Influence of maggot excretions/secretions on the anti-Pseudomonas aeruginosa effect of neutrophils in patients with diabetic foot ulcer
-
摘要:
目的 探讨医用蛆虫分泌排泄物（ES）对糖尿病足溃疡（DFU）患者中性粒细胞吞噬及杀菌作用的影响。 方法 采用实验研究的方法。选择东部战区空军医院内分泌科，糖尿病足中心2020年6—12月收治的符合入选标准的30例DFU患者［男16例、女14例，年龄（64±7）岁］。Percoll非连续性密度梯度离心法分离中性粒细胞，将每例患者细胞分别纳入生理盐水组、蛆虫ES组（每组30孔），分别加入无菌生理盐水和终质量浓度357 μg/mL（下同）蛆虫ES。培养1、2 h，行瑞氏染色观察并计算细胞吞噬率和吞噬指数。选择10例患者，将每例患者细胞分别纳入铜绿假单胞菌+中性粒细胞组、铜绿假单胞菌+中性粒细胞+蛆虫ES组（每组10孔），分别进行相应处理；另设置单纯铜绿假单胞菌组、铜绿假单胞菌+蛆虫ES组（每组10孔），分别加入铜绿假单胞菌+RPMI 1640培养液+无菌生理盐水、铜绿假单胞菌+RPMI 1640培养液+蛆虫ES。培养2 h，平板菌落计数法计数活菌菌落。分别选择6、6、3例患者，同前将每例患者细胞分别纳入蛆虫ES组和生理盐水组（每组6、6、3孔）并行相应处理。培养6 h，采用实时荧光定量反转录PCR法检测细胞中白细胞介素1β（IL-1β）、IL-6、溶菌酶mRNA表达，采用酶联免疫吸附测定法检测细胞培养上清液中IL-1β、IL-6含量，采用免疫荧光法观察溶菌酶表达阳性细胞情况。对数据行单因素方差分析、配对样本
t 检验、LSD检验及Wilcoxon符号秩和检验。 结果 培养1 h，蛆虫ES组细胞吞噬率、吞噬指数［53.5%（49.7%，58.0%）、3.18（2.96，3.32）］分别与生理盐水组［52.0%（47.5%，55.2%）、3.15（2.96，3.25）］相近（
Z =-1.701、-1.092，
P >0.05）；培养2 h，蛆虫ES组患者细胞吞噬率、吞噬指数［70.0%（66.7%，72.0%）、4.47（4.22，4.96）］分别明显高于生理盐水组［58.0%（55.0%，60.0%）、4.11（3.52，4.24），
Z =-4.786、-4.279，
P <0.01］。培养2 h，铜绿假单胞菌+中性粒细胞组活菌菌落数明显低于单纯铜绿假单胞菌组（
P <0.01），铜绿假单胞菌+中性粒细胞+蛆虫ES组活菌菌落数明显低于铜绿假单胞菌+蛆虫ES组和铜绿假单胞菌+中性粒细胞组（
P <0.01）。培养6 h，蛆虫ES组细胞IL-1β、IL-6、溶菌酶mRNA表达均较生理盐水组明显上调（
t= -3.279、-4.273、-4.763，
P <0.05或
P <0.01），蛆虫ES组细胞培养上清液中IL-1β、IL-6含量分别明显高于生理盐水组（
t= -9.526、-6.447，
P <0.01），蛆虫ES组溶菌酶阳性细胞明显多于生理盐水组。 结论 蛆虫ES可通过促进DFU患者中性粒细胞免疫防御相关因子和溶菌酶的产生，从而增强中性粒细胞对铜绿假单胞菌的吞噬、杀菌作用。
Abstract:Objective To investigate the effects of medical maggot excretions/secretions (ES) on neutrophils phagocytosis and bactericidal effect in patients with diabetic foot ulcer (DFU). Methods The experimental research method was used. Thirty DFU patients (16 males and 14 females, aged (64±7) years)who were admitted to the Diabetes Foot Center, the Department of Endocrinology of Air Force Hospital of Eastern Theater Command from June to December 2020 and met the inclusion criteria were recruited. Discontinuous percoll gradient centrifugation method was used to separate the neutrophils. Cells from each patient were enrolled into normal saline group and maggot ES group (30 wells in each group), respectively; sterile normal saline and ES with a final mass concentration of 357 μg/mL (the same as below) were added, respectively. After 1 and 2 hour(s) of culture, the phagocytosis rate and phagocytic index of cells were observed and counted under Wright's staining. Ten patients were selected, then the cells of each patient were enrolled into
Pseudomonas aeruginosa +neutrophils group and
Pseudomonas aeruginosa +neutrophils+maggot ES group (10 wells in each group) and were treated corresponding, respectively.
