| Hair follicle is an important accessory structure of skin. It is widely distributed in the surface of body. Besides the function of defence and protection, hair have the effect of beauty and information exchange for inner and outer part of the skin. The hair follicle is a complex structure with the function of controlling the growth of hair shaft. The stem cells of hair follicle located in hair follicle have the function of participating the regeration and repair of epithelial cells of skin. Hair follicle has the character of cyclical growth, including growth (anagen), regression (catagen) and rest(telogen). Generally, hair follicle, stimulated by some factors will transform from telogen, into hair follicle in anagen. Hair growth and cyclicity is the result of multipotent epidermal stem cells and mesenchymal cells around the stem cells.Activated signal from mesenchymal cells induce the migration and differentiation of multipotent stem cells, and the maintance of the hair growth and improve cyclicality.The hair growth and periodic self-renewal depend on periodic proliferation and differentiation of hair follicle stem cells. Trouble in any step of hair follicle cyclicality shift would disturb hair growth and induce related diseases of hair follicle, for example alopecia or alopecia areata,et al.Hair follicle stem cell is a kind of progenitor cells located in middle part area of hair follicle outer root sheath, which is one of area epidermal stem cells located. It is reported that the stem cells in bulge area participate in skin wound healing, which supply stems for basal layer of epidermis. It is generally reported that hair follicle stem cells is mainly located in bulge area.Cdc42 is a ubiquitously expressed small GTPase belonging to the Rho family. It exists in an active GTP-bound and an inactive GDP-bound form. Cdc42 is a molecular switch in cell signal transduction pathway. Only in its active form Cdc42 can interact with different effectors, which in turn regulate cell growth, differentiation,apoptosis, cell cycle and a series of other cellular processes. The most important function is regulation of the restructure of the actin cytoskeleton.Recent studies have found that Rho family in small GTPase plays an important role in regulating keratinocytes proliferation,differentiation and apoptosis. The previous studies confirmed that the Rho A can regulate keratinocytes proliferation and differentiation and promote skin wound healing through MEKK1/JNK signaling pathway. But, there are few researches focused on the effect of Cdc42 in hair follicle stem cells and the crucial role in skin wound healing. Recently, using Cre/loxp recombinase system, I generated mice with the keratinocytes-restricted deletion of the Cdc42 gene. Result unexpectedly is postnatal 49 Cdc42 gene knockout mice(KO mice) progressively loss of hair from head to tail end, and to postnatal 64, compared with control group WT mice, KO mice hair clearly sparse throughout the body. In addition, periodic regeneration of hair dispeared. The result indicate that Cdc42 play important role in hair follicle stem cells activation. Using the model of transgenic mice, we investigate the impact of the keratinocytes-restricted deletion of the Cdc42 gene on the hair follicle development and skin wound healing as well as the related signaling mechanism. I hope the research results would provide theoretical basis for the alopecia and skin wounding healing.1. Generation of the mice with the keratinocytes-restricted deletion of the Cdc42 gene and the impact of Cdc42 gene knockout on hair follicle stem cells(1) object: To generate build the animal model of keratinocytes special Cdc42 deletion (2) Methods: Using Cre/loxp system, the mice analyzed in this study were generated by crossing Cdc42loxp/loxp mice with K5-Cre transgenic mice. From the pup ,the Cdc42loxp/+/-K5-Cre~+ mice were then crossed with Cdc42loxp/loxp mice to generate 1/4 KO mice that its genotype is Cdc42loxp/loxp/-K5-Cre~+ and 1/2 WT mice that its genotype is Cdc42loxp/+/-K5-Cre- or Cdc42loxp/loxp/-K5-Cre-. The genotypes are identified by polymerase chain reaction (PCR) technology and immunohistochemical method. Continuously observe skin and hair development for postnatal KO mice and WT mice. We Sampling skin tissue of KO and WT mice from postnatal 24 days to 70 days, and put these samples in 4% paraformaldehyde for prepared, then, the sample were used as follows:(1) General observation: Continuously observe skin and hair appearance and integrity especially observing the difference of hair cyclicality between postnatal KO mice and WT mice. (2) Histomorphology: observing hair follicle transformation and differences in different hair cycle stage between KO mice and WT mice by H&E staining. (3) Immunohistochemical method of K15 to compare the difference of hair follicle stem cells between KO mice and WT mice and identify the impact of Cdc42 gene knockout on maintenance and stability of hair follicle stem cells. (4) Investigate the impact of Cdc42 gene knockout on proliferation of hair follicle stem cells by the stimulation of depilation approach.