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自噬在小鼠皮肤创面修复中作用的初步研究
Role of autophagy during cutaneous wound healing in mice
- 第三军医大学学报 2019年41卷第1期 页码：25-32
- 作者机构：
400038 重庆，陆军军医大学(第三军医大学)军事预防医学系防原医学教研室，全军复合伤研究所，创伤、烧伤与复合伤国家重点实验室，重庆市纳米医学工程研究中心
- 作者简介：
[ 王涛，E-mail: wangtmmu@hotmail.com WANG Tao, E-mail: wangtmmu@hotmail.com
- 基金信息：国家自然科学基金面上项目(81372061);国家自然科学基金面上项目(81673089);重庆市基础与前沿研究计划项目(CSTC2016jcyjA0381)
DOI：10.16016/j.1000-5404.201808194
中图分类号： R322.99;R329.28;R641收稿：2018-08-30，
修回：2018-10-19，
纸质出版：2019-01-15
- 稿件说明：
移动端阅览
赵娜, 刘登群, 汪国建, 等. 自噬在小鼠皮肤创面修复中作用的初步研究[J]. 第三军医大学学报, 2019,41(1):25-32.
Na ZHAO, Dengqun LIU, Guojian WANG, et al. Role of autophagy during cutaneous wound healing in mice[J]. Journal of Third Military Medical University, 2019, 41(1): 25-32.
赵娜, 刘登群, 汪国建, 等. 自噬在小鼠皮肤创面修复中作用的初步研究[J]. 第三军医大学学报, 2019,41(1):25-32. DOI： 10.16016/j.1000-5404.201808194.
Na ZHAO, Dengqun LIU, Guojian WANG, et al. Role of autophagy during cutaneous wound healing in mice[J]. Journal of Third Military Medical University, 2019, 41(1): 25-32. DOI： 10.16016/j.1000-5404.201808194.
摘要
目的
2
观察自噬在小鼠皮肤创面愈合中的发生情况，探讨其在创面修复中的可能作用。
方法
2
以GFP-LC3转基因小鼠制备皮肤后背部创面，在致伤后0、3、7、14 d取皮肤创面组织制作石蜡切片进行GFP免疫荧光染色标记自噬点，观察自噬在创面愈合过程中的动态变化；采用GFP抗体与α-SMA、F4/80抗体进行荧光双标，明确肉芽间质中发生自噬的主要细胞类型。而后将GFP-LC3小鼠分为自噬抑制剂3-甲基腺嘌呤(3-MA)处理组与对照组，伤后连续10 d腹腔注射3-MA(10 mg/kg)，动态观察创面大体愈合情况，在0、7、10 d取创面皮肤，组织学固定染色，评价自噬对创面愈合的影响；同时采用定量PCR(qRT-PCR)检测α-SMA、TGF-β1、PAI-1、Ⅰ型和Ⅲ型胶原等分子的mRNA表达，评价自噬对创面修复过程中胶原与胞外基质表达水平的影响。
结果
2
创面愈合过程中自噬水平先增强后逐渐回落，在增殖期(7 d)的自噬水平最高；肉芽角质细胞自噬强于间质细胞，肉芽间质中发生自噬的细胞主要为α-SMA阳性肌成纤维细胞；与对照组相比，3-MA处理组在伤后7 d与10 d创面肉芽的面积、宽度与表皮厚度差异均无统计学意义，而处理组7 d表皮基底细胞增殖活力增强(
P
<
0.05)；两组α-SMA、胶原、TGF-β1与PAI-1等分子的mRNA水平差异均无统计学意义。
结论
2
皮肤创面愈合过程中增殖期存在高自噬水平，但抑制小鼠创面愈合过程中的自噬水平对创面愈合未见显著影响。
Abstract
Objective
2
To observe the dynamic changes in autophagy during cutaneous wound healing and investigate its possible contributions to wound repair.
Methods
2
Two skin wound of 6 mm in size were inflicted on the back of GFP-LC3 transgenic mice. In 0
3
7 and 14 d after skin injury
the skin samples were harvested to prepare paraffin-embedded sections
and GFP immunohistochemical (IHC) staining with anit-LC3 antibody was performed for the dynamic changes of autophagy during wound healing. Double-labeling immunofluorescence assay was utilized for identifying cell types of autophgy in granulation tissues by staining both GFP and macrophage marker F4/80 or myofibroblasts marker α-SMA. The mice were divided into skin defect group and injured mice with 3-methyladenine (3-MA
10 mg/kg
intra-peritoneal injection for 10 continuous days) treatment group. The dynamic changes of the wound were observed
and the skin tissues were collected in 0
7 and 10 d after injury to evaluate the effect of autophagy on wound healing after fixation and staining. qRT-PCR was employed to detect the expression levels of α-SMA
TGF-β1
PAI-1
type Ⅰ and Ⅲ collagen in the wound samples.
Results
2
The autophagic activity was enhanced and then declined in the process of wound healing
and its strongest activity was obse
rved in proliferative phase (7 d). In the granulation tissues
the activity was stronger in the keratinocytes than the interstitial cells
and the autophagic cells were mainly α-SMA-positive myofibroblasts. There were no significant differences in wound area and width
and epidermis thickness in 7 and 10 d after skin injury between the 3-MA treatment and untreated mice
but the former group had significantly increased proliferation activity of epidermal basal cells (
P
<
0.05) at 7 d after injury. However
no significant differences were seen in the mRNA levels of α-SMA
TGF-β1
PAI-1 and collagen molecules between the 2 groups of mice.
Conclusion
2
There exists stronger autophagic activity in the proliferative phase of cutaneous wound healing
but inhibiting autophagy shows little impact on the process.
关键词
Keywords
references
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