HaCaT and human dermal fibroblasts HDFs was purchased from Chinese Academy of Medical Sciences, China. Human bone marrow mesenchymal stem cells (hBM-MSCs) were generously provided by Dr. Yi Wang (Jilin University, Changchun, China), P3-5 lines of hBM-MSCs were used in the experiments. Cells cultured in DMEM (Gibco, Grand island, USA) supplemented with 10% FBS (Gibco, Grand island, USA), humidified 5% CO2 atmosphere at 37 °C.
Cell Proliferation Assay
HaCaT and HDFs were cultured until cells grew to 70 ~ 80% confluence, then seeded in 96-well plates at a density of 4,000 cells per well. Then, we prepared hBM-MSCs-Ex which purification and characterization in our previous published paper. Supplemented the cells with 100µL hBM-MSCs-Ex (25 µg/mL) or PBS (Invitrogen, Shanghai, China), then incubated at 37 °C with 5% CO2 for 5 days. The cell viability was examined by CCK-8 (Sigma, San Francisco, U.S.), and corresponding OD value measured at the 490 nm wavelength.
Immunofluorescence Staining (IF)
HaCaT and HDFs cultured by hBM-MSCs-Ex (25 µg/mL) or PBS were incubated in 24-well plate for 24 h. When cells reached 60 ~ 70% confluence, incubated with 4% paraformaldehyde for 10 minutes, and incubated with 1% bovine serum albumin (BSA; Biosharp, Hefei, China) for 30 minutes. Then, cells were incubated with antibodies against PCNA (1:100 dilution, BD Biosciences, Franklin Lakes, NJ, U.S.), and isotype-matched rabbit or mouse IgG/IgM (1:100 dilution, Abcam, Cambridge, UK) served as the negative controls. Next, incubated with the secondary antibody (anti-rabbit IgG, 1:500 dilution, Abcam, Cambridge, UK) for 2 h, and the nuclei were labeled with DAPI (Thermo Scientific, Waltham, U.S.) for 5 min. Lastly, the intensity was examined by a fluorescence microscopy (EVOS, Thermo Scientific, Waltham, U.S.), and the PCNA positive cells were analyzed in ten random optical fields.
Animals And Treatments
The 8-week old, female Sprague-Dawley (200 g) rats were purchased from Jilin Biotechnology Co., Ltd. (Changchun, China). All animal experiments were performed in accordance with the guidelines of the Animal Experiment Ethic Committee of Jilin University. The animal model was made according to previously published methods. Briefly, rats were anesthesia and shaved the dorsal hair. Then, we made one full-thickness skin excisional wounds of 10 mm in diameter circular holes in each rats. The rats were randomly divided into three groups (n = 8/group): PBS group; hBM-MSCs group (intravenous injection with 1 × 106 cells/ rat); hBM-MSCs-Ex group (250 mg, harvested from 1 × 106 hBM-MSCs). The skin damage was treatment and recorded photographically every four days. The wound area was calculated out using Adobe Photoshop CS6, lasso tool was used to trace and circle the edge of wound on photograph, then calculate the circled area based on the pixels of that area. Rats were euthanized on day 16 and collected the healed tissue.
Skin tissue sections were cut at 4 µm thickness and used for histological examination. Hematoxylin and eosin (H&E) staining was performed following the manufacturer’s standardized protocols (Sigma, San Francisco, U.S.). Immunohistochemistry (IHC) was carried using the Kit (Maixin KIT-9710, Fuzhou, China) following the manufacturer’s instructions. Briefly, the sections were deparaffinized, rehydrated and incubated in a 99 °C water bath for 15 min, 3% H2O2 was added for 15 min, and blocked with 10% normal goat serum for 1 h at 37 °C. Next, the sections were incubated with primary antibody anti-α-SMA with1:500 dilution (Abcam, Cambridge, UK) overnight at 4 °C. Next, these sections were incubated with biotinylated goat-anti-rabbit IgG antibody for 2 h. Then, we used diaminobenzidine solution as the chromogenic agent at 37 °C for 15 min, and incubated with avidin peroxidase reagent sequentially. Lastly, we used hematoxylin was for counterstaining. These sections were photographed using a microscope (EVOS, Thermo Scientific, Waltham, U.S.).
Proteins were extracted from the skin healed tissue in thelysis buffer. The protein samples in SDS sample buffer were heated to 95 °C for 10 min, and separated on SDS-polyacrylamide gels. Resolved proteins were then electro blotted onto nitrocellulose membranes and probed with antibody against TGF-β1, TGF-β3, Smad2, Smad3, Smad4, Smad7 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000 dilution, Abcam, Cambridge, UK) overnight at 4 °C. Then, the samples followed by secondary antibodies HRP-conjugated goat anti-rabbit IgG (1:1000 dilution, Abcam, Cambridge, UK), and visualized by chemiluminescent detection according to the manufacturer’s instructions (Immobilon western chemiluminescent HRP substrate, Millipore).
Real-time PCR Assay
Total RNA from skin healed tissue was extracted with Trizol (Invitrogen, Shanghai, China) according to the manufacturer's protocol. SYBR Green I dye was used for reverse transcription in an ABI 7500 fluorescence quantitative PCR instrument, and the mRNA levels of TGF-β1, TGF-β3, Smad2, Smad3, Smad4, Smad7 and GAPDH were measured, and the primers were added in supplementary Table S1. The thermocycler conditions as follow: initial step at 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. Expression levels were recorded as cycle threshold (Ct). Data were acquired using the 7500 Software (Applied Biosystems Life Technologies, Foster City, CA, U.S.). All reactions were performed in triplicate and the data were analyzed using the 2−ΔΔCt method.
Statistical analysis was performed using Prism 6 (Graph Pad software) and Image J. One-way ANOVA with Dunnett’s multiple comparisons test was used to test for statistically significant differences. All quantitative data were given as the mean ± SD. for at least three independent experiments, and p < 0.05 was considered to be statistically significant.