The Study Of The Effects Of Thymosin β4 On Hair Follicle Reconstitution

Background:Hair loss is a problem that plagues many people, but there is rare breakthrough in their treatment for decades. Current drugs to treat hair loss are mostly doing their roles by slowing down the loss of hair follicles or by stimulating hair growth, another solution-hair transplant is just moving hair follicles of other parts to defective part, both of which cannot solve the problem fundamentally. So, promotion of hair follicle regeneration is the key to solve hair loss. Domestic and international research on hair follicle reconstruction showed that, factors that impact hair follicle reconstruction mainly include seed cells of epidermal and dermal sources, and their microenvironment. Currently, hair follicles can be successfully rebuilt with the use of seed cells from mice, but the use of human origin seed cells mostly cannot reconstruct a complete hair follicle. Thymosin 34 is a polypeptide consisting of 43 amino acid residues, originally isolated and purified from thymus, was widely distributed in the body. Thymosin β4 had an important role in the regulation of immune and neurological system, can promote angiogenesis and wound healing, and also can promote hair growth in mice and rats. However, the effects of thymosin 04 on human hair follicle regeneration remained poorly understood. In this study, thymosin 34 was used to stimulate human outer root sheath cells and dermal papilla cells in vitro, to explore its function on seed cells of hair follicle reconstruction; at the same time, we established hair follicle regeneration model by chamber method and applied this model to evaluate the effects of thymosin β4 on murine hair follicle remodeling.Objective:1. To detect effects of thymosin β4 on human hair follicle outer root sheath cells and dermal papilla cells cultured in vitro2. To build a hair follicle regeneration model by chamber method3. To evaluate effects of thymosin β4 on murine hair follicle regeneration by chamber modelMethods and Results:1. Isolation, culture and identification of human outer root sheath cells and dermal papilla cellsMethods:Acquire the fat tissue layer containing dermal papillae from scalp tissue disposed during cosmetic surgery, digest the tissue with 0.2% type I collagenase, pick droplet-like dermal papillae under microscope and culture them adherently; cut the remaining tissue into 1-2mm thin strips, soak them in 0.25% dispase solution overnight, pull out the hair and pick the middle fragment, get and culture human outer root sheath cells after trypsin digestion. Observe the cell morphology of outer root sheath cells and dermal papilla cells, and identify respective cells by immunofluorescence staining, flow cytometry and special staining.Results:Human outer root sheath cells with strong proliferative ability can be obtained by microdissection; A small amount of adherent cells can be found at 2-3 hours after inoculation, with cobblesone-like morphology, which was in line with epithelial-like typical characteristics, and cell fusion can be seen after 7-9 days; Immunofluorescence staining and flow cytometry results show that the cells have positive expression of Sox9 and CD49f. Human dermal papilla cells with high purity and less pollution can be obtained by the method of one-step enzymatic digestion and dermal papillae selection under microscope; dermal papilla cells was fusiform, its movement from dermal papillae was visible at 2-3 days after inoculation, and emigration increased with longer culture time; alkaline phosphatase staining showed that the cells express iconic gene ALP of dermal papilla cells.2. Detection of thymosin β4’s effects on human hair follicle outer root sheath cells cultured in vitroMethods:Thymosin β4 was added to culture medium of human outer root sheath cells, with a final concentration of 10ng/mL,100ng/mL and 1000ng/mL, then observe alterations of cellular morphology after 24h, get cells and extract total RNA, detect the expression of human outer root sheath cells in hair regeneration related genes by Real-Time PCR.Results:There is no significant morphologically difference between human hair follicle outer root sheath cells with and without treatment of Thymosin β4, no significant difference in expression of hair follicle stem cell iconic gene Sox9、 EPCAM、 TCF3, cellular-microenvironmental interaction related gene VEGF、 Integrina3、 Integrina5, hair regeneration associated signaling pathway key gene Lef1、 EDA、 CCN2、PDGFA、 Follistatin、 Nfatcl between human outer root sheath cells with and without Thymosin (β4’s treatment, and no significant difference in each group.3. Detection of thymosin 04’s effects on human dermal papilla cells cultured in vitroMethods:Thymosin β4 was added to culture medium of human dermal papilla cells, with a final concentration of 10ng/mL, 100ng/mL and 1000ng/mL, then observe alterations of cellular morphology after 24h, get cells and extract total RNA, detect the expression of human dermal papilla cells in hair regeneration related genes by Real-Time PCR, and verify the significantly altered genes by Western BlotResults:There is no significant morphologically difference between human dermal papilla cells with and without treatment of Thymosin β4, no significant difference in expression of hair regeneration-induced dermal papilla cell related gene AlP、 α-SMA、 ACTG2、 TRPS1、 SERPINF1、 PTGER3, cellular-microenvironmental interaction related gene VEGF、 HGF、 Laminin5, and no significant difference in each group. The expression of fibronectin (FN) was statistically significant higher in human dermal papilla cells under thymosin β4’s treatment than control group, and its upregulation was validated by Western Blot.4. Construction of hair follicle regeneration model by chamber methodMethods:Apply 1cm diameter polyethylene bottle stopper as chamber, make a 1cm full-thickness skin wound in the back of nude mice and implant the chamber into the wound with several sutures to secure it. Isolate epidermal and dermal cells from C57BL/6 newborn mice, mix them and inject into chamber. Observe the wound site after the removal of chamber at 7 days. Acquire and HE stain the skin from wound site at 30 days, observe its morphological structure.Results:Hair regeneration was visible at the wound site, and new hair was no significantly different from normal hair for gross appearance and microscopic observation, and it had well-maintained polarity and growth density. Analyze the wound site without hair regeneration, it was found that chamber implantation position has an important influence on the success of hair follicle regeneration.5. Evaluation of Thymosin β4’s effects on murine hair follicle regeneration by chamber modelMethods:Establish chamber model by protocols above, add thymosin β4 to cells and mix them during cellular resuspension, with the final concentration of 10ng/mL, 100ng/mL, 1000ng/mL, and continue adding this drug into chamber in the next 6 days. Observe the wound site after removal of chamber at 7 days. Acquire and HE stain the skin from wound site at 30 days, observe its morphological structure.Results:There is no significant difference in hair follicle reconstruction between wound site with and without thymosin 34 addtion, and no significant difference in each group.Conclusion:1. Thymosin P4 may act on hair follicle reconstruction by upregulating the expression of Fibronectin in human dermal papilla cells.2. Chamber model is an effective model for hair follicle reconstruction; Chamber implant location can influence the effects of building hair follicle reconstruction model by chamber method.