Research BackgroundHair transplantation is currently the only long-term technique for treating baldness.Its essence is the redistribution of its own dominant hair.However,due to the limited amount of hair in the autologous donor area,the application of hair transplantation technology in the treatment of large areas of hair loss is limited.In order to obtain enough advantageous hair follicle tissues for transplantation,the researchers established a target project to reconstruct hair follicle tissues in vitro,aiming to find suitable cells,and through tissue engineering and cloning techniques,cultivate mature hair with normal growth cycle in vitro.According to the morphological maintenance of hair follicles and the cycle growth depends on the complex interaction between epidermis and dermis.Therefore,you can choose true epidermal cells,hair papillary cells,or pluripotent stem cells or totipotent stem cells that can differentiate into target cells.At present,the commonly used cell compatibility is: the compatibility of epidermal cells,dermal cells and hair papilla cells(the mature hair follicles have been successfully cultivated in nude mice);there are also related studies using dermal mesenchymal stem cells,epidermal stem cells,and even bone marrow stem cells Report.But this design is only suitable for basic experimental research,not for clinical practice.Current research shows that hair nipple may play a decisive role in the growth of hair follicles.Hair follicle stem cells have the potential for multi-directional differentiation.Hair papillary cells can provide a good growth environment for hair follicle stem cells.The mixed culture of the two in vitro has the potential to differentiate into new mature hair follicles.In theory,as long as a small amount of hair is taken,hair papillary cells and hair follicle stem cells are extracted and purified,and then the hair follicle stem cells and hair papillary cells are expanded and cloned through tissue engineering,a sufficient amount of hair follicle structures for treating large-scale hair loss can be induced.However,at present,there is no mature and standardized method for the extraction,culture,purification,expansion,and identification of hair follicle stem cells,and there is no successful precedent for the mixed culture of human hair follicle stem cells and human hair papillary cells to produce human hair.Research purposes1.Explore the possibility and specific scheme of human hair papillary cells in combination with human hair follicle stem cells for inducing human hair follicle regeneration.2.Looking for a set of experimental protocols suitable for the extraction,cultivation,purification,expansion and identification of human-derived hair follicle stem cells.Research methods1.Two-step enzymatic extraction of human-derived hair follicle stem cells,using K-SFM containing growth factors for cell cultivation and passage,to observe cell growth in vitro.2.Screening and identification of hair follicle stem cells by immunofluorescence technology and cell flow cytometry3.Extract and cultivate homologous human hair papillary cells by enzymatic hydrolysis,and observe the growth of the cells in vitro.4.Try different types of cells in different proportions,and implant them into nude mice by injection method and miniature chamber method to observe whether there are new hair follicles.5.Observe the new hair follicle tissue by HE staining of pathological section.Research result1.Hair follicle stem cell cytology experiment results: two-step enzyme digestion method was used to obtain hair follicle stem cells,then inoculated in a10 cm culture flask and cultured with serum-free keratinocyte culture medium.It can be seen under the microscope that the exfoliated cells not only contain hair follicle stem cells but also contain a small amount of external roots Sheath cells,impurities and keratinocytes.Incubate in a 37 ° C constant temperature incubator.On day 3,most of the cells adhered to the hair follicle stem cell microscope.It is paving stone-like,rich in cytoplasm,and large and round nuclei are hair follicle stem cells,but still contain more impurity cells.On the 10 th day,when the proliferation and fusion reached about 90%,the cells could be passaged to continue the culture.After the passage,the cells could still proliferate normally,and the proportion of hair follicle stem cells increased.After passage to the third passage,the proliferation rate slowed down and could hardly grow adherently.Occasionally,adherent small clump cells could not proliferate and produce more cell populations,and the cell proliferation ability decreased.2.The results of immunofluorescence experiments on hair follicle stem cells:K15 can show strong double-positivity in primary cultured cells and cells cultured for 7 days in subculture.After cells were subcultured for 10 days,K15 could show a certain double positive,and the density was significantly reduced.In the primary culture of hair follicle stem cells,K19 can show a certain double positive,and the sensitivity is lower than that of K15.3.Flow cytometry test results of hair follicle stem cells: Cell flow cytometry to detect CD200,integrin α6 double antibody labeled passage 7 days of hair follicle stem cells in the flow cytometry analysis chart,that is,hair follicle stem cell double antibody labeled positive throughout The proportion of cells is about 28.3%.CD200 single antibody-positive cells accounted for approximately 62.5%.According to previous research standards,positive CD200 antibody labeling is considered to be defined as hair follicle stem cells,the total proportion of hair follicle stem cells can reach 90.8%.According to strict double-label positive identification,hair follicle stem cells account for about 28.3%,which are both proportion.4.Results of hair papillary cell cytology experiment: the hair follicle can be observed under the microscope.The outer part is a spherical structure surrounded by a layer of protein sheath,which mainly contains hair papillary cells,and also contains a small amount of hair matrix cells.After enzyme digestion,it can be seen that more cells in this area come out.Culture at 37 ℃ in whole culture medium containing fetal bovine serum.Around 24 to 48 hours,the structure and cells of the hair nipple are visible at the bottom of the bottle,and the hair nipple is radiated out radially.The cells that have accumulated in small clusters adhere to the wall and begin to break down into single cells.The shape is round or round.,The cells are small and the cells are viable.After culturing for about 1 week,the cells proliferated and stretched into a fusiform shape.The morphology of the hairy nipple passage cells gradually stabilized to form a spindle-like fibrous shape,with a part of round cells interspersed in radial,bundle or vortex arrangement.The cytoplasm is rich and the core is large and round.The proliferation rate is relatively fast,and the cells can be full in 7-10 days and can be passaged.After passaging,the observation continued and the hair papilla cells had a strong growth activity.The cells were continuously passaged to the 5th to 7th generation,and a large amount of secretions appeared in the cells under the microscope.Continued observation showed that a large amount of apoptosis and weakened proliferation capacity were observed.5.Animal experiment results: the hair follicle stem cells and hair papillary cell suspensions were mixed in a ratio of 1: 2,and the cell density was 1 * 107 / ul,which were planted on the back of nude mice using injection method and miniature chamber method respectively.It can be seen that after the injection method for 7days,more white fluff grows at the injection site,and after 21 days for the chamber method,the white fluff grows at the planting site,which is slightly thicker than the surrounding area,but none of the human black hair grows.The villi tissue grown and the untreated back skin tissue were stained with pathological sections by HE staining,which can be seen under the microscope,and the results of mature hair follicles can also be seen in the blank control group.However,the density of the hair follicle structure was significantly higher in the small chamber method.Although the injection method did not show more hair follicle structure,the concentric tissue structure in the subcutaneous tissue increased significantly.Analysis conclusion1 Hair follicle stem cells are mixed with hair papillary cells in proportion to transplantation in nude mice,which may promote the generation of new mature hair follicles in nude mice.2.Human hair follicle stem cells can be cultured with K-SFM medium containing growth factors.After subculture,the proportion of hair follicle stem cells is significantly increased,which can achieve the purpose of cell purification.3.Hair follicle stem cells can be identified with K15 / k19 antibody for immunofluorescence identification,and CD200 / integrin-6 antibody can be used for cell flow cytometry identification.