(3) Results:? By PCR genotyping identification,K5-Cre~+ only displays a 667bp specific band. Cdc42loxp/loxp displays 700bp band or 700bp and 200bp two specific bands. KO mice not only have K5-Cre~+ but also have Cdc42loxp/loxp. According to the immunohistochemical results we could identify that Cdc42 protein is not expression in skin keratinocyte in KO group and Cdc42 is successful knocked out in keratinocyte of skin.? Observing results in general: Comparing with WT mice there are no clear differences in bodily form, in weight and in skin integrity of KO mice. Compared with WT mice, the KO mice have no clear differences in hair re-growth, in density and length of hair re-growth of KO mice before postnatal 49 days. After postnatal 49 days , the difference begin to be present between KO mice and WT mice. Clearly progressive depilation could be found in KO mice from head to tail after postnatal 49 days. Compared with WT mice, KO mice hair is clearly sparse to postnatal 64 days and hair periodic growth disappeared.? Morphologic observation of hair follicle: Hair of KO mice rest on telogen permanently after depilation phenotype can be found.? Immunohistochemical detection: The result of K15 immunohistochemistry stain showed that after postnatal 49 days hair follicle stem cells in bulge area of KO mice almost exhausted. The cyclicity of KO mice disappeard rest on the second telogen, and the abilities of natural and stimulated proliferation receded or lost. The re-growth of hair shaft is blocked in KO mice after pluck out hairs 10 days.(4) Conclusion: ? Mice with keratinocytes special deletion of the Cdc42 gene were successfully generated.? Cdc42 has the improved impacts of maintenance and proliferation on hair follicle stem cells.? With the K15 positive hair follicle stem cells disappeared, the hair regenerate was inhibited by keratinocytes-restricted deletion of the Cdc42 gene.2. The impact on skin wound healing of Cdc42 gene knockout in keratinocyte(1) Object: Skin is the biggest organ in the body, which the important role that can isolate the body from the external environment and protecting and remain the stability of the inter environment of the body. Skin wound is very common which was caused by injury, burn, infection tumor and other metabolic diseases in clinical. As a result,wound healing was dramatically focused by many researchers and clinical professors in recent researches.Skin wound-healing includes the regeneration of the epidermis and dermis, and the former is more important, which so called skin re-epithelialization.Re-epithelialization is a key process for wound, which delay would lead to excessive fibrillation, and further produce hypertrophic scars. In the epidermis regeneration process: the keratinocyte migration to the wound of epithelial and ancillary structures around the wound, series of phenotypic changes in cells migration process. Two core issues of wounds in the skin areas: one is the speed of wound healing; the other one is wound healing quality problems. Rapid healing of wounds can reduce wound infection and reduce the formation of hypertrophic scars, so to explore how to promote the proliferation and migration of keratinocytes around wound is the crux of the problem during wound healing.Wound healing is one of most complex processes in the body, in which multiple biological signals pathways are activated and participating different function. MAPK(mitogen activated protein kinases, MAPK) is an important signaling pathway system including ERK, JNK and p38 signal. Skin development, hair follicle periodical re-growth and wound healing are directly related with Wnt signaling pathway. It is reported that Wnt signaling involved in wounding, and P-catenin is a key role for Wnt signaling pathway.Mice with keratinocytes special Cdc42 deletion were found a series of hair follicle developmental disorder including progressive depilation, periodic re-growth disappeared and hair follicle stem cells in bulge area exhausted. In order to investigate the impact of keratinocyt especial deletion of the Cdc42 gene on skin wound healing, and investigate the impact of hair follicle development disorders on wound healing and possible mechanism, we then produced the model of skin wound-healing for this research.