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. 2026 May 20;42(5):467–476. [Article in Chinese] doi: 10.3760/cma.j.cn501225-20250402-00158

大鼠骨髓间充质干细胞来源外泌体对高糖环境下大鼠Fb的影响及其机制

Influence and mechanism of exosomes derived from rat bone marrow mesenchymal stem cells on rat Fbs under high glucose conditions

Yue Wu 1, Wenhui Yin 1, Miao Yu 2, Da Wang 1, Jian Wu 1,*
PMCID: PMC13234708  PMID: 42209185

Highlights

(1) It was confirmed that microRNA-140-3p derived from rat bone marrow mesenchymal stem cells inhibited high-glucose-induced rat fibroblast pyroptosis by negatively regulating the NOD-like receptor pyrin domain-containing protein 3/cysteine aspartic acid specific protease-1/gasdermin D signaling pathway.

(2) The strategy of using exosomes to deliver microRNA for intervening in cell pyroptosis provided an alternative approach that can circumvent the risks associated with stem cell therapy for refractory diabetic wounds.

Keywords: Mesenchymal stem cells, Exosomes, Diabetes mellitus, Pyroptosis, Fibroblasts, MicroRNAs

Abstract

Objective

To investigate the influence and mechanism of exosomes derived from rat bone marrow mesenchymal stem cells (BMSCs) on rat fibroblasts (Fbs) under high glucose conditions, with the aim of exploring a potential novel strategy for the treatment of diabetic wounds.

Methods

This study was designed as grouped experimental study. The exosomes derived from BMSCs (BMSC-Exos) were extracted from the rat primary BMSCs and were identified successfully. The BMSC-Exos were divided into control group and high-glucose group. The BMSC-Exos in control group were cultured routinely, while the BMSC-Exos in high-glucose group were cultured in DMEM medium containing glucose at a final molarity of 30 mmol/L (hereinafter referred to as high-glucose medium). The eukaryotic mRNA sequencing was performed on BMSC-Exos in both groups, combined with multi-database prediction and enrichment analysis, differentially expressed genes that strongly interacted with the classical pyroptosis signaling pathway were screened and identified. The rat BMSCs of passages 1 to 3 were divided into microRNA-140-3p (miR-140-3p) mimic control group, miR-140-3p mimic group, miR-140-3p inhibitor control group, and miR-140-3p inhibitor group according to the random number table method, then the corresponding miR-140-3p mimic control, miR-140-3p mimic, miR-140-3p inhibitor control, and miR-140-3p inhibitor were transfected into cells, respectively, after 24 hours of culture. The BMSC-Exos were extracted at 24 hours post-transfection, and the expression of miR-140-3p in BMSC-Exos was detected by real-time fluorescence quantitative polymerase chain reaction. The rat Fbs in the logarithmic growth phase were divided into miR-140-3p mimic control group, miR-140-3p mimic group, miR-140-3p inhibitor control group, and miR-140-3p inhibitor group. After 24 hours of culture in high-glucose medium, the Fbs were added with the exosomes secreted by BMSCs after being transfected with miR-140-3p mimic control, miR-140-3p mimic, miR-140-3p inhibitor control, and miR-140-3p inhibitor, respectively (the same grouping and treatment below). At 24 hours post-transfection, the cell absorbance value was detected using cell counting kit-8, representing cell proliferation activity. The rat Fbs in the logarithmic growth phase were grouped and treated, then the cell migration rate at 24 hours after scratching was detected by scratch test. At 24 hours post-transfection, the protein expression levels of pyroptosis-related protein, including interleukin-1β (IL-1β), IL-18, NOD-like receptor pyrin domain-containing protein 3 (NLRP3), cysteine aspartic acid specific protease-1 (caspase-1), and gasdermin D in cells were detected by Western blotting. The sample size was 3.

