| ObjectiveThere are significant differences of skin wound healing during the different periods of body development. The full-thickness skin defect of the fetus is mainly repaired in completely regenerative manner at the second trimester, and with the development of the fetus to the various periods after birth, the healing mode of the skin would change with the degree of development. The homologous box is a major regulatory gene for animal development, and the degree of expression is different at different stages of development. The expression of homologous box genes in normal fetuses and adult skin tissues is different, especially the PRX-2 gene, which is much higher in expression in fetal skin than in adult skin. The epidermal stem cells (epidermal stem cells, ESCs) is mainly located in the basal layer of epidermis. But whether the PRX-2 gene regulation of epidermal stem cell expression result in the ifference between adult and fetal wound healing is still indistinct, the biological effects of PRX-2 on ESCs is unclear.In this study, the target gene encoding PRX-2 was introduced into the lentiviral vector by transgenic technology, and exploring the effect of the transfected adult human ESCs on the biological behavior of adult ESCs and a reasonable wound healing model was established to explore the effect of PRX-2 gene on wound healing in adult epidermal stem cells.Methods1. Type IV collagen were used to screen epidermal stem cells, and the related surface markers were identified by immunofluorescence staining. the PRX-2 gene lentiviral vector was constructed in vitro and MOI value was determined, The PRX-2 gene was lentivirus transfected into epidermal stem cells. The effect of PRX-2 gene on epidermal stem cell proliferation was detected by MTT assay.2. The epidermal stem cells and fibroblasts were cultured with different PRX-2 gene levels with silk fibroin / chitosan (SFCS) as cell scaffolds. The normal structure of the skin was used to construct tissue-engineered skin. Animal model was built with nude mice with full -thickness skin to defect the contraction of the wound. Observing the effects of different PRX-2 gene levels on the repair of wound and the effect of SFCS on wound repair.3. Exploring the differences among the four animal wound healing groups with taking pictures, drawing and visual observation at different time points (0,3,7,10,14 days). The image was analyzed using Image Pro image analysis software to calculate the wound healing rate and compare the differences between groups. HE staining and MASSON staining were used to observe wound healing and collagen regeneration.Results1. The lentiviral vector containing PRX-2 gene was isolated from human tissue successfully,and epidermal stem cells could be stably inherited by PRX-2 gene. PRX-2 gene has a certain effect on the proliferation of epidermal stem cells.2. Epidermal stem cells and fibroblasts could grow well on scaffolds of silk fibroin / chitosan(SFCS), and the animal model of skin defects in nude mice can effectively reduce the contraction of the wound and imitated of human skin well, ensuring the scientific nature of the experimental data.3. After 7 day treatment, there were significant differences in the wound healing rate between the transfection group and the control group, and the material group and the blank group. HE staining showed that the granulation tissue of the wound was normal, the new skin was moved to the wound, the wound was slowly covered, and some skin accessories were produced. The process was the experimental group> the control group>the material group> the blank group. Masson staining showed that the collagen formation in the experimental group was more regular and the wound healing is better.Conclusions1. Constructed the lentiviral vector containing PRX-2 gene successfully, and established epidermal stem cells with capable of stably inheriting PRX-2 gene overexpressing PRX-2 gene. PRX-2 gene has a certain effect on the proliferation of epidermal stem cells.2. Silk fibroin / chitosan (SFCS) as cell scaffold material, can culture epidermal stem cells and fibroblasts well, providing a good extracellular matrix environment, it’s a robust method for studying on tissue engineering skin. Animal model of full-thickness skin defects contraction of the wound in nude mice is a good animal model for wound repair studies.3. PRX-2 gene can promote wound repairing and contribute for the repairing of nude mice wound. But the mechanism remains to be further studied, and the composite scaffold material itself can promote the role of wound repair. |
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