Abstract:
Objective To explore effects of allogeneic skin fibroblast (Fb) on promoting wound healing of diabetic mice and the mechanism.
Methods (1) Experiment 1. Ten diabetic mice and ten normal mice were chosen and sacrificed to collect back skin tissue. Suspension of the fourth generation of normal skin Fb and diabetic skin Fb were made. Another 27 diabetic mice were collected and divided into phosphate buffered saline (PBS) group, normal skin Fb group, and diabetic skin Fb group with random number table, with 9 mice in each group. Full-thickness skin defect wounds with area of 1 cm×1 cm were made on back of each mouse. Immediately after injury, 4 corners of wound of mice in normal skin Fb group and diabetic skin Fb group were injected with normal skin Fb and diabetic skin Fb suspension of 200 μL, respectively. Mice in PBS group were injected with the same amount of PBS at the same position. On post injury day (PID) 3, 7, 10, 14, and 17, surviving mice in the three groups were collected for gross wound observation and wound healing rate was calculated. On PID 7 and 14, 3 mice in each group were taken after gross wound observation to collect wound skin tissue. Percentage of Ki67 positive cell in wound tissue was detected by immunofluorescence method. Microvessel density (MVD) of wound tissue was detected by immunohistochemistry. Collagen fiber deposition of wound tissue was detected by Masson staining. (2) Experiment 2. Ten diabetic mice and ten normal mice were collected to make primary and the fourth generation normal skin Fb, and primary and the fourth generation diabetic skin Fb with the same method as in experiment 1. Apoptosis rate of Fb was detected by flow cytometry. The mRNA expressions and protein expressions of transforming growth factor β
1 (TGF-β
1), advanced glycation end products (AGE), matrix metalloproteinase 9 (MMP-9), and neurokinin 1 (NK-1) of Fb were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Data were processed with analysis of variance of factorial design, one-way analysis of variance, and LSD-
t test.
Results (1) The drying and scab growing speeds of wounds of mice in normal skin Fb group and diabetic skin Fb group at each time point post injury were faster than those of mice in PBS group. On PID 17, wound healing rate of mice in normal skin Fb group was close to that of mice in PBS group (
t=3.45,
P>0.05). At other time points, wound healing rate of mice in normal skin Fb group and diabetic skin Fb group was significantly higher than that of mice in PBS group, respectively (
t=9.15, 10.25, 35.28, 6.79, 8.37, 10.69, 22.53, 6.70, 4.47,
P<0.05 or
P<0.01). On PID 7 and 14, wound healing rate of mice in normal skin Fb group was significantly higher than that of mice in diabetic skin Fb group (
t=4.41, 4.16,
P<0.05). On PID 7 and 14, percentages of Ki67 positive cells in wound tissue of mice in normal skin Fb group and diabetic skin Fb group were significantly higher than that of mice in PBS group (
t=20.89, 31.82, 4.86, 29.53,
P<0.05 or
P<0.01); percentages of Ki67 positive cells in wound tissue of mice in normal skin Fb group were significantly higher than those of mice in diabetic skin Fb group (
t=8.78, 13.51,
P<0.05 or
P<0.01). On PID 7 and 14, MVD of wound tissue of mice in normal skin Fb group and diabetic skin Fb group was significantly higher than that of mice in PBS group (
t=26.92, 56.42, 10.36, 26.85,
P<0.01). On PID 14, MVD of wound tissue of mice in normal skin Fb group was significantly higher than that of mice in diabetic skin Fb group (
t=8.61,
P<0.01). On PID 7 and 14, the amount of collagen fiber deposition of wound tissue of mice in normal skin Fb group was significantly higher than that of mice in diabetic skin Fb group and PBS group, respectively (
t=10.09, 5.48, 4.77, 3.14,
P<0.05 or
P<0.01). (2) Apoptosis rate of primary normal skin Fb was (5.61±0.18)%, which was close to that of normal skin Fb of the fourth generation [(6.48±0.16)%,
t=1.44,
P=0.06]. Apoptosis rate of primary diabetic skin Fb was (26.25±0.56)%, which was significantly higher than that of primary normal skin Fb (
t=36.61,
P<0.01) and close to that of diabetic skin Fb of the fourth generation [(25.68±0.93)%,
t=0.91,
P=0.41]. The mRNA expressions of TGF-β
1 and NK-1 of primary normal skin Fb were significantly higher than those of primary diabetic skin Fb (
t=25.25, 273.30,
P<0.01). The mRNA expressions of AGE and MMP-9 of primary normal skin Fb were significantly lower than those of primary diabetic skin Fb (
t=23.01, 8.84,
P<0.05 or
P<0.01). The mRNA expressions of TGF-β
1, AGE, and NK-1 in primary diabetic skin Fb were significantly higher than those of diabetic skin Fb of the fourth generation (
t=4.34, 22.84, 12.10,
P<0.05 or
P<0.01). The protein expression of TGF-β
1 and NK-1 of primary normal skin Fb were significantly higher than those of primary diabetic skin Fb (
t=4.61, 8.53,
P<0.05). The protein expressions of AGE and MMP-9 of primary normal skin Fb were significantly lower than those of primary diabetic skin Fb (
t=10.22, 29.90,
P<0.01). The protein expressions of AGE and NK-1 of primary diabetic skin Fb were significantly higher than those of diabetic skin Fb of the fourth generation (
t=8.09, 4.36,
P<0.05 or
P<0.01).
Conclusions Allogeneic skin Fb can promote wound healing through promoting Fb proliferation, angiogenesis, collagen fiber deposition in wound of diabetic mice. When diabetic skin Fb of mice is cultured in vitro away from diabetic microenvironment, cell activity can′t return to normal levels, and the effects of diabetic skin Fb on promoting wound healing is not as good as normal skin Fb.
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