Results

Compared with that in control group, the expressions of miR-140-3p and miR-542-5p were significantly upregulated in BMSC-Exos of high-glucose group. MiR-140-3p was identified as the differentially expressed gene that strongly interacted with the classical pyroptosis signaling pathway. At 24 hours post-transfection, the expression of miR-140-3p in BMSC-Exos of miR-140-3p mimic group was significantly higher than that in miR-140-3p mimic control group (P < 0.05), and the expression of miR-140-3p in BMSC-Exos of miR-140-3p inhibitor group was significantly lower than that in miR-140-3p inhibitor control group (P < 0.05). At 24 hours post-transfection, the absorbance value of Fbs in miR-140-3p mimic group was 0.940±0.031, which was significantly higher than 0.781±0.020 in miR-140-3p mimic control group (P < 0.05); the absorbance value of Fbs in miR-140-3p inhibitor group was 0.510±0.041, which was significantly lower than 0.822±0.061 in miR-140-3p inhibitor control group (P < 0.05). The Fb migration rate at 24 hours after scratching in miR-140-3p mimic group was significantly higher than that in miR-140-3p mimic control group (P < 0.05), and the Fb migration rate at 24 hours after scratching in miR-140-3p inhibitor group was significantly lower than that in miR-140-3p inhibitor control group (P < 0.05). At 24 hours post-transfection, the protein expressions of NLRP3, IL-18, IL-1β, caspase-1, and gasdermin D in Fbs of miR-140-3p mimic group were significantly lower than those in miR-140-3p mimic control group (P < 0.05); the protein expressions of NLRP3, IL-18, IL-1β, caspase-1, and gasdermin D in Fbs of miR-140-3p inhibitor group were significantly higher than those in miR-140-3p inhibitor control group (P < 0.05).

Conclusions

The rat BMSC-Exos can deliver miR-140-3p to promote the proliferation and migration of rat Fbs under high-glucose conditions, inhibit the expression of pyroptosis-related protein, and alleviate cell pyroptosis. This study provides a promising therapeutic target for diabetic wound repair.

Keywords: Mesenchymal stem cells, Exosomes, Diabetes mellitus, Pyroptosis, Fibroblasts, MicroRNAs

随着糖尿病的发病率不断上升，高血糖环境对机体组织的损伤日益受到关注。作为重要的细胞类型，Fb在组织修复和再生过程中发挥着关键作用。然而，高糖环境会导致Fb出现功能障碍，进而影响创面愈合和组织再生。因此，探索高糖环境下Fb的调控机制，对于改善糖尿病相关并发症具有重要意义[1-2]。近年来，骨髓间充质干细胞（bone marrow mesenchymal stem cell，BMSC）因良好的再生能力和免疫调节特性而受到广泛关注。BMSC通过分泌外泌体释放多种生物活性分子，参与细胞间的信号传递和调控[3-4]。外泌体中的微小RNA（microRNA，miR）被认为是其主要的生物活性成分之一，能够通过调节靶基因的表达，影响细胞的增殖、迁移和凋亡等[5]，但其在高糖环境下对Fb的影响及其机制尚不明确。因此，本研究旨在探讨BMSC来源外泌体（exosome derived from bone marrow mesenchymal stem cell，BMSC-Exo）中的miR对高糖环境下大鼠Fb的作用及其潜在机制。

1. 材料与方法

本研究为成组设计实验研究。

1.1. 主要材料来源

大鼠原代BMSC、大鼠原代Fb和RAT-iCell-s010 Fb培养基均购自赛百慷（上海）生物技术股份有限公司，DMEM培养基购自广州创融生物科技有限公司，鬼笔环肽购自翌圣生物科技（上海）股份有限公司，兔来源的IL-1β、IL-18、含pyrin结构域的NOD样受体蛋白3（NOD-like receptor pyrin domain-containing protein 3，NLRP3）、胱天蛋白酶-1（cysteine aspartic acid specific protease-1，caspase-1）、消皮素D、CD9、CD63、CD81、β肌动蛋白单克隆抗体购自武汉三鹰生物技术有限公司，辣根过氧化物酶标记的山羊抗兔IgG多克隆抗体购自武汉阿斯本生物技术有限公司，外泌体示踪染料PKH26购自上海懋康生物科技有限公司，4'，6-二脒基-2-苯基吲哚购自美国Sigma公司，胰蛋白酶购自浙江吉诺生物医药技术有限公司。miR-140-3p模拟物对照剂、miR-140-3p模拟物、miR-140-3p抑制剂对照剂、miR-140-3p抑制剂购自武汉金开瑞生物工程有限公司。细胞计数试剂盒-8购自广州赛国生物科技有限责任公司。