Pseudomonas aeruginosa alone group and
Pseudomonas aeruginosa +maggot ES group (10 wells in each group) were set up respectively;
Pseudomonas aeruginosa +RPMI 1640 culture medium+sterile normal saline and
Pseudomonas aeruginosa +RPMI 1640 culture medium+maggot ES were added, respectively. After 2 hours of culture, the number of viable bacteria colony was counted by plate colony number method. Six, six, and three patients were selected respectively, and the cells of each patient were respectively enrolled into maggot ES group and normal saline group (6, 6, and 3 wells in each group, respectively) and treated accordingly. After 6 hours of culture, real-time fluorescent quantitative reverse transcription polymerase chain reaction was used to detect the mRNA expressions of interleukin 1β (IL-1β), IL-6, and lysozyme in cells, the content of IL-1β and IL-6 in cell culture supernatant were determined by enzyme-linked immunosorbent assay, and the positive cells expressing lysozyme were observed with immunofluorescence method. Data were statistically analyzed with one-way analysis of variance, paired sample
t test, least significant difference test, and Wilcoxon rank sum test. Results After 1 hour of culture, the phagocytosis rate and phagocytic index of cells in maggot ES group (53.5% (49.7%, 58.0%) and 3.18 (2.96, 3.32)) were similar to 52.0% (47.5%, 55.2%) and 3.15 (2.96, 3.25) of normal saline group (
Z =-1.701, -1.092,
P >0.05). After 2 hours of culture, the phagocytosis rate and phagocytic index of cells in maggot ES group (70.0% (66.7%, 72.0%) and 4.47 (4.22, 4.96)) were significantly higher than 58.0% (55.0%, 60.0%) and 4.11 (3.52, 4.24) in normal saline group (
Z =-4.786, -4.279,
P <0.01). After 2 hours of culture, the number of viable bacteria colony in
Pseudomonas aeruginosa +neutrophils group was significantly lower than that in
Pseudomonas aeruginosa alone group (
P <0.01), and the number of viable bacteria colony in
Pseudomonas aeruginosa +neutrophils+maggot ES group was significantly lower than that in
Pseudomonas aeruginosa +maggot ES group and
Pseudomonas aeruginosa +neutrophils group (
P <0.01). After 6 hours of culture, the mRNA expressions of IL-1β, IL-6, and lysozyme of cells in maggot ES group were significantly higher those in normal saline group (
t =-3.279, -4.273, -4.763,
P <0.05 or
P <0.01); the concent of IL-1β and IL-6 in cell culture supernatant of maggot ES group were significantly higher than those of normal saline group (
t =-9.526, -6.447,
P <0.01); there were significantly more positive cells expressing lysozyme in maggot ES group than in normal saline group. Conclusions Maggot ES can enhance the phagocytosis and bactericidal effect of neutrophils on Pseudomonas aeruginosa by promoting the production of neutrophils immune defense related cytokines and lysozyme in DFU patients.
-
Key words:
- Diabetic foot /
- Foot ulcer /
- Pseudomonas aeruginosa /
- Maggot excretions/secretions /
- Neutrophil
-
参考文献
(26) -
计量
- 文章访问数: 854
- HTML全文浏览量: 28
- PDF下载量: 17
- 被引次数: 0
下载:
