Objective: To explore effects of dendritic epidermal T cells (DETCs) and Vγ4 T lymphocytes on proliferation and differentiation of mice epidermal cells and the effects in wound healing of mice. Methods: (1) Six C57BL/6 male mice aged 8 weeks were collected and divided into control group and wound group according to random number table (the same grouping method below), with 3 mice in each group. A 4 cm long straight excision with full-thickness skin defect was cut on back of each mouse in wound group, while mice in control group received no treatment. On post injury day (PID) 3, mice in 2 groups were sacrificed, and skin within 5 mm from the wound margin on back of mice in wound group and normal skin on corresponding part of mice in control group were collected to make single cell suspensions. The percentage of Vγ4 T lymphocyte expressing interleukin-17A (IL-17A) and percentage of DETCs expressing insulin-like growth factor Ⅰ (IGF-Ⅰ) were detected by flow cytometer. (2) Ten C57BL/6 male mice aged 8 weeks were collected and divided into control group and Vγ4 T lymphocyte depletion group with 5 mice in each group. Mice in Vγ4 T lymphocyte depletion group were injected with 200 g Vγ4 T lymphocyte monoclonal neutralizing antibody of Armenian hamster anti-mouse intraperitoneally, and mice in control group were injected with the same amount of Armenian hamster Ig intraperitoneally. One hole with full-thickness skin defect was made on each side of spine of back of each mice. The wound healing was observed on PID 1-8, and percentage of remaining wound area was calculated. (3) Six C57BL/6 male mice aged 8 weeks were grouped and treated in the same way as in experiment (2), with 3 mice in each group. On PID 3, expressions of IL-17A and IGF-Ⅰ in epidermis on margin of wound were detected with Western blotting. (4) Thirty C57BL/6 male mice aged 3 days were sacrificed, and epidermal cells were extracted. The keratin 14 positive cell rate was examined by flow cytometer (the same detecting method below). (5) Another batch of mouse epidermal cells were collected and divided into control group, IGF-Ⅰ group, and IL-17A group, with 3 wells in each group (the same well number below). Cells in IGF-Ⅰ group and IL-17A group were added with 1 mL recombinant mouse IGF-Ⅰ and IL-17A with final mass concentration of 100 ng/mL respectively, while cells in control group were added with the same amount of sterile phosphate buffered saline (PBS). On post culture day (PCD) 5, keratin 14 negative cell rate was examined. Another batch of mouse epidermal cells were collected, grouped, and treated in the same way as aforementioned experiment, and keratin 10 positive cell rate was examined on PCD 10. (6) Another batch of mouse epidermal cells were collected and added with 4 mmol/L 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) solution, and divided into control 0 d group, control 7 d group, IGF-Ⅰ group, and IL-17A group. Cells in IGF-Ⅰ group and IL-17A group were treated in the same way as the corresponding groups in experiment (5), and cells in control 0 d group