树突状表皮T淋巴细胞对小鼠创缘表皮细胞增殖和凋亡的影响

刘勉; 朱海杰; 杨加彩; 李雅舒; 胡晓红; 张小容; 贺伟峰; 罗高兴

doi:10.3760/cma.j.issn.1009-2587.2020.02.008

树突状表皮T淋巴细胞对小鼠创缘表皮细胞增殖和凋亡的影响

doi: 10.3760/cma.j.issn.1009-2587.2020.02.008

陆军军医大学(第三军医大学)第一附属医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆市疾病蛋白质组学重点实验室,重庆 400038

基金项目:

国家自然科学基金（31872742）

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出版历程
- 收稿日期: 2019-05-29
- 网络出版日期: 2021-10-28
- 刊出日期: 2020-02-20

Effects of dendritic epidermal T cells on proliferation and apoptosis of epidermal cells in wound margin of mice

State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing Key Laboratory for Disease Proteomics, Chongqing 400038, China

摘要

摘要:
目的了解树突状表皮T淋巴细胞(DETC)对小鼠创缘表皮细胞增殖和凋亡的影响及其在创面愈合中的作用。方法选取无特殊病原体(SPF)级野生型C57BL/6健康8～12周龄雄性小鼠28只、SPF级8～12周龄T淋巴细胞受体δ敲除(TCR δ^－/－)雄性小鼠60只用于如下实验。(1)采用随机数字表法取8只野生型小鼠分离表皮细胞、原代培养DETC，并于培养即刻及培养15、30 d行DETC形态学观察和采用流式细胞仪检测纯度。(2)采用随机数字表法取5只野生型小鼠、5只TCR δ^－/－小鼠，分别设为野生型对照组、TCR δ^－/－组，每只小鼠背部制作直径6 mm圆形全层皮肤缺损创面。观察伤后即刻及伤后2、4、6、8、10 d创面愈合情况、计算剩余创面面积百分比。(3)同实验(2)取小鼠，分组并制作全层皮肤缺损模型。伤后3 d取创缘组织进行苏木精-伊红染色，检测新生上皮长度。(4)同实验(2)取小鼠，分组并制作全层皮肤缺损模型。伤后3 d取创缘表皮组织，采用蛋白质印迹法检测增殖细胞核抗原(PCNA)的表达，评估表皮细胞增殖情况。(5)同实验(2)取小鼠，分组并制作全层皮肤缺损模型。伤后3 d取创缘的表皮组织消化成单细胞悬液，采用流式细胞仪检测细胞凋亡情况。(6)取40只TCR δ^－/－小鼠，同前行实验(2)～(5)检测，并采用随机数字表法分为TCR δ^－/－对照组和TCR δ^－/－＋DETC组，每个实验每组5只小鼠。其中小鼠背部同前造成全层皮肤缺损，TCR δ^－/－＋DETC组小鼠伤后即刻于创缘多点注射1×10⁵个DETC(溶解于100 μL磷酸盐缓冲液，纯度超过90%)，TCR δ^－/－对照组小鼠同法注射等体积磷酸盐缓冲液。对数据行重复测量方差分析、
t
检验及Bonferroni校正。结果 (1)随着培养时间的延长，DETC树突状突起的数量逐渐增多。培养即刻，4.67%的细胞是T淋巴细胞，这部分T淋巴细胞中94.10%为DETC。培养15 d时DETC纯度达18.50%，培养30 d时DETC纯度达98.70%。(2)伤后即刻，TCR δ^－/－组、野生型对照组小鼠的创面情况相似；TCR δ^－/－组小鼠伤后2～10 d的创面愈合速度慢于野生型对照组。与野生型对照组小鼠比较，TCR δ^－/－组小鼠伤后2、4、6、8、10 d剩余创面面积百分比均明显升高(
t
＝3.492、4.425、4.170、4.780、7.318,
P
<0.01)。(3)TCR δ^－/－组小鼠伤后3 d创缘新生上皮长度为(359±15)μm，明显短于野生型对照组的(462±26)μm(
t
＝3.462，
P
<0.01)。(4)TCR δ^－/－对照组小鼠伤后即刻的创面情况与TCR δ^－/－＋DETC组相似；TCR δ^－/－＋DETC组小鼠伤后2～10 d的创面愈合速度明显快于TCR δ^－/－对照组。与TCR δ^－/－对照组比较，TCR δ^－/－＋DETC组小鼠伤后2、4、6、8、10 d剩余创面面积百分比明显降低(
t
＝2.308、3.725、2.698、3.707、6.093,
P
<0.05或
P
<0.01)。(5)TCR δ^－/－＋DETC组小鼠伤后3 d创缘新生上皮长度为(465±31)μm，明显长于TCR δ^－/－对照组的(375±21)μm(
t
＝2.390，
P
<0.05)。(6)伤后3 d，TCR δ^－/－组小鼠创缘表皮细胞的PCNA表达量为1.25±0.04，明显低于野生型对照组的2.01±0.09(
t
＝7.415，
P
<0.01)。(7)伤后3 d，TCR δ^－/－＋DETC组小鼠创缘表皮细胞的PCNA表达量为1.62±0.08，明显高于TCR δ^－/－对照组的1.05±0.14(
t
＝3.561，
P
<0.05)。(8)伤后3 d，TCR δ^－/－组小鼠创缘表皮细胞凋亡率为(16.1±1.4)%，明显高于野生型对照组的(8.1±0.6)%(
t
＝5.363，
P
<0.01)。(9)伤后3 d，TCR δ^－/－＋DETC组小鼠创缘的表皮细胞凋亡率为(11.4±1.0)%，明显低于TCR δ^－/－对照组的(15.4±1.4)%(
t
＝2.377，
P
<0.05)。结论 DETC可通过促进小鼠创缘表皮细胞的增殖、抑制其凋亡，参与创面愈合过程。
- 伤口愈合 /
- 细胞增殖 /
- 细胞凋亡 /
- 树突状表皮T淋巴细胞 /
- 表皮细胞
Abstract:
Objective To explore the effects of dendritic epidermal T cells (DETC) on proliferation and apoptosis of epidermal cells in wound margin of mice and its effects on wound healing. Methods Twenty-eight healthy specific pathogen free (SPF) C57BL/6 wild-type (WT) male mice aged 8-12 weeks and 60 SPF T lymphocyte receptor δ-knockout (TCR δ^-/-) male mice aged 8-12 weeks were selected to conduct the following experiments. (1) Eight WT mice were selected to isolate epidermal cells and primarily culture DETC according to the random number table. Morphological observation and purity identification of DETC by flow cytometer were detected immediately after culture and on culture day (CD) 15 and 30, respectively. (2) According to the random number table, 5 WT mice and 5 TCR δ^-/- mice were selected and enrolled into WT control group and TCR δ^-/- group. Round full-thickness skin defect with diameter of 6 mm was made on the back of each mouse. The wound healing condition was observed immediately after injury and on post injury day (PID) 2, 4, 6, 8, 10, and the percentage of residual wound area was calculated. (3) Mice were selected to group and reproduce model of full-thickness skin defect as in experiment (2). On PID 3, the tissue of wound margin was collected for hematoxylin eosin staining, and the length of new epithelium was measured. (4) Mice were selected to group