Regulation mechanism of E2F1 transcription factor on M2 macrophages in full-thickness skin defect wounds of mice
10.3760/cma.j.issn.1009-2587.2019.02.005
- VernacularTitle: E2F1转录因子对小鼠全层皮肤缺损创面中M2型巨噬细胞的调节机制
- Author:
Hui XIAO
1
;
Zhen YI
;
Changchun YANG
;
Ning ZENG
;
Yi XU
;
Pei DENG
;
Haiping WANG
;
Yiping WU
;
Min WU
Author Information
1. Department of Plastic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China (Xiao Hui is now working at the Department of Breast Surgery, Henan Tumor Hospital, Affiliated Tumor Hospital of Zhengzhou University, Zhengzhou 463100, China)
- Publication Type:Journal Article
- Keywords:
Wounds and injuries;
Wound healing;
E2F1 transcription factor;
Peroxisome proliferator-activated receptors;
M2 macrophages
- From:
Chinese Journal of Burns
2019;35(2):104-109
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the regulatory mechanism of E2F1 transcription factor on M2 macrophages in full-thickness skin defect wounds of mice.
Methods:E2F1 gene knockout heterozygotes C57BL/6 mice and wild-type C57BL/6 mice were introduced and self-reproduced. Two weeks after birth, E2F1 gene knockout homozygotes mice and wild-type mice were identified by polymerase chain reaction (PCR). Twelve identified 6-8 weeks old male E2F1 gene knockout homozygotes C57BL/6 mice and wild-type C57BL/6 mice were selected respectively according to the random number table and set as E2F1 gene knockout group and wild-type group. A full-thickness skin defect wound was made on the back of each mouse. On post injury day (PID) 2 and 7, 6 mice in each group were selected according to the random number table and sacrificed, and the wound tissue was excised. The expression of CD68 and CD206 double positive M2 macrophages was observed by immunofluorescence method, and the percentage of CD206 positive cells was calculated. The protein expression of CD206 was detected by Western blotting. The mRNA expression of arginase 1 was detected by real-time fluorescent quantitative reverse transcription PCR (RT-PCR). Wound tissue specimens of the two groups on PID 7 were obtained, and the protein and mRNA expressions of peroxisome proliferator-activated receptor gamma (PPAR-γ) were detected by Western blotting and real-time fluorescent quantitative RT-PCR respectively. The above-mentioned experiments were repeated four times. Three specimens of wound tissue of mice in wild-type group on PID 7 were obtained to detect the relationship between E2F1 and PPAR-γ by co-immunoprecipitation and Western blotting, and this experiment was repeated two times. Data were processed with unpaired t test.
Results:The size of PCR products of E2F1 gene knockout homozygotes C57BL/6 mice and wild-type C57BL/6 mice were 227 and 172 bp respectively, which were the same as those of the designed DNA fragments. On PID 2 and 7, the number of CD68 and CD206 double positive M2 macrophages in the wound tissue of mice in E2F1 gene knockout group was more than that of wild-type group, and the percentages of CD206 positive cells in the wound tissue of mice in E2F1 gene knockout group were (0.234±0.032)% and (0.584±0.023)% respectively, which were significantly higher than (0.129±0.017)% and (0.282±0.071)% of wild-type group (t=3.29, 3.54, P<0.05). On PID 2 and 7, the protein expression of CD206 in the wound tissue of mice in E2F1 gene knockout group were 1.00±0.23 and 1.63±0.26 respectively, which were significantly higher than 0.43±0.06 and 0.97±0.08 of wild-type group (t=2.41, 2.45, P<0.05). On PID 2 and 7, the mRNA expressions of arginase 1 in the wound tissue of mice in E2F1 gene knockout group were 0.482±0.105 and 0.195±0.031 respectively, which were significantly higher than 0.163±0.026 and 0.108±0.017 of wild-type group (t=3.04, 2.86, P<0.05). On PID 7, the protein and mRNA expressions of PPAR-γ in the wound tissue of mice in E2F1 gene knockout group were 0.61±0.12 and 0.51±0.13 respectively, which were significantly higher than 0.20±0.04 and 0.20±0.04 of wild-type group (t=3.36, 2.86, P<0.05). On PID 7, detection of the wound tissue of mice in wild-type group showed that PPAR-γ had unidirectional effect on E2F1.
Conclusions:E2F1 transcription factor affects the polarization of M2 macrophages by inhibiting the expression of PPAR-γ, thereby inhibiting the healing process of full-thickness skin defect wounds in mice.
