Analysis the mechanism IL-36α in Wound-Induced Hair Follicle Neogenesis
The mice were under isoflurane (RWD Life Science Co., Shenzhen, China) inhalation anesthesia and the dorsal hair was shaved. Skin wounds were made by excising a 1.5 cm2 square of full-Thickness dorsal skin (used for investigating wound healing and related hair neogenesis as indicated). For WIHN promotion experiment, 50µL of mrIL-36α (1ng/μL) or PBS was injected into healing wound (under scab) from post-wound day (PWD) 7 to PWD 14. Healed skin was taken at PWD 28 and incubated in 5% dispase (Gibco, Japan) at room temperature overnight. Remove the epidermal portion under a dissecting microscope. Fixed the dermal portion in formalin for 10 minites. Rinsed with PBS three times and incubated with BCIP/NBT (BCIP/NBT Alkaline Phosphatase Color Development Kit, Jiangsu, China) for 30 minites. Finally, rinsed with distilled water twice to stop the reaction. Hair follicle counting was under the dissecting microscope. Total RNA was extracted with miRNeasy mini kit (Qiagen, Germany) following the manufacturer’s instructions. cDNA was synthesized from RNA by GoScriptTM Reverse Transcription System (Promega, USA). RT-PCR was performed in 96-well plates by 7900HT Fast RTPCR system (Applied Biosystems, USA). As an internal control, GAPDH levels were quantified in parallel with target genes. Normalization and fold changes were calculated using the △△Ct method. Mouse dorsal skin was harvested. Fat underlying the subcutis was mechanically scraped and the skins were incubated in dispase (2mg/ml) for 40min at 37℃. Epidermis was harvested by mechanical scraping with a scalpel and incubated in digestive fluid (0.05% Trypsin, 100μg/ml DNase) for 40min at 37℃. Added complete medium with equal volume to stop digestion. Cells were strained through 150 and 300 mesh filters. Resulting solution was centrifuged at 1200r for 8min. The supernatant was discarded and adult mouse-derived primary skin epithelial cells (MPSECs) were washed twice with phosphate-buffered-saline (PBS, contained 1% fetal calf serum). MPSECs in single cell suspensions in PBS were exposed to antibodies directly coupled with a fluorochrome for 25 min at 4℃. After washing twice, the cells were resuspended in PBS. FACS analyses were performed using a Flow Cytometer (BD LSRFortessa, USA). Total protein was extracted from mice skin in RIPA lysis buffer (Beyotime Biotechnology, China) with protease inhibitors and phosphatase inhibitors (Beyotime Biotechnology, China). Protein concentration was determined using the BCA assay (Thermo Scientific, USA). Samples of 30 μg of protein were applied to SDS-PAGE gels (Invitrogen) and transferred to PVDF membranes. Membranes were incubated with primary antibodies overnight at 4 °C, then incubated with horseradish peroxidase–conjugated secondary antibodies for 2 hour at room temperature. Detection was conducted using BeyoECL Plus (Beyotime Biotechnology, China).