聚糖抗菌成膜喷剂通过调控自然杀伤细胞和淋巴细胞亚群缓解大鼠皮肤损伤的机制

Mechanism of glycan antibacterial film-forming spray by regulating natural killer cells and lymphocyte subsets to alleviate rat skin damage

目的探究聚糖抗菌成膜喷剂通过调控自然杀伤细胞和淋巴细胞亚群缓解大鼠皮肤损伤的机制。方法选择24只成年雄性Sprague-Dawley大鼠用于本研究。共建模成功18只皮肤损伤模型,将其根据实验目的分为3组:对照组(皮肤损伤大鼠模型,无敷料覆盖,n=6);传统换药组(皮肤损伤大鼠模型,乙醇消毒后,无菌辅料覆盖,n=6)和聚糖喷剂组(皮肤损伤大鼠模型,乙醇消毒后,聚糖抗菌成膜喷剂治疗,n=6)。在术后1、3、7、10和14 d,用数码相机记录每组伤口的总体外观,并通过Image J软件量化伤口面积。通过流式细胞术分析CD4^(+)和CD8^(+)细胞百分比。通过免疫荧光染色检测淋巴细胞标志物CD3和血管生成标志物CD31在损伤部位的表达。蛋白质印迹法检测不同处理组生长因子[血管内皮生长因子(VEGF)、表皮细胞生长因子(EGF)、转化生长因子β(TGF-β)和碱性成纤维细胞生长因子(b FGF)]蛋白质水平。实时荧光定量聚合酶链式反应检测不同处理组炎症因子[肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-1β和IL-6)]mRNA水平。结果术后10、14 d,传统换药组和聚糖喷剂组伤口面积比例较对照组减少,且聚糖喷剂组较传统换药组减少,差异均有统计学意义(P<0.05)。传统换药组和聚糖喷剂组真皮高度和再上皮化百分比较对照组升高,且聚糖喷剂组较传统换药组升高,差异均有统计学意义(P<0.05)。传统换药组和聚糖喷剂组CD4^(+)细胞百分比和CD4^(+)/CD8^(+)较对照组升高,CD8^(+)细胞百分比较对照组降低,且聚糖喷剂组CD4^(+)细胞百分比和CD4^(+)/CD8^(+)较传统换药组升高,CD8^(+)细胞百分比较传统换药组降低,差异均有统计学意义(P<0.05)。聚糖喷剂组和传统换药组CD3^(+)和CD31^(+)淋巴细胞表达较对照组升高,且聚糖喷剂组高于传统换药组,差异均有统计学意义(P<0.05)。传统换药组和聚糖喷剂组VEGF、EGF、TGF-β和b FGF蛋白质水平均较对照组升高,且聚糖喷剂组均较传统换药组升高,差异均有统计学意义(P<0.05)。传统换药组和聚糖喷剂组TNF-α和IL-1β和IL-6 mRNA水平均较对照组降低,且聚糖喷剂组均较传统换药组降低,差异均有统计学意义(P<0.05)。结论聚糖抗菌成膜喷剂通过调控自然杀伤细胞和淋巴细胞亚群,增加生长因子表达水平,降低炎症因子表达水平,从而加速皮肤损伤大鼠的愈合过程。

Objective To explore the mechanism of glycan antibacterial film-forming spray by regulating natural killer cells and lymphocyte subsets to alleviate rat skin damage.Methods Twenty-four adult male Sprague-Dawley rats were selected for this study.A total of 18 skin injury models were successfully modeled,and they were divided into 3 groups according to the purpose of the experiment:control group(skin injury rat model,no dressing,n=6);traditional dressing group(skin Injury rat model,after alcohol disinfection,covered with sterile excipients,n=6)and glycan spray group(skin injury rat model,after alcohol disinfection,glycan antibacterial film-forming spray treatment,n=6).On the1,3,7,10,and 14 days after operation,the overall appearance of each group of wounds was captured with a digital camera,and the wound area was quantified by Image J software.The percentage of CD4^(+)and CD8^(+)cells was analyzed by flow cytometry.The expression of lymphocyte marker CD3 and angiogenesis marker CD31 at the injury site were detected by immunofluorescence staining.The levels of growth factors[vascular endothelial growth factor(VEGF),epidermal growth factor(EGF),transforming growth factorβ(TGF-β)and basic fibroblast growth factor(bFGF)]protein in different treatment groups were detected by Western blotting.The levels of inflammatory factors[tumor necrosis factor alpha(TNF-α),interleukin(IL)-1βand IL-6]mRNA in different treatment groups were detected by real-time polymerase chain reaction.Results On the 10 and 14 days after operation,the ratio of wound area in the traditional dressing change group and the glycan spray group was lower than that in the control group,and the ratio of wound area in the glycan spray group was lower than that in the traditional dressing group,the difference was statistically significant(P<0.05).The dermal height and the percentage of re-epithelialization in the traditional dressing change group and the glycan spray group were higher than those in the control group,and the dermal height and the percentage of re-epithelialization in the glycan spray group were higher than those in the traditional dressing group,the differences were statistically significant(P<0.05).The percentage of CD4^(+)cells and the ratio of CD4^(+)/CD8^(+)in the traditional dressing change group and the glycan spray group were higher than those in the control group,and the percentage of CD8^(+)cells was lower than that in the control group,and the percentage of CD4^(+)cells and CD4^(+)/CD8^(+)ratio in the glycan spray group were higher than those in the traditional dressing change group,the percentage of CD8^(+)cells was lower than that in the traditional dressing change group,the differences were statistically significant(P<0.05).The expression of CD3^(+)and CD31^(+)lymphocytes in the traditional dressing change group and the glycan spray group was higher than that in the control group,and the expression of CD3^(+)and CD31^(+)lymphocytes in the glycan spray group was higher than that in the traditional dressing group,the differences were statistically significant(P<0.05).The levels of VEGF,EGF,TGF-βand bFGF protein in the traditional dressing change group and the glycan spray group were higher than those in the control group,and the levels of VEGF,EGF,TGF-βand bFGF protein in the glycan spray group were higher those in the traditional dressing change group,the differences were statistically significant(P<0.05).The levels of TNF-α,IL-1βand IL-6 mRNA in the traditional dressing change group and the glycan spray group were lower than those in the control group,and the levels of TNF-α,IL-1βand IL-6 mRNA in the glycan spray group were lower than those in the traditional dressing change group,the differences were statistically significant(P<0.05).Conclusion Glycan antibacterial film-forming sprays can increase the expression levels of growth factors and reduce inflammatory factors expression level,thereby accelerating the healing process of rats with skin injury.