QuantStudio 6 Flex型荧光定量PCR仪购自美国赛默飞世尔科技公司，Multiskan FC型酶标仪购自赛默飞世尔科技（中国）有限公司，Tecnai G2 Spirit BioTwin型透射电子显微镜购自美国FEI公司，ZetaVIEW S/N 17-310型纳米颗粒跟踪分析仪购自美国Particle Metrix公司，SU8010型扫描电子显微镜购自日本日立公司，Eclipse Ci-L型荧光显微镜购自日本尼康公司，IX51型倒置显微镜购自日本奥林巴斯公司。

1.2. BMSC-Exo的提取与鉴定

将大鼠原代BMSC采用DMEM培养基于37 ℃、含体积分数5%二氧化碳培养箱中常规培养（常规培养条件下同）至生长达60%~70%融合时，更换为含体积分数10%胎牛血清的DMEM培养基继续培养24 h；随后小心收集上清液，采用超高速离心法提取BMSC-Exo。取适量BMSC-Exo，于透射电子显微镜40 000倍放大倍数下观察形态，采用纳米颗粒跟踪分析仪检测粒径，另采用蛋白质印迹法检测BMSC和BMSC-Exo中外泌体标志物CD9、CD63和CD81的表达。其中一抗为兔来源的CD9、CD63、CD81单克隆抗体（稀释比均为1∶1 000），二抗为辣根过氧化物酶标记的山羊抗兔IgG多克隆抗体（稀释比为1∶5 000）。

1.3. Fb对BMSC-Exo的内化情况

取100 μL 1.2制备的BMSC-Exo，加入4 μL PKH26进行标记，室温避光孵育5 min后，于4 ℃下以10 000×g离心1 h，获取沉淀，然后采用100 μL PBS重新悬浮备用。调整Fb浓度为5×104个/mL，按照每孔100 μL接种于96孔板中，采用RAT-iCell-s010 Fb培养基常规培养至细胞生长达70%融合后，加入前述20 μL PKH26标记的BMSC-Exo悬液，避光共培养24 h后用PBS清洗3次。用40 g/L多聚甲醛固定15 min后，用鬼笔环肽行细胞骨架染色，用4'，6-二脒基-2-苯基吲哚行细胞核染色。在荧光显微镜400倍放大倍数下观察细胞对PKH26标记的BMSC-Exo的吞噬情况。其中外泌体阳性染色为红色，细胞核阳性染色为蓝色，细胞骨架阳性染色为绿色。

1.4. 真核mRNA测序及分析

取适量1.2制备的BMSC-Exo，分成对照组和高糖组，对照组外泌体常规培养，高糖组外泌体用含终物质的量浓度30 mmol/L葡萄糖的DMEM培养基（以下简称高糖培养基）进行常规培养。每组各取1 mL BMSC-Exo行RNA测序。提取外泌体总RNA，分离出mRNA并将其断裂成300 bp左右的小片段。利用随机引物，以mRNA为模板反转录合成一链互补DNA，随后进行二链合成，形成双链结构。对连接接头后的产物进行片段分选，随后进行PCR扩增，得到最终的文库，采用Illumina PE150平台进行测序。

利用fastp软件把每条读段首尾不确定的N、低于Q20的低质碱基以及残留接头序列全部剪掉或过滤掉，然后用FastQC软件评估质量值。利用bowtie短序列比对工具比对Rfam库，过滤非目标RNA，分别采用miRDeep2软件和DESeq2软件完成miR定量及差异表达分析。结合多数据库预测与富集分析，以|log₂（差异倍数）| > 1且P < 0.05为标准，采用R语言clusterProfiler包进行基因本体论和京都基因与基因组百科全书富集分析。

1.5. BMSC-Exo中miR-140-3p的表达检测

取第1~3代BMSC，以每孔1×105个的密度接种至96孔板中，常规培养至细胞生长达40%融合时，采用随机数字表法将其分为miR-140-3p模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组，培养24 h后分别转染miR-140-3p模拟物对照剂、miR-140-3p模拟物、miR-140-3p抑制剂对照剂、miR-140-3p抑制剂。