and control 7 d group were treated in the same way as the control group in experiment (5). The CFSE fluorescence peaks were examined on PCD 0 of control 0 d group and PCD 7 of the other 3 groups. (7) Another batch of mouse epidermal cells were collected and divided into control group and IGF-Ⅰ group. Cells in IGF-Ⅰ group were added with 1 mL recombinant mouse IGF-Ⅰ with final mass concentration of 100 ng/mL, and cells in control group were added with the same amount of sterile PBS. On PCD 5, cells were underwent keratin 14 staining and CFSE staining as aforementioned, and keratin 14 negative cell rate of CFSE positive cells was examined. Another batch of mouse epidermal cells were collected and divided into control group and IL-17A group. Cells in IL-17A group were added with 1 mL recombinant mouse IL-17A with final mass concentration of 100 ng/mL, and cells in control group were added with the same amount of sterile PBS. On PCD 5, keratin 14 negative cell rate of CFSE positive cells was examined. Data were processed with one-way analysis of variance and t test. Results: (1) On PID 3, percentage of DETC expressing IGF-Ⅰ in normal epidermis of control group was (9.9±0.8)%, significantly lower than (19.0±0.6)% of epidermis around margin of wound group (t=8.70, P<0.01); percentage of Vγ4 T lymphocyte expressing IL-17A in normal epidermis of control group was (0.123±0.024)%, significantly lower than (8.967±0.406)% of epidermis around margin of wound group (t=21.77, P<0.01). (2) On PID 1-4, there was obvious inflammatory reaction around wounds of mice in control group, and on PID 5-8, the wound area was still large. On PID 1-4, there was slight inflammatory reaction around wounds of mice in Vγ4 T lymphocyte depletion group, and on PID 5-8, the wound area was significantly reduced. On PID 3-7, percentages of residual wound area in Vγ4 T lymphocyte depletion group were significantly lower than those in control group (t=5.92, 5.74, 7.17, 5.38, 5.57, P<0.01), while percentages of residual wound area in two groups on PID 1, 2, 6 were similar (t=1.46, 3.17, 3.10, P>0.05). (3) On PID 3, compared with those in control group, expression of IL-17A and IGF-Ⅰ in epidermis around wound margin of mice in Vγ4 T lymphocyte depletion group was markedly decreased and increased respectively (t=8.47, 19.24, P<0.01). (4) The keratin 14 positive cell rate of mouse epidermal cells was 94.7%. (5) On PCD 5, the keratin 14 negative cell rate of mice in control group was markedly higher than that of IGF-Ⅰ group, while significantly lower than that of IL-17A group (t=7.25, 5.64, P<0.01). On PCD 10, the keratin 10 positive cell rate of mice in control group was significantly higher than that of IGF-Ⅰ group, while significantly lower than that of IL-17A group (t=3.99, 10.82, P<0.05 or P<0.01). (6) Compared with that of control 0 d group, CFSE fluorescence peaks of mouse epidermal cells in control 7 d group, IGF-Ⅰ group, and IL-17A group on PCD 7 shifted to the left. Compared with that of control 7 d group, CFSE fluorescence peaks of mouse epidermal cells in IGF-Ⅰ group