蛆虫清创疗法促进糖尿病足溃疡患者创面血管新生的机制

Mechanism of maggot debridement therapy in promoting wound angiogenesis in patients with diabetic foot ulcer

目的探讨蛆虫清创疗法(MDT)促进糖尿病足溃疡(DFU)患者创面血管新生的机制。方法(1)将东部战区空军医院2018年6月—2019年6月收治的符合入选标准的12例应用MDT(疗程为3 d)的DFU患者[男6例、女6例,年龄(56±12)岁]、12例无糖尿病的急性外伤患者[男6例、女6例,年龄(53±10)岁]分别设为DFU组与外伤无糖尿病组。应用MDT前后观察DFU组患者创面大体情况并留取创面组织,留取外伤无糖尿病组患者首次就诊时清创前创面组织,采用免疫组织化学法检测DFU组患者应用MDT前后创面组织中血管新生标志物CD31的表达,分别采用蛋白质印迹法、实时荧光定量反转录PCR(RT-PCR)法检测DFU组患者应用MDT前后及外伤无糖尿病组患者清创前创面组织中脂肪酸合成酶(FAS)的蛋白、mRNA表达。(2)用含体积分数10%胎牛血清的内皮细胞培养基体外培养人脐静脉内皮细胞(HUVEC),取第3~6代对数生长期细胞进行以下实验。取3 d龄无菌丝光绿蝇幼虫,提取分泌排泄物(ES)用于后续实验。取3个批次细胞,均分为磷酸盐缓冲液(PBS)对照组、单纯高糖组、高糖+5μg/mL蛆虫ES组和高糖+10μg/mL蛆虫ES组,分别加入PBS、终物质的量浓度20 mmol/L葡萄糖、终物质的量浓度20 mmol/L葡萄糖+终质量浓度5μg/mL蛆虫ES、终物质的量浓度20 mmol/L葡萄糖+终质量浓度10μg/mL蛆虫ES处理,各组试剂总体积相同。培养48 h,分别采用蛋白质印迹法、实时荧光定量RT-PCR法和免疫荧光法检测各批次细胞中FAS的蛋白、mRNA表达情况及细胞内FAS蛋白表达与定位情况,其中mRNA表达样本数为3。(3)取2个批次细胞,均分为单纯小干扰RNA(siRNA)对照组、siRNA对照+蛆虫ES组、siRNA-FAS+蛆虫ES组,分别转染终物质的量浓度为100μmol/L的无意义对照siRNA、无意义对照siRNA、siRNA-FAS 4~6 h,之后分别加入PBS、终质量浓度10μg/mL蛆虫ES、终质量浓度10μg/mL蛆虫ES常规培养24 h,各组试剂的总体积相同。1个批次细胞进行划痕试验,划痕后24 h观察划痕宽度,检测细胞迁移能力;1个批次细胞进行成管实验,培养24 h后观察细胞小管生成情况。划痕试验和成管实验样本数均为3。对数据行t检验、单因素方差分析、LSD检验、重复测量方差分析及Bonferroni法。结果(1)与应用MDT前比较,DFU组患者应用MDT后创面新鲜肉芽组织明显增多,坏死组织明显减少;创面组织中CD31表达明显增多。DFU组患者应用MDT前创面组织中FAS蛋白表达较外伤无糖尿病组清创前明显减少,DFU组患者应用MDT后创面组织中FAS蛋白表达较应用MDT前明显增多。DFU组患者应用MDT前创面组织中FAS mRNA表达量为1.00±0.17,较外伤无糖尿病组清创前的3.87±1.02明显减少(t=9.808,P<0.01);DFU组患者应用MDT后创面组织中FAS mRNA表达量为1.85±0.31,较应用MDT前明显增多(t=-10.853,P<0.01)。(2)培养48 h,蛋白质印迹法检测显示,单纯高糖组细胞中FAS蛋白表达较PBS对照组明显减少,高糖+5μg/mL蛆虫ES组、高糖+10μg/mL蛆虫ES组细胞中FAS蛋白表达较单纯高糖组明显增多。实时荧光定量RT-PCR法检测显示,单纯高糖组细胞中FAS mRNA相对表达量为0.392±0.073,较PBS对照组的1.000±0.085明显减少(P<0.01);高糖+5μg/mL蛆虫ES组和高糖+10μg/mL蛆虫ES组细胞中FAS mRNA相对表达量分别为0.561±0.047、0.687±0.013,组间比较差异有统计学意义(P<0.05),且均较单纯高糖组显著增多(P<0.01)。免疫荧光法检测结果显示,各组细胞内FAS蛋白主要定位在细胞质中,表达情况与蛋白质印迹法检测结果相近。(3)划痕后24 h,siRNA对照+蛆虫ES组、siRNA-FAS+蛆虫ES组细胞划痕未愈合宽度明显窄于单纯siRNA对照组(P<0.01),siRNA-FAS+蛆虫ES组细胞划痕未愈合宽度明显宽于siRNA对照+蛆虫ES组(P<0.01)。培养24 h,siRNA对照+蛆虫ES组、siRNA-FAS+蛆虫ES组细胞小管生成数明显多于单纯siRNA对照组(P<0.05或P<0.01),siRNA-FAS+蛆虫ES组细胞小管生成数明显少于siRNA对照+蛆虫ES组(P<0.05)。结论MDT通过蛆虫ES上调FAS的表达水平促进血管内皮细胞活力,从而促进DFU患者创面血管新生。