(2) Methods:Skin wound model: postnatal 7 weeks - postnatal 8 weeks,keratinocytes-restricted deletion of the Cdc42 gene mice, 1cm×1cm square incision on back right.The mice were divided two Group:Control (wild type mice, WT mice)Experimental group (keratinocytes-special deletion of the Cdc42 gene mice, KO mice)Detection indicators: (1) Histomorphology : mice skins were harvested in different time point after wound: 3 days, 5 days, 7 days, 9 days and 11 days. The wound re-epithelialization were detected through the H&E staining; (2) Macrophage infiltration: on 3 days, 5 days and 7 days after wound, the expression of the F4/80 ,which is the marker of macrophage was detected, through immunohistochemistry and the statistical analysis were implemented; (3) The sum of regenerate vascular endothelial cell in granulation tissue: on 5 days, 7days and 9 days after wound, the expression of CD31, which is the special marker of vascular endothelial cell were detected through immunohistochemistry and statistical analysis were carried out; (4) collagen staining: on 7 days, 9days and 11 days after wound,paraffin sections Masson three-color stain were implemented to detect the expression of collagen in the tissue around the wound incision; (5) Detection of proliferation and differentiation for keratinocyte by immunohistochemical methords of K6 and K1 and statistical analysis; (6) Cell proliferation in re-epithelialization. The Detectoin of Brdu- positive cells in regenerate epidermis and hair follicle residual in tissue around wound and statistical analysis; (7) signal mechanisms: the expression of phosphorylation of these protein-kinases, including the p-JNK, p-ERK, p-p38 and p-ELK were detected by immunohistochemistry and statistical analysis were implented. The Wnt signaling pathway including the ?-catenin and the E-cadeherin also detected.(2) Results:The morphological observation: wound re-epithelialization was observed under the light microscope during postoperative day 3, 5, 7 and 9 days,respectively,the tissues were harvested from the area 0.5cm distant from the edge of the wound..Compared with the KO group, more cell layers were seen at WT group wound edge on 3 days after wound through H&E staining. and more keratinocytes migrated to the wound center to form the epithelium belt (re-epithelialization), but the proliferation of keratinocytes and the belt is not obvious in KO group. On day 5 there are more and more cell layers at WT group wound edge, and the belt is longer than KO group. And,the re-epithelialization of wound were finished completely for WT group on day 7,but the re-epithelialization were delayed and comlpeted until the day 9 after wound. f.The results suggested that keratinocytes-special cdc42 gene deletion in mice delayed wound re-epithelialization.Detection indicators: (1) Macrophage infiltration: On day 3 and 5 after wound,the expression of F4/80 showed that KO group more macrophages infiltration than WT group; (2) The sum of regenerated endothelial cells in granulation tissue: on 5 days and 7 days after wound, the expression of CD31 showed that the sum of regenerated vascular endothelial cells in granulation tissue was in WT group clearly more than KO group. (3) Collagen staining: on 7 days, 9 days and 11 days after wound, that the WT group promote the generation and degradation of collagen. (4)The proliferation and differentiation of keratinocytes: on 3 days, 5 days and 7 days after wound, the expression of K6 showed more proliferation of keratinocyteson WT group than KO group. The expression of K1 showed that on 5 days and 7 days after wound more differentiation of keratinocytes on WT group than KO group. (5)Through the detection of Brdu positive stain cells in regenerated epidermis and in hair follicle around tissue, Brdu positive stain cells was had clearly increased on WT group compared with KO group on 3 days 5 days and 7 days after wound. (6) Signal mechanisms: On 3 days, 5 days, 7 days and 9 days after wound, the expression of p-ERK signaling is increased in WT group than in KO group. The downstream signaling of p-ERK is p-ELK, expression of these two cytokines is consistent with p-ERK. (7) The detection of Wnt signal pathway. The results showed that on 3 days, 5 days, 7 days and 9 days after wound, compared with WT group, the expression of?-catenin in cytoplasma is greatly increased in KO group. The similar result of E-cadeherin expression was displayed.Conclusions:? Keratinocytes special cdc42 gene deletion delayed skin wound-healing,especially delay the process of re-epithelialization, which may be result from hair follicle stem cells in bulge area deficiency after second anagen.? Keratinocytes special cdc42 gene deletion delayed the process of re-epithelialization in vivo, the impact maybe was preformed by the inhibition of ERK-ELK expression in MAPK signal pathway. In addition,?-catenin in Wnt signaling pathway may also be involved in these influence of delaying the re-epithelialization. |
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