and IL-17A group on PCD 7 shifted to the left. (7) On PCD 5, keratin 14 negative cell rate of CFSE positive cells of mice in control group was significantly higher than that in IGF-Ⅰ group (t=9.91, P<0.01), and keratin 14 negative cell rate of CFSE positive cells of mice in control group was markedly lower than that in IL-17A group (t=6.49, P<0.01). Conclusions: In the process of wound healing, IGF-Ⅰ secreted by DETC can promote the proliferation of mouse keratin 14 positive epidermal cells and inhibit their terminal differentiation, while IL-17A secreted by Vγ4 T lymphocyte can promote the proliferation and terminal differentiation of mouse keratin 14 positive epidermal cells, thus both IGF-Ⅰ and IL-17A can affect wound healing.
目的：探讨树突状表皮T淋巴细胞(DETC)和Vγ4 T淋巴细胞对小鼠表皮细胞增殖分化的影响及其在创面愈合中的作用。 方法：(1)取6只8周龄C57BL/6雄性小鼠，采用随机数字表法(分组方法下同)分为对照组和创面组，每组3只。于创面组小鼠背部剪1条长4 cm深达皮肤全层的直线切口，对照组小鼠不行任何处理。伤后3 d，断颈处死2组小鼠，取创面组小鼠背部距创缘5 mm内的皮肤，取对照组小鼠相应部位正常皮肤制成单细胞悬液，流式细胞仪检测表达白细胞介素17A(IL－17A)的Vγ4 T淋巴细胞的百分比及表达胰岛素样生长因子Ⅰ(IGF－Ⅰ)的DETC百分比。(2)取10只8周龄C57BL/6雄性小鼠，分为对照组和Vγ4 T淋巴细胞清除组，每组5只。Vγ4 T淋巴细胞清除组小鼠腹腔注射200 g亚美尼亚仓鼠抗小鼠Vγ4 T淋巴细胞单克隆中和抗体，对照组小鼠腹腔注射等量亚美尼亚仓鼠Ig，分别于背部脊柱两侧各打1个深达皮肤全层的孔，观察伤后1～8 d创面愈合情况，并计算剩余创面面积百分比。(3)取6只8周龄C57BL/6雄性小鼠，同实验(2)进行分组及处理，每组3只。伤后3 d，蛋白质印迹法检测创缘表皮组织IL－17A和IGF－Ⅰ的蛋白表达。(4)取30只3 d龄C57BL/6雄性小鼠，断颈处死后提取表皮细胞，流式细胞仪(下同)检测角蛋白14阳性细胞率。(5)另取小鼠表皮细胞，分为对照组、IGF－Ⅰ组与IL－17A组，每组3孔(下同)。IGF－Ⅰ组与IL－17A组细胞分别加入1 mL终质量浓度为100 ng/mL的重组小鼠IGF－Ⅰ和重组小鼠IL－17A，对照组细胞加入等量无菌磷酸盐缓冲液(PBS)。培养5 d后，检测角蛋白14阴性细胞率。另取小鼠表皮细胞，同前进行分组及处理，培养10 d后，检测角蛋白10阳性细胞率。(6)另取小鼠表皮细胞，加入4 mmol/L羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)染液，分为对照0 d组、对照7 d组、IGF－Ⅰ组、IL－17A组。IGF－Ⅰ组、IL－17A组细胞同实验(5)对应组进行处理，对照0 d组、对照7 d组细胞同实验(5)对照组进行处理。对照0 d组细胞培养0 d，余3组细胞培养7 d，检测CFSE荧光波峰位置。(7)另取小鼠表皮细胞，分为对照组、IGF－Ⅰ组。IGF－Ⅰ组细胞加入1 mL终质量浓度为100 ng/mL重组小鼠IGF－Ⅰ，对照组细胞加入等量无菌PBS，培养5 d后，同前进行角蛋白14和CFSE染色，检测CFSE阳性细胞角蛋白14阴性细胞率。另取小鼠表皮细胞，分为对照组与IL－17A组，IL－17A组细胞加入1 mL终质量浓度为100 ng/mL重组小鼠IL－17A，对照组细胞加入等量无菌PBS，培养5 d后，检测CFSE阳性细胞角蛋白14阴性细胞率。对数据行单因素方差分析和t检验。 结果：(1)伤后3 d，对照组小鼠正常表皮组织中表达IGF－Ⅰ的DETC百分比为(9.9±0.8)%，明显低于创面组小鼠创缘表皮组织中的(19.0±0.6)%，t＝8.70，P<0.01；对照组小鼠正常表皮组织中表达IL－17A的Vγ4 T淋巴细胞百分比为(0.123±0.024)%，明显低于创面组小鼠创缘表皮组织中的(8.967±0.406)%，t＝21.77，P<0.01。(2)对照组小鼠伤后1～4 d创面周围有明显的炎症反应；伤后5～8 d，创面面积仍较大。Vγ4 T淋巴细胞清除组小鼠伤后1～4 d创面周围炎症反应较轻；伤后5～8 d，创面面积明显缩小。伤后3～7 d，Vγ4 T淋巴细胞清除组小鼠剩余创面面积百分比明显低于对照组(t＝5.92、5.74、7.17、5.38、5.57，P<0.01)；培养1、2、8 d，2组小鼠剩余创面面积百分比相近(t＝1.46、3.17、3.10，P>0.05)。(3)伤后3 d，与对照组比较，Vγ4 T淋巴细胞清除组小鼠创缘表皮组织的IL－17A蛋白表达明显下降，IGF－Ⅰ蛋白表达明显升高(t＝8.47、19.24，P<0.01)。(4)小鼠表皮细胞角蛋白14阳性细胞率为94.7%。(5)培养5 d后，对照组小鼠表皮细胞角蛋白14阴性细胞率明显高于IGF－Ⅰ组，明显低于IL－17A组(t＝7.25、5.64，P<0.01)。培养10 d后，对照组小鼠表皮细胞角蛋白10阳性细胞率明显高于IGF－Ⅰ组，明显低于IL－17A组(t＝3.99、10.82，P<0.05或P<0.01)。(6)相较于对照0 d组，对照7 d组、IGF－Ⅰ组和IL－17A组小鼠表皮细胞培养7 d后CFSE荧光波峰左移。相较于对照7 d组，IGF－Ⅰ组和IL－17A组小鼠表皮细胞培养7 d后CFSE荧光波峰均左移。(7)培养5 d后，对照组CFSE阳性小鼠表皮细胞角蛋白14阴性细胞率明显高于IGF－Ⅰ组(t＝9.91，P<0.01)，对照组CFSE阳性小鼠表皮细胞角蛋白14阴性细胞率明显低于IL－17A组(t＝6.49，P<0.01)。 结论：在创面愈合过程中，DETC分泌的IGF－Ⅰ促进小鼠角蛋白14阳性表皮细胞增殖并抑制其终末分化，Vγ4 T淋巴细胞分泌的IL－17A促进其增殖及终末分化，从而影响创面愈合。.
Keywords: Cell differentiation; Cell proliferation; Insulin-like growth factor Ⅰ; Interleukin-17; T-lymphocytes; Wound healing; Wounds and injuries.