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中华临床医师杂志(电子版)

中华临床医师杂志(电子版) ›› 2019, Vol. 13 ›› Issue (06) : 401 -408. doi: 10.3877/cma.j.issn.1674-0785.2019.06.001

所属专题： 文献；

临床研究

不同Wagner分级糖尿病足患者血清外泌体miR-3976的表达及机制
刘静1, 陈红星1, 刘兴州1, 吴雨茜1, 蓝碧云1, 麦梨芳1, 杨川1, 严励1, 任萌1,†()   
  1. 1. 510120　广州，中山大学孙逸仙纪念医院内分泌科
  • 收稿日期:2018-12-03 出版日期:2019-03-15
  • 通信作者: 任萌
  • 基金资助:
    国家自然科学基金项目(81870571)

Serum exosomal miR-3976 in patients with diabetic foot of different Wagner grades: expression and possible mechanism involved

Jing Liu1, Hongxing Chen1, Xingzhou Liu1, Yuxi Wu1, Biyun Lan1, Lifang Mai1, Chuan Yang1, Li Yan1, Meng Ren1,†()   

  1. 1. Department of Endocrinology, Sun Yat-sen Memorial Hospital, Zhongshan University, Guangzhou 510120, China
  • Received:2018-12-03 Published:2019-03-15
  • Corresponding author: Meng Ren
  • About author:
    Corresponding author: Ren Meng, Email:
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引用本文：

刘静, 陈红星, 刘兴州, 吴雨茜, 蓝碧云, 麦梨芳, 杨川, 严励, 任萌. 不同Wagner分级糖尿病足患者血清外泌体miR-3976的表达及机制[J]. 中华临床医师杂志(电子版), 2019, 13(06): 401-408.

Jing Liu, Hongxing Chen, Xingzhou Liu, Yuxi Wu, Biyun Lan, Lifang Mai, Chuan Yang, Li Yan, Meng Ren. Serum exosomal miR-3976 in patients with diabetic foot of different Wagner grades: expression and possible mechanism involved[J]. Chinese Journal of Clinicians(Electronic Edition), 2019, 13(06): 401-408.

目的

观察不同Wagner分级的糖尿病患者血清及血清外泌体内microRNA-3976（miR-3976）的表达变化，并探讨其表达异常的可能机制。

方法

选取2018年3~6月中山大学孙逸仙纪念医院内分泌科28例糖尿病患者，通过逆转录-实时荧光定量聚合酶链式反应（RT-qPCR）法检测血清及血清外泌体内miR-3976的表达水平，采用t检验比较不同Wagner分级糖尿病患者血清及血清外泌体miR-3976的表达差异。体外培养皮肤成纤维细胞和角质形成细胞，分别给予高糖、甘露醇及正常糖浓度培养，RT-qPCR法检测miR-3976的表达水平，采用单因素方差分析比较3种处理情况下细胞内miR-3976的表达差异。对miR-3976进行生物信息学分析。

结果

Wagner 3~4级糖尿病足患者与Wagner 0~2级患者相比，血清miR-3976表达水平显著升高（1.588±1.118 vs 1.068±0.108），差异具有统计学意义（t=3.256，P=0.0031），且血清外泌体中的miR-3976升高更显著（2.885±0.742 vs 1.156±0.220），差异具有统计学意义（t=2.435，P=0.0255）；高糖培养的成纤维细胞中miR-3976表达较正常糖及等渗甘露醇组显著升高（3.749±1.177 vs 1.000；3.749±1.177 vs 2.060±0.475），差异均具有统计学意义（q=6.497、3.992，P均<0.05），而HaCaT细胞在高糖、等渗甘露醇及正常糖培养情况下miR-3976表达差异无统计学意义（F=0.249，P=0.789）。miR-3976的靶基因KEGG信号通路分析结果显示靶基因在细胞间紧密连接信号通路上富集程度最高。

结论

Wagner 3~4级的糖尿病足患者血清中升高的miR-3976可能主要以外泌体的形式存在，且可能主要来源于伤口局部的成纤维细胞，并通过外泌体将表达增高的miR-3976传递至皮肤修复细胞，影响细胞紧密连接分子表达，从而抑制伤口愈合。

关键词: 糖尿病足, Wagner分级, 外泌体, miRNA, 生物信息学
Objective

To observe the changes of expression of microRNA-3976 (miR-3976) in serum and serum exosomes of patients with different Wagner grades of foot ulcers and explore the potential mechanism involved.

