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.
目的： 探讨异体皮肤成纤维细胞(Fb)在促进糖尿病小鼠创面愈合中的作用及其机制。 方法： (1)实验1。取糖尿病小鼠及健康小鼠各10只，处死后取背部皮肤组织，制备第4代正常皮肤Fb及糖尿病皮肤Fb悬液。另取27只糖尿病小鼠，按随机数字表法分为磷酸盐缓冲液(PBS)组、正常皮肤Fb组、糖尿病皮肤Fb组，每组9只，于背部制作面积为1 cm×1 cm的全层皮肤缺损创面。伤后即刻，分别于正常皮肤Fb组、糖尿病皮肤Fb组小鼠创面4个角注射制备的正常皮肤Fb和糖尿病皮肤Fb悬液各200 μL，PBS组小鼠分别于相同位置注射等量PBS。伤后3、7、10、14、17 d，取3组存活小鼠进行创面大体观察并计算创面愈合率。伤后7、14 d，每组各取行创面大体观察后的3只小鼠处死，取创面组织。免疫荧光法检测创面组织Ki－67阳性细胞百分比，免疫组织化学法检测创面组织微血管密度(MVD)，Masson染色检测创面组织胶原纤维沉积量。(2)实验2。取糖尿病小鼠及健康小鼠各10只，同实验1制备原代、第4代正常皮肤Fb及原代、第4代糖尿病皮肤Fb。采用流式细胞仪检测细胞凋亡率，实时荧光定量反转录PCR法和蛋白质印迹法分别检测细胞转化生长因子β(1)(TGF－β(1))、晚期糖基化终末产物(AGE)、基质金属蛋白酶9(MMP－9)、神经激肽1的mRNA和蛋白表达。对数据行析因设计方差分析、单因素方差分析及LSD－t检验。 结果： (1)正常皮肤Fb组、糖尿病皮肤Fb组小鼠伤后各时间点创面干燥、结痂速度较PBS组快。伤后17 d，正常皮肤Fb组小鼠创面愈合率与PBS组相近(t＝3.45，P>0.05)，其余各时间点正常皮肤Fb组、糖尿病皮肤Fb组小鼠创面愈合率明显高于PBS组(t＝9.15、10.25、35.28、6.79、8.37、10.69、22.53、6.70、4.47，P<0.05或P<0.01)。伤后7、14 d，正常皮肤Fb组小鼠创面愈合率明显高于糖尿病皮肤Fb组(t＝4.41、4.16，P<0.05)。伤后7、14 d，正常皮肤Fb组、糖尿病皮肤Fb组小鼠创面组织Ki－67阳性细胞百分比明显高于PBS组(t＝20.89、31.82、4.86、29.53，P<0.05或P<0.01)，正常皮肤Fb组小鼠创面组织Ki－67阳性细胞百分比明显高于糖尿病皮肤Fb组(t＝8.78、13.51，P<0.05或P<0.01)。伤后7、14 d，正常皮肤Fb组、糖尿病皮肤Fb组小鼠创面组织MVD明显高于PBS组(t＝26.92、56.42、10.36、26.85，P<0.01)。伤后14 d，正常皮肤Fb组小鼠创面组织MVD明显高于糖尿病皮肤Fb组(t＝8.61，P<0.01)。伤后7、14 d，正常皮肤Fb组小鼠创面组织胶原纤维沉积量明显高于糖尿病皮肤Fb组及PBS组(t＝10.09、5.48、4.77、3.14，P<0.05或P<0.01)。(2)原代正常皮肤Fb凋亡率为(5.61±0.18)%，与第4代正常皮肤Fb凋亡率相近[(6.48±0.16)%，t＝1.44，P＝0.06]。原代糖尿病皮肤Fb凋亡率为(26.25±0.56)%，明显高于原代正常皮肤Fb(t＝36.61，P<0.01)，与第4代糖尿病皮肤Fb相近[(25.68±0.93)%，t＝0.91，P＝0.41]。原代正常皮肤Fb的TGF－β(1)及神经激肽1 mRNA表达量明显高于原代糖尿病皮肤Fb(t＝25.25、273.30，P<0.01)，AGE及MMP－9 mRNA表达量明显低于原代糖尿病皮肤Fb(t＝23.01、8.84，P<0.05或P<0.01)。原代糖尿病皮肤Fb的TGF－β(1)、AGE及神经激肽1 mRNA表达量明显高于第4代糖尿病皮肤Fb(t＝4.34、22.84、12.10，P<0.05或P<0.01)。原代正常皮肤Fb的TGF－β(1)及神经激肽1蛋白表达量明显高于原代糖尿病皮肤Fb(t＝4.61、8.53，P<0.05)，AGE及MMP－9蛋白表达量明显低于原代糖尿病皮肤Fb(t＝10.22、29.90，P<0.01)。原代糖尿病皮肤Fb的AGE及神经激肽1蛋白表达量明显高于第4代糖尿病皮肤Fb(t＝8.09、4.36，P<0.05或P<0.01)。 结论： 异体皮肤Fb可通过促进创面Fb增殖、血管生成、胶原纤维沉积等促进糖尿病小鼠创面愈合，而小鼠糖尿病皮肤Fb在脱离糖尿病微环境进行体外培养后，细胞活性低于正常皮肤Fb，因而促进创面愈合的作用不如正常皮肤Fb。.
Keywords: Cell therapy; Diabetes mellitus; Fibroblasts; Wound healing.