转染24 h后提取并纯化各组细胞分泌的外泌体，采用实时荧光定量PCR法检测BMSC-Exo中miR-140-3p的表达。引物序列由上海生工生物工程有限公司合成。miR-140-3p的上游引物为5'-TACCACAGGGTAGAACCACGG-3'，下游引物为5'-CTCAACTGGTGTCGTGGAGTC-3'，产物大小为70 bp；U6的上游引物为5'-CCTGCTTCGGCAGCACAT-3'，下游引物为5'-AACGCTTCACGAATTTGCGT-3'，产物大小为100 bp。以U6为内参照，基于Δ循环阈值对miR-140-3p的表达进行定量。样本数为3。

1.6. BMSC-Exo中miR-140-3p对Fb增殖的影响

取对数生长期Fb，以每孔6×104个的密度接种至加入高糖培养基的6孔板中，分成miR-140-3p模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组，培养24 h后分别加入转染miR-140-3p模拟物对照剂后的BMSC分泌的终质量浓度为1 μg/mL（后同）的外泌体、转染miR-140-3p模拟物后的BMSC分泌的外泌体、转染miR-140-3p抑制剂对照剂后的BMSC分泌的外泌体、转染miR-140-3p抑制剂后的BMSC分泌的外泌体。于转染24 h后，根据细胞计数试剂盒-8说明书，用酶标仪测量各组细胞在波长450 nm处的吸光度值，以此表示细胞增殖活力。样本数为3。

1.7. BMSC-Exo中miR-140-3p对Fb迁移的影响

取对数生长期Fb，以每孔6×104个的密度接种至加入高糖培养基的6孔板中，同1.6分组与处理后，用规格为1 mL的移液器枪头垂直在各孔单层细胞上划1条直线。吸弃原有培养基后采用PBS冲洗细胞3次，去除划下的细胞。分别于划痕后0（即刻）、24 h，于倒置显微镜100倍放大倍数下拍照并计算划痕后24 h细胞迁移率。划痕后24 h细胞迁移率=（划痕后0 h划痕面积-划痕后24 h划痕面积）÷划痕后0 h划痕面积×100%。样本数为3。

1.8. BMSC-Exo中miR-140-3p对Fb中焦亡相关蛋白的蛋白表达的影响

取对数生长期Fb，同1.6分组与处理。于转染24 h后，采用蛋白质印迹法检测细胞中焦亡相关蛋白IL-1β、IL-18、NLRP3、caspase-1和消皮素D的蛋白表达。一抗为兔来源的IL-1β、IL-18、NLRP3、caspase-1、消皮素D、β肌动蛋白单克隆抗体（稀释比均为1∶1 000），二抗为辣根过氧化物酶标记的山羊抗兔IgG多克隆抗体（稀释比为1∶5 000）。以β肌动蛋白为内参照，计算各焦亡相关蛋白的蛋白表达。样本数为3。

1.9. BMSC-Exo中miR-140-3p对Fb形态的影响

取对数生长期Fb，同1.6分组与处理。于转染24 h后，取各组细胞样品经体积分数2.5%戊二醛固定、乙醇梯度脱水、临界点干燥后，离子溅射镀金，采用扫描电子显微镜在2 000倍放大倍数下观察细胞焦亡的形态表现（气球样突起）。

1.10. 统计学处理

采用SPSS 21.0统计软件进行数据分析。计量资料数据均符合正态分布，以x ± s表示。组间总体比较行单因素方差分析，组间多重比较行LSD检验。以上检验均为双侧检验，P < 0.05为差异有统计学意义。

2. 结果

2.1. BMSC-Exo的鉴定

培养24 h后，外泌体粒径主要分布在50~250 nm，主要形态为椭圆形或杯状，符合BMSC-Exo的粒径和形态；BMSC-Exo中检测到外泌体标志物CD9、CD63和CD81。BMSC-Exo鉴定成功。见图 1。

图 1.

大鼠BMSC-Exo的鉴定。1A.外泌体粒径主要分布在50~250 nm；1B.外泌体呈杯状透射电子显微镜×40 000；1C.蛋白质印迹法检测显示BMSC-Exo表达外泌体标志物CD9、CD63、CD81

Identification of rat BMSC-Exos

注：BMSC-Exo为骨髓间充质干细胞来源外泌体；条带上方1、2分别指示骨髓间充质干细胞、BMSC-Exo

图 1

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2.2. Fb对BMSC-Exo的内化情况

共培养24 h后，可见Fb的细胞核周围呈现红色荧光，说明BMSC-Exo被摄入Fb内。见图 2。

图 2.

