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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.