Toll样受体4基因敲除延迟皮肤创面愈合及促进早期瘢痕形成

Toll-like receptor 4 knockout delays skin wound healing and promotes scar formation in mice

目的探索Toll样受体4(TLR4)在小鼠皮肤缺损创面愈合及早期瘢痕形成的作用。方法将36只购自南京大学动物模式研究所的小鼠分为野生型组(WT)和TLR4基因敲除组(TLR4KO)切除部分背部全层皮肤建立模型,每组18只,观察小鼠创面愈合及瘢痕形成变化。实时定量聚合酶链反应(Real-timePCR)检测造模后7、14、21、28dTLR4,11d和33d白细胞介素(IL)-6、IL-10mRNA水平,苏木精-伊红(HE)和天狼星红染色评估33d创面及变化。组间比较采用t检验。结果在创面愈合过程中,TLR4KO创面闭合时间高于WT[(25.91±2.81)d比(19.55±2.42)d,t=5.690,P<0.01],TLR4K0 IL-10 mRNA指标低于WT(0.005比1.000,t=37.070,P<0.01),TLR4KOIL-6mRNA指标高于WT(2.650比1.000,t=19.290,P<0.01)。在瘢痕形成早期,TLR4K0表皮厚度高于WT[(45.83±1.61)μm比(22.79±2.07)μm,t=7.645,P<0.01],TLR4KOI型/Ⅲ型胶原比值高于WT(24.54±1.27比7.93±1.76,t=10.820,P<0.01),TLR4K0 IL-6mRNA指标高于WT(3.190比1.000,t=9.865,P<0.01),TLR4K0与WTIL-10mRNA指标差异无统计学意义(1.108比1.000,t=0.960,P>0.05)。结论TLR4功能完全丧失导致皮肤创面延迟愈合,进而导致TLR4基因敲除小鼠早期瘢痕明显,可能与炎性反应失调有关。

Objective To explore the role of Toll-like receptor 4(TLR4)in wound healing and early scar formation of skin defects in mice.Methods The mice purchased from the Institute of Animal models of Nanjing University were divided into wild-type(WT)group and TLR4 gene knockout(TLR4KO)group.The full thickness skin of the back was removed to establish the model,and the changes of wound healing and scar formation were observed.The real-time quantitative polymerase chain reaction(Real-time PCR)was used to detect the level of TLR4 at 7,14,21 and 28 days after modeling,and the levels of in-terleukin(IL)-6 and IL-10 in wound and scar tissue at 11 and 33 days after injury.Hematoxylin and eosin(HE)staining was used to evaluate the wound healing and scar changes at 33 days after injury,and Sirius red staining was used to evaluate the scar formation at 33 days after injury.T-test was used to analyze the data.Results In the process of wound healing,the wound closure time in TLR4KO group was longer than that in WT group[(25.91±2.81)d vs.(19.55±2.42)d,t=5.690,P<0.01].The expression of IL-10 mRNA in TLR4KO group was lower than that in WT group(0.005 vs.1.000,t=37.070,P<0.01),and the expression of IL-6 mRNA in TLR4KO group was higher than that in WT group(2.650 vs.1.000,t=19.290,P<0.01).In the early stage of scar formation,the epidermal thickness in TLR4KO group was greater than that in WT group[(45.83±1.61)μm vs.(22.79±2.07)μm,t=7.645,P<0.01],and the ratio of type I/type II collagen in TLR4KO group was higher than that in WT group(24.54±1.27 vs.7.93±1.76,t=10.820,P<0.01).The expression of IL-6 mRNA in TLR4KO group was higher than that in WT group(3.190 vs.1.000,t=9.865,P<0.01).There was no significant difference in the expression of IL-6 between the TLR4KO group and the WT group(1.108 vs.1.000,t=0.960,P>0.05).Conclusion The complete loss of TLR4 function leads to delayed healing of skin wounds,which leads to obvious scar in the early stage of TLR4 gene knockout mice,which may be related to the imbalance of inflammatory response.