共培养24 h后大鼠Fb摄入大鼠BMSC-Exo的情况PKH26-鬼笔环肽-4'，6-二脒基-2-苯基吲哚×400。2A、2B、2C、2D.分别为Fb中的细胞核染色、外泌体染色、细胞骨架染色及复合染色情况，可见大鼠BMSC-Exo被大鼠Fb摄入

The uptake of rat BMSC-Exos by rat Fbs after 24 hours of co-culture

注：Fb为成纤维细胞，BMSC-Exo为骨髓间充质干细胞来源外泌体；Fb细胞核阳性染色为蓝色，外泌体阳性染色为红色，细胞骨架阳性染色为绿色

图 2

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2.3. 真核mRNA测序及分析

与对照组相比，高糖组BMSC-Exo中有16个差异表达基因（P < 0.05），其中miR-140-3p、miR-542-5p的表达显著上调。结合多数据库预测与富集分析，鉴定出与经典焦亡信号通路高度互相作用的差异表达基因是miR-140-3p。

2.4. BMSC-Exo中miR-140-3p的表达

转染24 h后，miR-140-3p模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组BMSC-Exo中miR-140-3p表达分别为1.041±0.030、2.442±0.071、1.070±0.021、0.531±0.040，组间总体比较，差异有统计学意义（F=1 062.839，P < 0.001）。miR-140-3p模拟物组BMSC-Exo中miR-140-3p的表达明显高于miR-140-3p模拟物对照组（P < 0.001）；miR-140-3p抑制剂组BMSC-Exo中miR-140-3p表达明显低于miR-140-3p抑制剂对照组（P < 0.001）；miR-140-3p模拟物对照组与miR-140-3p抑制剂对照组BMSC-Exo中miR-140-3p表达比较，差异无统计学意义（P=0.327）。

2.5. BMSC-Exo中miR-140-3p对Fb增殖的影响

转染24 h后，miR-140-3p模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组Fb吸光度值分别为0.781±0.020、0.940±0.031、0.822±0.061、0.510±0.041，组间总体比较，差异有统计学意义（F=121.269，P < 0.001）。miR-140-3p模拟物组Fb吸光度值明显高于miR-140-3p模拟物对照组（P < 0.001）；miR-140-3p抑制剂组Fb吸光度值明显低于miR-140-3p抑制剂对照组（P < 0.001）；miR-140-3p模拟物对照组与miR-140-3p抑制剂对照组Fb吸光度值比较，差异无统计学意义（P=0.132）。

2.6. BMSC-Exo中miR-140-3p对Fb迁移的影响

miR-140-3p模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组Fb划痕后24 h迁移率分别为（35.93±0.79）%、（55.70±0.88）%、（35.45±1.49）%、（11.18±0.19）%，组间总体比较，差异有统计学意义（F=1 090.674，P < 0.001）。miR-140-3p模拟物组Fb划痕后24 h迁移率明显高于miR-140-3p模拟物对照组（P < 0.001）；miR-140-3p抑制剂组Fb划痕后24 h迁移率明显低于miR-140-3p抑制剂对照组（P < 0.001）；miR-140-3p模拟物对照组与miR-140-3p抑制剂对照组Fb划痕后24 h迁移率比较，差异无统计学意义（P=0.560）。见图 3。

图 3.

4组大鼠Fb划痕后各时间点迁移情况倒置显微镜×100。3A、3B、3C、3D.分别为miR-140-3p模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组划痕后0 h（即刻）的划痕面积，均相近；3E、3F、3G、3H.分别为miR-140-3p模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组划痕后24 h的划痕面积，其中图 3E划痕面积明显大于图 3F，图 3G划痕面积明显小于图 3H

Migration of rat Fbs in four groups at different time points after scratching under high-glucose conditions

注：微小RNA-140-3p（miR-140-3p）模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组大鼠成纤维细胞（Fb）均经高糖培养24 h后分别加入转染miR-140-3p模拟物对照剂后的大鼠骨髓间充质干细胞（BMSC）分泌的外泌体、转染miR-140-3p模拟物后的BMSC分泌的外泌体、转染miR-140-3p抑制剂对照剂后的BMSC分泌的外泌体、转染miR-140-3p抑制剂后的BMSC分泌的外泌体

