窄带LED无创光疗对糖尿病足真皮成纤维细胞增殖及分泌功能的影响

Regulatory Effect of Narrow-Band LED-Based Non-Damage Phototherapy on Dermal Fibroblasts of Diabetic Foot

目的研究窄带发光二极管(light emitting diode,LED)无创光疗对糖尿病足真皮成纤维细胞(diabetic foot dermal fibroblasts,DFFs)增殖和分泌功能的影响。方法将糖尿病足溃疡患者的足部皮肤标本使用酶消化法原代分离培养获得DFFs,采用细胞免疫荧光实验进行成纤维细胞鉴定。以DFFs为研究对象,实验分为两部分:(1)窄带LED对DFFs细胞增殖情况的影响。细胞增殖实验分为3组,分别为非光疗组(A组)即仅接种细胞但不进行LED光照;光疗组(B组),采用波长630 nm,带宽16 nm,功率密度10 mW/cm^(2)的LED光分别照射60 s、100 s、500 s、1000 s、2000 s和2500 s。空白对照组(C组)即不接种细胞也不进行LED光照。各组处理后24 h检测细胞吸光度计算各组细胞活力以评估增殖情况。(2)窄带LED对DFFs细胞因子分泌的影响。实验分为光疗组和非光疗组两组,优选促进DFFs增殖作用最强的光剂量作为光疗组的光剂量;非光疗组不进行光照,两组干预后12 h,比较两组DFFs分泌转化生子因子-β1(transforming growth factor-beta 1,TGF-β1)、基质金属蛋白酶抑制剂1(matrix metalloproteinase inhibitor 1,TIMP1)和血管生成素(angiopoietin 1,Ang1)的差异。结果(1)细胞免疫荧光鉴定发现酶消化法分离培养的原代DFFs中成纤维细胞纯度接近100%。(2)窄带LED照射DFFs细胞后细胞增殖情况。光疗组(B组)的细胞增殖随照射时间的增加先升高达到最大值后开始降低。光疗组(B组)中以波长630nm,功率密度10 mW/cm^(2)的LED照射500 s和1000 s时能够促进原代培养的DFFs的增殖,与非光疗组(A组)比较差异具有统计学意义(P<0.05)。(3)窄带LED照射DFFs细胞后细胞因子分泌的情况。光疗组以波长630 nm,功率密度10 mW/cm^(2)的LED照射1000 s时,能促进DFFs分泌TGF-β1和Ang1,与非光疗组比较差异具有统计学意义(P<0.05),但对TIMP1因子的分泌无影响(P>0.05)。结论窄带LED无创光疗可以促进DFFs增殖,并增加与成纤维细胞增殖相关的因子TGF-β1、血管生成相关的因子Ang1的分泌。

Objective To investigate the effects of non-damage phototherapy using narrow-band light emitting diodes(LED)on the proliferation and secretion functions of diabetic foot dermal fibroblasts(DFFs).Methods DFFs were isolated and cultured from skin samples of diabetic foot ulcer patients using an enzymatic digestion method,and fibroblasts were identified through cell immunofluorescence experiments.The study was divided into two parts:(1)The effect of narrow-band LED on the proliferation of DFFs.The cell proliferation experiment was divided into three groups:the non-phototherapy group(Group A),which involved only cell seeding without LED exposure;the phototherapy group(Group B),which was exposed to LED light at a wavelength of 630 nm,bandwidth of 16 nm,and power density of 10 mW/cm^(2)for durations of 60 s,100 s,500 s,1000 s,2000 s,and 2500 s;and the blank control group(Group C),which neither had cells seeded nor received LED exposure.After 24 hours of treatment,the absorbance of cells in each group was measured to calculate the cell viability of each phototherapy group in order to assess proliferation.(2)The fect of narrow-band LED on cytokine secretion by DFFs.The experiment was divided into the phototherapy group and the non-phototherapy group,with the most effective light dose for promoting DFF proliferation chosen for the phototherapy group's exposure parameters;the non-phototherapy group did not receive light exposure.12 hours after intervention,the differences in the secretion of transforming growth factor-beta 1(TGF-β1),matrix metalloproteinase inhibitor 1(TIMP1),and Angiopoietin 1(Ang1)between the two groups were compared.Results(1)Immunofluorescence detection showed that the purity of fibroblasts in the primary cultured DFFs was close to 100%.(2)Cell proliferation in DFFs after narrow-band LED irradiation.In the phototherapy group(Group B),cell proliferation increased with irradiation time,reaching a peak before beginning to decrease.The phototherapy group(Group B),with LED irradiation at a wavelength of 630 nm and power density of 10 mW/cm^(2)for 500 s and 1000 s,significantly promoted the proliferation of primary cultured DFFs compared to the non-phototherapy group(Group A)(P<0.05).(3)Cytokine secretion in DFFs after narrow-band LED irradiation.In the phototherapy group,LED irradiation at a wavelength of 630 nm and power density of 10 mW/cm^(2)for 1000 s significantly promoted the secretion of TGF-β1 and Ang1 compared to the non-phototherapy group(P<0.05),but had no effect on the secretion of the TIMP1 factor(P>0.05).Conclusions Non-damage phototherapy with narrow-band LED can promote the proliferation of DFFs and increase the secretion of factors related to fibroblast proliferation(TGF-β1)and factors related to angiogenesis(Ang1).