Objective To investigate the mechanism of maggot debridement therapy(MDT)in promoting wound angiogenesis in patients with diabetic foot ulcer(DFU).Methods(1)From June 2018 to June 2019,the patients admitted to Nanjing Junxie Hospital who met the inclusion criteria were recruited,including 12 DFU patients given MDT for three days[6 males and 6 females,aged(56±12)years]and 12 acute trauma patients without diabetes mellitus[6 males and 6 females,aged(53±10)years],who were enrolled into DFU group and non-diabetic trauma group respectively.Before and after application of MDT,the wound characteristics of patients in DFU group were observed and the wound tissue samples were taken.The wound tissue in non-diabetic trauma group was taken at patient′s first visit before debridement.The expression of angiogenesis marker CD31 in the wound tissue of patients in DFU group was detected by immunohistochemistry before and after application of MDT.Western blotting and real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR)were used respectively to detect the protein and mRNA expressions of fatty acid synthase(FAS)in wound tissue of patients in DFU group before and after application of MDT and in non-diabetic trauma group before debridement.(2)Human umbilical vein endothelial cells(HUVECs)were cultured in endothelial cell culture medium containing 10%fetal bovine serum.The 3rd to 6th passages of cells in logarithmic growth phase were used in the following experiments.Excretions/secretions(ES)were extracted from 3-day-old sterile Lucilia sericata larvae for subsequent experiments.Three batches of cells were divided into phosphate buffer solution(PBS)control group,high glucose alone group,high glucose+5μg/mL maggot ES group,and high glucose+10μg/mL maggot ES group,which were treated with PBS,glucose in final molarity concentration of 20 mmol/L,glucose in final molarity concentration of 20 mmol/L+maggot ES in final mass concentration of 5μg/mL,and glucose in final molarity concentration of 20 mmol/L+maggot ES in final mass concentration of 10μg/mL respectively.The total volume of reagents in each group was the same.After 48 hours of culture,Western blotting,real-time fluorescent quantitative RT-PCR and immunofluorescence method were used to detect the protein and mRNA expressions of FAS in each batch of cells and the expression and localization of FAS protein in cells respectively.The number of samples for mRNA expression was 3.(3)Two batches of cells were divided into small interference RNA(siRNA)alone group,siRNA control+maggot ES group and siRNA-FAS+maggot ES group,which were transfected with 100μmol/L(final molarity concentration)insignificant control siRNA,insignificant control siRNA,and siRNA-FAS for 4-6 h respectively,and then they were routinely cultured for 24 h with PBS added,maggot ES in final mass concentration of 10μg/mL,and maggot ES in final mass concentration of 10μg/mL respectively.The total volume of reagents in each group was the same.One batch of cells was used for scratch test,the scratch width was observed at 24 hour after scratching to detect the cell migration ability;one batch of cells was subjected to tube forming experiment,and the formation of cell tubules was observed after 24 hours of culture.The number of samples was 3 in scratch test and tube forming experiments.Data were statistically analyzed with t test,one-way analysis of variance,least significant difference test,analysis of variance for repeated measurement,and Bonferroni method.Results(1)Compared with those before application of MDT,fresh granulation tissue significantly increased and necrotic tissue decreased obviously in wound,and the expression of CD31 significantly increased in wound tissue of patients in DFU group after application of MDT.The expression of FAS protein in wound tissue of patients in DFU group before application of MDT was significantly lower than that in non-diabetic trauma group before debridement,and the expression of FAS protein in wound tissue of patients in DFU group after application of MDT was significantly higher than that before application of MDT.The expression of FAS mRNA in wound tissue of patients in DFU group before application of MDT was 1.00±0.17,which was significantly less than 3.87±1.02 in non-diabetic trauma group before debridement(t=9.808,P<0.01).The expression of FAS mRNA in wound tissue of patients in DFU group after application of MDT was 1.85±0.31,which was significantly higher than that before application of MDT(t=-10.853,P<0.01).(2)After 48 hours of culture,Western blotting detection showed that the expression of FAS protein in cells in high glucose alone group was significantly less than that in PBS control group,and the expressions of FAS protein in cells in high glucose+5μg/mL maggot ES group and high glucose+10μg/mL maggot ES group were significantly higher than the expression in high glucose alone group.Real-time fluorescent quantitative RT-PCR determination showed that the expression of FAS mRNA in cells in high glucose alone group was 0.392±0.073,which was significantly lower than 1.000±0.085 in PBS control group(P<0.01);there was statistically significant difference between the expression of FAS mRNA in cells in high glucose+5μg/mL maggot ES group(0.561±0.047)and that in high glucose+10μg/mL maggot ES group(0.687±0.013)(P<0.05),both of which were significantly higher than the expression in high glucose alone group(P<0.01).The results of immunofluorescence detection showed that FAS protein was mainly located in the cytoplasm of cells in each group,and its expression was similar to that detected by Western blotting.(3)At 24 hour after scratch,the uncured widths of cell scratch in siRNA control+maggot ES group and siRNA-FAS+maggot ES group were significantly narrower than the uncured width in siRNA alone control group(P<0.01),and the uncured width of cell scratch in siRNA-FAS+maggot ES group was significantly wider than that in siRNA control+maggot ES group(P<0.01).After 24 hours of culture,the numbers of tubules in siRNA+maggot ES group and siRNA-FAS+maggot ES group were significantly more than the number in siRNA alone control group(P<0.05 or P<0.01),and the number of tubules in siRNA-FAS+maggot ES group was obviously less than that in siRNA control+maggot ES group(P<0.05).Conclusions MDT up-regulates the expression of FAS through maggot ES,which promotes the activity of vascular endothelial cells,thus promoting the wound angiogenesis in patients with DFU.