摘要目的 探讨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.方法 引进E2F1基因敲除杂合子C57BL/6小鼠、野生型C57BL/6小鼠,自行繁殖并于小鼠出生后2周通过PCR法鉴定出E2F1基因敲除纯合子小鼠和野生型小鼠.采用随机数字表法分别选取12只经鉴定后生长至6～8周龄的雄性E2F1基因敲除纯合子C57BL/6小鼠、野生型C57BL/6小鼠,设为E2F1基因敲除组与野生型组,在每只小鼠背部制成1个全层皮肤缺损创面.伤后2、7d,每组分别采用随机数字表法选取6只小鼠处死,切取创面组织,采用免疫荧光法观察CD68和CD206双阳性M2型巨噬细胞表达并计算CD206阳性细胞百分比,蛋白质印迹法检测CD206蛋白表达,实时荧光定量反转录PCR(RT-PCR)法检测精氨酸酶1 mRNA表达.另取2组伤后7d创面组织标本,分别采用蛋白质印迹法和实时荧光定量RT-PCR法检测过氧化物酶体增殖物激活受体γ(PPAR-γ)的蛋白和mRNA表达.前述实验均重复4次.另取野生型组小鼠伤后7d创面组织标本3个,采用免疫共沉淀法及蛋白质印迹法检测E2F1与PPAR-γ的关系,重复2次.对数据行非配对t检验. 结果 E2F1基因敲除纯合子C57BL/6小鼠和野生型C57BL/6小鼠PCR产物大小分别为227、172 bp,与所设计DNA片段大小一致.伤后2、7d,与野生型组比较,E2F1基因敲除组小鼠创面组织中CD68和CD206双阳性M2型巨噬细胞较多;与野生型组的(0.129±0.017)％、(0.282±0.071)％比较,E2F1基因敲除组小鼠创面组织中CD206阳性细胞百分比[(0.234±0.032)％、(0.584±0.023)％]明显增加(t=3.29、3.54,P＜0.05).伤后2、7d,与野生型组的0.43±0.06、0.97±0.08比较,E2F1基因敲除组小鼠创面组织中CD206蛋白表达(1.00 ±0.23、1.63 ±0.26)明显增加(t=2.41、2.45,P＜0.05).伤后2、7d,与野生型组的0.163 ±0.026、0.108±0.017比较,E2F1基因敲除组小鼠创面组织中精氨酸酶1 mRNA表达(0.482 ±0.105、0.195±0.031)明显增加(t=3.04、2.86,P＜0.05).伤后7d,与野生型组的0.20±0.04、0.20±0.04比较,E2F1基因敲除组小鼠创面组织中PPAR-γ蛋白与mRNA表达(0.61±0.12、0.51 ±0.13)均明显增加(t=3.36、2.86,P＜0.05).伤后7d,野生型组小鼠创面组织检测显示,PPAR-γ对E2F1存在单向作用. 结论 E2F1转录因子通过抑制PPAR-γ的表达影响M2型巨噬细胞的极化,从而抑制小鼠全层皮肤缺损创面愈合过程.

abstractsObjective 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 and0.97 ±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.