Expression And Effect Of Stromal Cell Derived Faceor-1α During Cutaneous Wound Healing In Mice

PART ONE EXPRESSION OF STROMAL CELL DERIVED FACTOR-1α(SDF-1α) DURING CUTANEOUS WOUND HEALING IN ADULT MICEObjective: To investigate the expression feature of stromal cell derived factor-1α(SDF-1α) during full-thickness cutaneous wound healing in adult mice.Methods: Thirty-two male Kunming mice were anaesthetized by intraperitoneal administration of pentobarbital sodium (30mg/kg body weight). Full-thickness skin wounds (1.5×1.5 cm) through the panniculus carnosus were made aseptically on the middle of backs, near the neck. The wounds were left unsutured and without dressing. Skin samples, including a 3-mm margin of unwounded skin from each wound, were collected at days 1, 2, 3, 4, 5, 7, 10 and 14 post-wounding. Each specimen was divided into two parts: one was fixed in 4% paraformaldehyde for histopathological analyses, and the other was frozen in liquid nitrogen for reverse transcription- polymerase chain reaction (RT-PCR) analysis of SDF-1αmRNA. Four animals for each experimental time point were collected.Results: The gene expressions of SDF-1αassayed by RT-PCR in mice wounds were significantly increased and peaked at 1 day and 5 day post-wounding (P<0.05), but reached the lowest level at 3 day. At day 10 post-wounding, the gene expression of SDF-1αremained elevated level (P<0.05). The protein expressions of SDF-1αwere predominantly expressed at hair follicle, dermal fibroblasts, vascular endothelial cells and re- epithelization cells.Conclusion: The profile of SDF-1αexhibited a double-peak expression indicated that SDF-1αmay take part in the process of cutaneous wound healing, and the inflammatory reaction may impact the SDF-1αexpression.PART TWO EFFECT OF INFLAMMATORY CYTOKINES ON THE EXPRESSION OF SDF-1αBY FIBROBLASTS OF SKINObjective: To investigate the expression of SDF-1αinduced by tumor necrosis factor-α(TNF-α) and interleukin-1 (IL-1) in mice skin fibroblasts. Methods: Fibroblasts of adult mice skin were isolated and cultured, then were treated with different concentrations of TNF-αand IL-1 (0.1ng/mL,1ng/mL,10ng/mL,100ng/mL) for 48h, respectively, but the control group was treated without the two agents. RT-PCR analysis was used for measure the SDF-1αmRNA expression.Results: SDF-1αwas constitutively expressed in adult mouse skin fibroblast. The low concentration of TNF-αand IL-1 treatment could effectively inhibit SDF-1αmRNA expression (P<0.05), and the inhibition was gradually enhanced with the augmentation of TNF-αand IL-1 concentration. There was no significant difference at identical concentration between TNF-αgroups and IL-1 groups (P>0.05).Conclusions: Pro-inflammatory cytokines (TNF-αand IL-1) could inhibit the SDF-1αgene expression of fibroblast, and the inhibition was notable in a dose-dependent fashion.PART THREE EFFECTS OF SDF-1αDURING CUTANEOUS WOUND HEALING IN MICEⅠ: Induction of SDF-1αon bone marrow derived cells recruitment during cutaneous wound healing Objective: To explore the effect of SDF-1αon inducing bone marrow derived cells (BMDCs) recruitment to wound area.Methods: BMDCs were isolated from bone marrow and cultured with routine method, then were identitied by CXCR4 antibody. Cells treatment with CXCR4 antibody (100 ng/mL) for 6 hours were labeled with Chloromethyl-1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per- chlorate (CM-DiI), then were injected into the tail vein of full-thickness incisional wound model as CXCR4 antibody group. BMDCs labeled with CM-DiI without antibody treatment was injected as BMDCs group, and injected Dulbecco's modified Eagle's medium/F12 (DMEM/F12) serum-free medium was as control group. The quantity of labeled BMDCs at the wound site and the percentage of wound closure were measured. Four animals for each experimental time point were collected.Results: (1) All BMDCs were expressed CXCR4. (2) The percentages of wound closure of BMDCs group were higher than that of control group and anti-CXCR4 group on days 7 and 14 (P<0.05), but the percentage of wound closure of anti-CXCR4 group was higher than that of control group on day 14 (P<0.05). (3) The quantities of CM-DiI labeled BMDCs at wound site in the BMDCs group were much more than that of anti-CXCR4 group on days 7 and 14 after trauma (P<0.05).Conclusions: BMDCs participated in the cutaneous wound healing. SDF-1αplays an important role on recruitmenting BMDCs to wound site. Ⅱ: Effect of SDF-1αon neovascularization during cutaneous wound healingObjective: To investigate the effect of SDF-1αon neovascularization during cutaneous wound healing.Methods: Mice full-thickness wound models (1.0×1.5cm) were prepared, and divided into 3 groups: control group (DMEM/F12 serum-free medium), antibody groupⅠ(10 ng/μL of anti-SDF-1αantibody) and antibody groupⅡ(20 ng/μL of anti-SDF-1αantibody), prepared with DMEM/F12 serum-free medium. DMEM/F12 serum-free medium and different concentrations of anti-SDF-1αantibody were injected into full-thickness wound site of mice, respectively, for successive 6 days after injury. Skin samples, including a 3-mm margin of unwounded skin from each wound, were collected and the percentages of wound closure were detected at days 3, 4, 5, 6 and 7 post-wounding. Four animals for each experimental time point were collected. Specimens were frozen in liquid nitrogen and fixed in 4% paraformaldehyde, respectively, which were used for RT-PCR analysis of CD146 mRNA and histopathological analyses.Results: (1) Compared with control group, the expressions of CD146 mRNA of the two antibody groups were much lower (P<0.05), but in antibody groupⅡ, the expressions of CD146 mRNA were much lower than that of antibody groupⅠon days 5, 6 and 7 (P<0.05). (2) Compared with control group, the percentages of wound closure of antibody groupⅠwere much lower on days 6 and 7 (P<0.05), and the percentages of wound closure of antibody groupⅡwere much lower on days 5, 6 and 7 (P<0.05), only on day 7 post-wounding, the percentage of wound closure of antibody groupⅠwas much higher than that of antibody groupⅡ(P<0.05). (3) Microvascular densities of the two antibody groups were much lower than that of control group (P<0.05), but in antibody groupⅡ, the microvascular densities were much lower than that of antibody groupⅠon days 5, 6 and 7 post-wounding (P<0.05).Conclusions: SDF-1αplays an important role on neovascularization of wound field, and has potential application value on improving healing velocity and elevating healing quality.