and reproduce model of full-thickness skin defect as in experiment (2). On PID 3, epidermal tissue of wound margin was collected to determine expression of proliferating cell nuclear antigen (PCNA) using Western blotting for evaluation of proliferation of epidermal cell. (5) Mice were selected to group and reproduce model of full-thickness skin defect as in experiment (2). On PID 3, epidermal tissue of wound margin was selected and digested into single-cell suspension, and apoptosis of cells was detected by flow cytometer. (6) Forty TCR δ^-/- mice were selected to carry out the same treatment as in experiments (2)-(5). According to the random number table, these mice were enrolled into TCR δ^-/- control group and TCR δ^-/-+ DETC group, with 5 mice in each group for each experiment. Round full-thickness skin defect was made on the back of each mouse. DETC in the number of 1×10⁵ (dissolution in 100 μL phosphate with buffer purity above 90%) were injected through multiple points of wound margin of mice in TCR δ^-/-+ DETC group immediately after injury, and equal volume of phosphate buffer was injected into mice of TCR δ^-/- control group with the same method as above. Data were processed with one-way analysis of variance for repeated measurement,
t
test, and Bonferroni correction. Results (1) Along with the culture time elapse, the number of dendritic structures of DETC increased gradually. The percentage of T lymphocytes was 4.67% and 94.1% of these T lymphocytes were DETC. The purity of DETC on CD 15 was 18.50% and the purity of DETC on CD 30 was 98.70%. (2) Immediately after injury, the wound healing condition of mice in WT control group and TCR δ^-/- group was similar. The wound healing speed of mice in TCR δ^-/- group was slower than that in WT control group on PID 2-10. The percentages of residual wound area of mice in TCR δ^-/- group on PID 2, 4, 6, 8, and 10 were increased significantly compared with those in WT control group (
t
=3.492, 4.425, 4.170, 4.780, 7.318,
P
<0.01). (3) The length of new epithelium of mice in TCR δ^-/- group on PID 3 was (359 ± 15) μm, which was obviously shorter than that in WT control group [(462±26) μm,
t
=3.462,
P
<0.01]. (4) Immediately after injury, wound condition of mice in TCR δ^-/-+ DETC group and TCR δ^-/- control group was similar. Compared with TCR δ^-/-+ DETC group, the wound healing speed of mice in TCR δ^-/- control group were obviously slower on PID 2-10. The percentages of residual wound area of mice in TCR δ^-/-+ DETC group on PID 2, 4, 6, 8, and 10 were decreased significantly compared with those in TCR δ^-/- control group (
t
=2.308, 3.725, 2.698, 3.707, 6.093,
P
<0.05 or
P
<0.01). (5) On PID 3, the length of new epithelium of mice in TCR δ^-/-+ DETC group was (465±31) μm, which was obviously longer than that in TCR δ^-/- control group [(375±21) μm,
t
=2.390,
P
<0.05]. (6) On PID 3, PCNA expression of epidermal cell in wound margin of mice in TCR δ^-/- group was 1.25±0.04, which was obviously lower than that in WT control group (2.01±0.09,
t
=7.415,
P
<0.01). (7) On PID 3, PCNA expression of epidermal cell in wound margin of mice in TCR δ^-/-+ DETC group was 1.62±0.08, which was significantly higher than that in TCR δ^-/- control group (1.05±0.14,
t
=3.561,
P
<0.05). (8) On PID 3, apoptosis rate of epidermal cell in wound margin of mice in TCR δ^-/- group was (16.1±1.4)%, which was higher than that in WT control group [(8.1±0.6)%,
t
=5.363,
P
<0.01]. (9) On PID 3, apoptosis rate of epidermal cell in wound margin of mice in TCR δ^-/-+ DETC group was (11.4±1.0)%, which was obviously lower than that in TCR δ^-/- control group [(15.4±1.4)%,
t
=2.377,
P
<0.05]. Conclusions DETC participates in the process of wound healing though promoting the proliferation of epidermal cells in wound margin and inhibit the apoptosis of these cells.
- Wound healing /
- Cell Proliferation /
- Apoptosis /
- Dendritic epidermal T cells /
- Epidermal cells