Methods

Peripheral blood serum samples of 28 patients with diabetic foot of different Wagner grades from Sun Yat-sen Memorial Hospital were used for RNA extraction or exosomal RNA extraction, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to verify the difference of miR-3976 expression among patients in different groups. The t-test was performed to compare serum and serum exosomal miR-3976 expression levels among different groups of patients. Fibroblasts and HaCaT cells were cultured in medium with mannitol or high or normal level of glucose, and the level of miR-3976 expression in these cells was further detected and analyzed by one-way ANOVA. Bioinformatic analysis was used to explore the function of miR-3976.

Results

The expression of miR-3976 increased significantly in serum samples of patients with Wagner grades 3 and 4 disease compared with those with Wagner grades 0-2 (1.588±1.118 vs 1.068±0.108, t=3.256, P=0.0031). Serum exosomal miR-3976 increased more significantly than serum miR-3976 in patients with Wagner grades 3 and 4 (2.885±0.742 vs 1.156±0.220, t=2.435, P=0.0255). The level of miR-3976 expression increased significantly in fibroblasts treated with high sugar than in those treated with normal sugar (q=6.497, P<0.05) or mannitol (q=3.992, P<0.05), while there was no difference in HaCaT cells among different groups (F=0.249, P=0.789). Kytoencyclopedia of genes and genomes (KEGG) pathway enrichment analysis of target genes of miR-3976 indicated that the target genes are enriched most in the pathway of tight junction.

Conclusions

The upregulated expression of miRNA-3976 in patients with Wagner grades 3 and 4 diabetic foot may occur mainly in exosomes, which might mainly come from fibroblasts in the local wound. Exosomal miR-3976 may be transferred to cells involved in skin recovery and affect the expression of junctional molecules, thus inhibiting wound healing.

Key words: Diabetic foot, Wagner grade, Exosomes, MicroRNAs, Bioinformatics analysis
表1 Wagner分级0级与1~4级糖尿病患者临床资料比较
临床资料 Wagner 0级（7例） Wagner 1~4级（21例） Z值 P值
女性［例（%）］ 1（14.29） 5（23.81） - 1.000
年龄（岁，±s） 59.57±10.64 63.67±15.82 88.50 0.435
体质量指数（kg/m2，±s） 20.59±8.98 24.02±3.87 69.50 0.836
收缩压（mmHg，±s） 126.14±19.68 127.48±28.27 71.00 0.917
舒张压（mmHg，±s） 76.43±7.83 71.33±10.59 47.50 0.172
空腹血糖（mmol/L，±s） 6.03±2.17 7.60±3.42 92.00 0.348
糖化血红蛋白（%，±s） 8.57±3.23 8.50±2.53 81.00 0.717
血肌酐（μmol/L，±s） 127.71±101.42 143.33±96.94 80.00 0.756
血三酰甘油（mmol/L，±s） 3.14±4.89 1.27±0.71 56.50 0.376
血胆固醇（mmol/L，±s） 111.57±28.95 97.43±21.58 59.00 0.466
低密度脂蛋白胆固醇（mmol/L，±s） 308.43±144.58 356.48±137.30 62.50 0.568
高密度脂蛋白胆固醇（mmol/L，±s） 8.27±2.13 9.48±3.58 65.50 0.678
红细胞数（×1012/L，±s） 3.86±0.75 3.54±0.75 53.00 0.296
白细胞数（×109/L，±s） 0.87±0.33 0.77±0.22 88.50 0.435
血小板数（×109/L，±s） 2.79±1.38 2.3±0.53 88.50 0.435
血红蛋白（g/L，±s） 4.63±2.20 3.52±0.71 47.00 0.172
图1 外泌体鉴定结果
图2 miR-3976靶基因的GO-BP（基因功能注释-生物过程）富集差异倍数（fold enrichment）条形分布图
图3 miR-3976靶基因KEGG信号通路分布气泡图　气泡越大表示在该信号通路富集基因数目越多，气泡颜色越红表示P值越小
图4 miR-3976靶基因在细胞连接信号通路中的分布情况
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