图 3

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2.7. BMSC-Exo中miR-140-3p对Fb中焦亡相关蛋白的蛋白表达的影响

转染24 h后，miR-140-3p模拟物组Fb中NLRP3、IL-18、IL-1β、caspase-1、消皮素D的蛋白表达均明显低于miR-140-3p模拟物对照组（P < 0.05），miR-140-3p抑制剂组Fb中NLRP3、IL-18、IL-1β、caspase-1、消皮素D的蛋白表达均明显高于miR-140-3p抑制剂对照组（P < 0.05）。见图 4、表 1。

图 4.

蛋白质印迹法检测的4组大鼠Fb转染24 h后焦亡相关蛋白的蛋白表达

The protein expression levels of pyroptosis-related protein in rat Fbs of four groups detected by Western blotting at 24 hours post-transfection

注：IL为白细胞介素，NLRP3为含pyrin结构域的NOD样受体蛋白3；条带上方1、2、3、4分别指示微小RNA-140-3p（miR-140-3p）模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组；miR-140-3p模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组大鼠成纤维细胞（Fb）均经高糖培养24 h后分别加入转染miR-140-3p模拟物对照剂后的大鼠骨髓间充质干细胞（BMSC）分泌的外泌体、转染miR-140-3p模拟物后的BMSC分泌的外泌体、转染miR-140-3p抑制剂对照剂后的BMSC分泌的外泌体、转染miR-140-3p抑制剂后的BMSC分泌的外泌体

图 4

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表 1.

4组大鼠Fb转染24 h后焦亡相关蛋白的蛋白表达比较（x ± s）

Comparison of the protein expression levels of pyroptosis-related protein in rat Fbs of four groups at 24 hours post-transfection

组别 样本数 消皮素D caspase-1 IL-1β IL-18 NLRP3
注：caspase-1为胱天蛋白酶-1，IL为白细胞介素，NLRP3为含pyrin结构域的NOD样受体蛋白3；微小RNA-140-3p（miR-140-3p）模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组大鼠成纤维细胞（Fb）均经高糖培养24 h后分别加入转染miR-140-3p模拟物对照剂后的大鼠骨髓间充质干细胞（BMSC）分泌的外泌体、转染miR-140-3p模拟物后的BMSC分泌的外泌体、转染miR-140-3p抑制剂对照剂后的BMSC分泌的外泌体、转染miR-140-3p抑制剂后的BMSC分泌的外泌体；F值、P值为组间各指标总体比较所得；P1值、P2值、P3值分别为miR-140-3p模拟物对照组与miR-140-3p模拟物组、miR-140-3p抑制剂对照组与miR-140-3p抑制剂组、miR-140-3p模拟物对照组与miR-140-3p抑制剂对照组各指标比较所得
miR-140-3p模拟物对照组 3 0.251±0.050 0.32±0.10 0.231±0.030 0.43±0.06 0.29±0.07
miR-140-3p模拟物组 3 0.073±0.021 0.11±0.05 0.070±0.023 0.30±0.06 0.10±0.05
miR-140-3p抑制剂对照组 3 0.252±0.033 0.33±0.10 0.235±0.027 0.44±0.08 0.28±0.09
miR-140-3p抑制剂组 3 0.811±0.123 0.66±0.11 0.751±0.100 0.66±0.06 0.62±0.13
F值 70.831 18.690 85.177 15.335 19.094
P值 < 0.001 0.001 < 0.001 0.001 0.001
P1值 0.011 0.024 0.007 0.035 0.026
P2值 < 0.001 0.002 < 0.001 0.004 0.001
P3值 0.958 0.946 0.938 0.893 0.947
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2.8. BMSC-Exo中miR-140-3p对Fb形态的影响

转染24 h后，miR-140-3p模拟物组Fb细胞膜表面气球样突起数量明显少于miR-140-3p模拟物对照组，miR-140-3p抑制剂组Fb细胞膜表面气球样突起数量明显多于miR-140-3p抑制剂对照组。见图 5。

图 5.

