1. Sample collection, primary fibroblasts culture, and reagents
Skin samples were obtained from 10 keloid patients and 9 healthy volunteers (9 males and 10 females, 18–55 years old) at cosmetic surgical operations (face or chest) after signing informed consents.
Samples were placed in 0.5% dispase (Invitrogen, Carlsbad, CA) at 4 °C, and the dermis was separated from the epidermis after overnight incubation with 0.1% collagenase (Invitrogen). The released FBs were cultured in DMEM containing 10% fetal bovine serum. FBs between the second and the sixth passages were used in subsequent experiments. FBs from healthy volunteers were sorted into Groups F1, F2, and F3. FBs from keloid tissues were grouped into F4, F5, and F6[13].
2. Adipose-derived stem cells isolation and identification
All human adipose tissues were from three volunteers (1 male and 2 females) without systemic diseases, aged 20 to 25 years old. They underwent liposuction surgery after providing their informed consent and with the due approval of the Research Ethical Committee of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University.
Fifty ml human lipoaspirate was washed three times in phosphate-buffered saline (PBS; Hyclone, USA) and finely minced, before being digested in 0.1% collagenase I (Sigma, USA) at 37.0 °C with continuous vibration for approximately 1 h. Digestion was terminated by adding Dulbecco’s Modified Eagle Medium/F12 (DMEM; Hyclone, USA) containing 10% Mesenclaymal stem cell Bovine Serum (MFBS; BI, USA). The tissue was centrifuged at 1,200 revolutions per minute for 5 min, at the temperature of 25 °C. Then, the supernatant was discarded, and the cell pellet was washed in PBS before being centrifuged at 1,000 revolutions per minute for 5 min, at the temperature of 25 °C. Cells were resuspended in common culture media (DMEM containing 10% MFBS) and counted; then, they were inoculated at a density of 1×109 cells per liter. The first culture medium change was conducted 24 h after inoculating and was routinely repeated every 72 h. When cells reached 80–90% confluence, the subculture was operated using 0.25% trypsin with 0.03% EDTA (Gibco, USA). Human ADSCs (hADSCs) between the third and the fourth passages were used in the subsequent experiments.
hADSCs (passage 2) were harvested and washed in PBS three times. Cell suspension was incubated with FITC-conjugated antibodies against CD45 (11-0459-42, eBioscience), CD34 (22-0349-42, eBioscience), PE-conjugated antibodies against CD105 (12-1057-42, eBioscience), PE Cyanine5-conjugated antibodies against HLA-DR (11-0459-42, eBioscience) at 37.0 °C for 15 min in the dark, followed by washing and resuspension in PBS. Then, it was finally detected by flow cytometer (BD Biosciences, USA).
3. Immunohistochemistry analysis
The paraffin-embedded samples were sliced into 4-µm sections and incubated with primary antibodies: Anti-Col-1 (ab34710, Abcam. Dilution 1:100), Anti-Col-3 (ab7778, Abcam. Dilution 1:100), Anti-CTGF (ab6992, Abcam. Dilution 1:100), and Anti-P4HA2 (ab211527, Abcam. Dilution 1:100) after dewaxing, hydration, antigen retrieval, and blocking. The expression was detected using the immunoperoxidase technique (DAB kit, China).
4. RNA Extraction
RNA extraction, reverse transcription, and qRT-PCR were conducted as described in [13].
5. Co-culture of fibroblast and ADSC, CCK8 assay for cell growth curve
Normal FBs (F1, F2, and F3) and keloid FBs (F4, F5, and F6) were co-cultured with ADSCs (grouped into A1, A2, and A3) in a 24-well plate (Table 1). The FB suspension was seeded in a 24-well plate (5×104/well). Then, 5×104/well ADSCs were plated onto the chamber (0.4 µM). The co-culture lasted for 48 h when 10% CCK8 solution was added to each well, and the plates were incubated for 1 h. Finally, the absorbance was measured at 450 nm using a microplate reader.
Table 1
Co-cultural scheme in a 24-well plate.
|
nFBs
|
kFBs
|
F1
|
F2
|
F3
|
F4
|
F5
|
F6
|
NC
|
F1 + NC
|
F2 + NC
|
F3 + NC
|
F4 + NC
|
F5 + NC
|
F6 + NC
|
A1
|
F1 + A1
|
F2 + A1
|
F3 + A1
|
F4 + A1
|
F5 + A1
|
F6 + A1
|
A2
|
F1 + A2
|
F2 + A2
|
F3 + A2
|
F4 + A2
|
F5 + A2
|
F6 + A2
|
A3
|
F1 + A3
|
F2 + A3
|
F3 + A3
|
F4 + A3
|
F5 + A3
|
F6 + A3
|
A: adipose-derived stem cells; F: fibroblasts; NC: normal control; nFBs: normal fibroblasts; kFBs: keloid fibroblasts. |
6. Cell apoptosis detection
After 48 h of co-culture with ADSCs, FBs were collected and resuspended in 1× binding buffer at a concentration of 1.0 × 106 cells/mL, and 100 µL of sample was mixed with 5 µL of FITC Annexin V and 5 µL of propidium iodide (PI). The sample was mixed gently and shielded from light at room temperature for 5 min. Then, 400 µL of 1× binding buffer was added. The sample was detected and analyzed within 1 h by flow cytometry.
7. In vitro wound healing assay
The wound-healing assay was conducted as described in[14].
8. Western blot analysis
Western blot was conducted as described in[14]. Antibodies against Col-1, Col-3, CTGF, and P-4-HB were purchased from Abcam, and antibodies against β-actin were purchased from Multi Sciences.
9. Statistical analysis
All of the experiments were performed in triplicate at minimum. Data were presented as the mean ± standard deviations from more than two independent experiments.
The Kolmogorov-Smirnov test using SPSS 16.0 (SPSS, Inc) software was used to determine whether or not data fitted normal distribution. GraphPad Prism 7 (GraphPad Software, USA) software was used to evaluate statistically significant differences by t-test and to generate graphs. Results were considered statistically significant when the probability was less than 5% (p < 0.05).