| Skin wound healing is divided into four phases, including Hemostasis Phase, Inflammation Phase, Proliferative Phase and Tissue remodeling Phase.Wound healing is very complicated, regulated by many factors. If any pathological abnormality occurs, the normal wound healing will not be completed. Chronic wounds, as a typical abnormal wound healing, bring inconvenience for patients’ daily life.Recently, Gene Knock-Out animal model has been widely used in the study of specific gene function during wound healing.Gpr48, also named as leucine-rich repeat-containing G protein-coupled receptor4(LGR4), belongs to a subfamily of G protein-coupled receptors(GPCR).Because of its unclear ligand, Gpr48is also been called" Orphan Receptor". The latest study revealed that Gpr48played very important roles in the development of various organs. We found that skin keratinocyte showed high expression of Gpr48, and hair follicle development as well as hair cycle were regulate by Gpr48. Therefore, Gpr48seems to play a role in maintaining skin function. We thus assume that:deficiency of Gpr48might affect wound healing.The purpose of this study is to research the effects of Gpr48deficiency on skin wound healing. By establishing full thickness skin wound model in Gpr48+/+and Gpr48-/-mice, we found that the deletion of Gpr48caused prolonged wound healing, abundant inflammation infiltration and evident angiogenesis. Transcription expression level of various inflammatory factor, chemotactic factor and growth factor were up-regulated and extended in Gpr48-/-mice. Cell signaling pathway including MAPK,NF-Kb and STAT were also abnormally activated in Gpr48-/-mice. In addition, we found that deletion of Gpr48obviously injury the migration ability of HaCaT cell in our vitro, further verifying that Gpr48could affect skin wound healing. Detailed study is listed as follow:1. Reproduction and genotyping of Gpr48Knock-Out mice:Adult Gpr48+/-female and male mice were raised and mate in one cage. The offspring of Gpr48+/-mice should have three genotype:Gpr48+/+,Gpr48+/-and Gpr48-/-respectively. By using RT-PCR, X-gal staining and eye observation, we identified the genotype of offsprings. Six pairs of littermate Gpr48+/+and Gpr48-/-mice were been selected and used in the follow-up experiments. The experiment results indicate that it is feasible to use RT-PCR、X-gal staining and phenotype observation to identify the genotype of Gpr48+/-offsprings.2. Establishment of full thickness skin wound model and wound observation:8to10week-old littermates of Gpr48+/+and Gpr48-/-mice were selected and created8.0mm diameter wound by using punching bear on the dorsal skin. Wound sizes were taken photograph and measured at Day1,3,5,8,13. The results show that the wound healing in Gpr48-/-mice is obviously delayed compare to Gpr48+/+mice.Especially at Day3,5,8,13, the difference is statistically significant (P<0.05). the results demonstrate the deletion of Gpr48delays wound healing process in mice.3. The effects of Gpr48gene deletion on wound healing and its possible mechanism at the histological and molecular levels:Skin samples were taken from wound site. Firstly, HE and immunohistochemical staining were taken to make morphological observation and the semiquantitation of the proteins related with inflammation and proliferation.Secondly, the expression of various inflammatory factor,chemotactic factor and growth factor were detected. Thirdly, the proteins of signaling pathway were investigated in affecting wound healing.Our results showed more severe inflammatory reactions in Gpr48-/-mice were observed compare to Gpr48+/+mice in any phases of wound healing, presenting aggregated inflammatory cells,accumulated granulation tissue, delayed re-epithelialization and reduced cell proliferation. In the later period of wound healing(Day8and Day13), lots of newly formed capillaries occurred in the wound site of Gpr48-/-mice.Immunocytochemistry result revealed that the number of leucocyte in Gpr48-/-mice dermis wound site was significantly highert than that in Gpr48+/+mice during the whole healing phases. In Proliferative Phase, the cell proliferation capability of epidermis and dermis is low in Gpr48-/-mice compare to Gpr48+/+mice, while the inflammatory reaction in Gpr48-/-keratinocyte is high Real-Time PCR result demonstrate that various inflammatory factors (IL-1,IL-6,TNF-a) and chemokine(CC motif and CXC motif) were significantly up-regulated in the wound site of Gpr48-/-mice compare to Gpr48+/+mice. Meanwhile, The activation of MAPK/ERK,NF-Kb and STAT signaling proteins were also strongly enhanced in Gpr48-/-mice.These results confirmed that Gpr48deficency could induce excessive inflammatory reaction and impaired skin wound healing. MAPK,NF-Kb and STAT signaling pathway might participate in the abnormal regulation of skin wound healing.4. Deletion of Gpr48gene impact migration capability of HaCaT cell:By using immunohistochemistry and Real-Time PCR,we aim to check out the expression and location of Gpr48gene in the skin.The result showed Gpr48mostly exists in epidermis and its appendage such as Hair Follicle, Sebaceous Gland and sweat gland. We then use RNAi technology to creat Gpr48Knockdown HaCaT cell line. Cells scratch experiment along with detection of ECM collagenase MMP1and its inhibitor TIMP1were made tp reveal the impact of Gpr48deletion on keratinocyte migration in vitro.We found the absence of Gpr48markedly reduced migration capability of HaCaT cell. The mRNA expression of MMP1is much lower than that in normal HaCaT cell. These results indicate the weak capability of ECM degradation of keratinocyte by GPR48deletion contributed to its reduced migration capability, which eventually leaded to impair wound healing. |
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