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Figures:
Figures 1. Construction and identification of RHC. (A) Construction schematic of recombinant pET3c-hlcollagen plasmid. (B) Nucleic acid electrophoresis of recombinant plasmid pET3C-HLC. M: DNA Ladder 2000; lane 1: monoclone of recombinant plasmid pET3C-HLC; lane 2: negative control. (C) Effect of temperature on RHC expression, as analyzed by SDS-PAGE. RHC was induced by IPTG for 4 h at either 37℃ or 30℃ or overnight at 20℃. M: middle molecular weight protein markers; lanes 2, 4, and 6: RHC after induction at 20℃, 30℃, or 37℃; lanes 1, 3, and 5: RHC before induction. (D) SDS-PAGE analysis of proteins during purification. M: middle molecular weight protein markers; lane 1: broken by PBS; lane 2: Ni-NTA spin columns; lane 3: washed protein through 80 mM imidazole. (E) Western blotting analysis. M: middle molecular weight protein markers; lane 1: RHC with anti-His antibody.
Figures 2. Cell biological activity of RHC/EGF (1:1). (A) MTT assay of NIH/3T3 cell proliferation rates on RHC, EGF, or RHC/EGF. (B) Images obtained at 0, 12, and 24 h after wound creation in vitro migration assay on RHC, EGF, or RHC/EGF. (C) Quantitative analysis of gap area of HaCaT cells cultured on RHC, EGF, or RHC/EGF. (D) Optical micrographs of crystal violet stained NIH/3T3 cells adhering to monolayers presenting. (E) Quantitative detection of the number of NIH/3T3 cells adhering. (F) Cytoskeleton staining. (G) Quantitative calculation of cell spread area. n = 3, means ± SD, *P<0.05, **P<0.01 vs control group, ns means no significant difference vs. control, P >0.05.
Figures 3. Characteristics of RHC and RHC/EGF. (A) Scanning electron micrographs of RHC and RHC/EGF freeze-dried dressing.
Figures 4. Experimental study of rat wound-healing model after treatment with physiological saline, RHC, EGF, or RHC/EGF. (A) Wounds were photographed at 0, 3, 5, 7, 10, 14, and 21 d. (B) H&E staining of rat wound-healing model after treatment for 3, 14, and 21 d. (C) Quantitative analysis of epithelial thickness at 14 and 21 d using ImageJ software. (D) Quantitative analysis of wound closure rate. (E) Masson’s trichrome staining of rat wound-healing model after treatment for 7 and 21 d. n = 6, means ± SD, *P<0.05, **P<0.01 vs control group, ns means no significant difference vs. control, P >0.05.
Figures 5. Immunofluorescence examination of Ki67 and PCNA expression. (A) Immunofluorescence examination of Ki67-positive cells after treatment for 7 and 21 d. Arrows indicate Ki67 expression. (B) Quantitative analysis of Ki67-expressing cells at 7 and 14 d measured using ImageJ software. (C) Immunofluorescence examination of PCNA-positive cells after treatment for 7 and 21 d. Arrows indicate PCNA expression. (B) Quantitative analysis of PCNA-expressing cells at 7 and 14 d measured using ImageJ software. n = 3, means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 vs control group, ns means no significant difference vs. control, P >0.05.
Figures 6. Immunohistochemical examination of CD31 and VEGF expression. (A) Immunohistochemical labeling of CD31-positive cells after treatment for 3 and 14 d. Arrows indicate CD31 expression. (B) Quantitative analysis of CD31-expressing cells at 3 and 14 d measured using ImageJ software. (C) Immunohistochemical labeling of VEGF-positive cells after treatment for 3 and 14 d. Arrows indicate VEGF expression. (D) Quantitative analysis of VEGF-expressing cells measured at 3 and 14 d using ImageJ software. n = 3, means SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs control group, ns means no significant difference vs. control, P >0.05.