4组大鼠Fb转染24 h后的焦亡表现扫描电子显微镜×2 000。5A、5B、5C、5D.分别为miR-140-3p模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组大鼠Fb，其中图 5B细胞膜表面气球样突起数量明显少于图 5A，图 5D细胞膜表面气球样突起数量明显多于图 5C

Pyroptotic manifestations of rat Fbs in four groups at 24 hours post-transfection

注：微小RNA-140-3p（miR-140-3p）模拟物对照组、miR-140-3p模拟物组、miR-140-3p抑制剂对照组、miR-140-3p抑制剂组大鼠成纤维细胞（Fb）均经高糖培养24 h后分别加入转染miR-140-3p模拟物对照剂后的大鼠骨髓间充质干细胞（BMSC）分泌的外泌体、转染miR-140-3p模拟物后的BMSC分泌的外泌体、转染miR-140-3p抑制剂对照剂后的BMSC分泌的外泌体、转染miR-140-3p抑制剂后的BMSC分泌的外泌体

图 5

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3. 讨论

糖尿病引起的高糖环境常导致创面修复障碍，这在糖尿病并发症中极具代表性[6-9]。糖尿病创面修复障碍的机制复杂多样，既涉及氧化应激加剧引发细胞损伤，又包括炎症因子的过度释放和细胞程序性死亡的异常激活。对细胞外囊泡（尤其是外泌体）在细胞间信号传递及疾病进程中作用的深入探究揭示，外泌体携带的miR可精细调控目标细胞的基因表达，从而影响组织损伤或修复的进程[10-13]。

本研究测序结果显示，大鼠BMSC-Exo中miR-140-3p表达在正常环境下和高糖环境下有明显差异，且结合蛋白质印迹法结果显示miR-140-3p在大鼠Fb焦亡相关蛋白调控中具有显著作用。焦亡是一种依赖于炎症小体激活的细胞程序性死亡，其主要特征在于钠离子内流导致细胞肿胀、膜孔形成，最终细胞发生裂解，伴有炎症因子如IL-1β、IL-18的活化与释放[14-16]。NLRP3炎症小体介导的经典信号是目前最为常见的焦亡机制，caspase-1前体被活化后，剪切IL-1β前体和IL-18前体，释放活性细胞因子并触发下游消皮素D裂解形成膜孔，这些过程与多种急慢性炎症疾病及高糖环境诱发的组织损伤密切相关[17-18]。本研究的测序结果提示，miR-140-3p或可通过作用于NLRP3/caspase-1/消皮素D信号通路来调节皮肤Fb的焦亡水平，为探究糖尿病创面难愈机制提供了新的分子靶点。

为了进一步阐明外泌体中携带的miR-140-3p在调控细胞功能与病理过程中的作用，本研究对大鼠BMSC转染miR-140-3p的模拟物和抑制剂，并对收集到的外泌体行实时荧光定量PCR，检测miR-140-3p的表达水平。结果显示，转染24 h后，miR-140-3p模拟物组BMSC-Exo中miR-140-3p的表达明显高于miR-140-3p模拟物对照组，miR-140-3p抑制剂组BMSC-Exo中miR-140-3p表达明显低于miR-140-3p抑制剂对照组。对大鼠BMSC转染miR-140-3p以过表达miR-140-3p时，细胞中miR-140-3p表达大幅提升，进而在分泌外泌体过程中，更多的miR-140-3p被包裹到外泌体内；而在miR-140-3p抑制剂组中，针对miR-140-3p特异性设计的抑制剂会降低大鼠BMSC及大鼠BMSC-Exo中的miR-140-3p表达。值得注意的是，外泌体具有良好的膜结构，能将所携带的核酸分子稳定地运输到靶细胞，从而在转录后水平上发挥调控作用。因此，通过转染供体细胞并测定其分泌的外泌体中miR表达，本研究不仅成功验证了大鼠BMSC-Exo中miR-140-3p的可塑性，还为在本研究中针对性地调控外泌体中miR的表达、探讨其在细胞功能方面的表现提供了操作依据和理论支持。

在功能学实验的层面，本研究表明，过表达miR-140-3p的大鼠BMSC-Exo显著增强了高糖环境下大鼠Fb的增殖和迁移能力；抑制miR-140-3p表达的大鼠BMSC-Exo则显著削弱了大鼠Fb的增殖和迁移能力。以上结果提示，提高miR-140-3p的水平能有效减轻高糖环境对Fb造成的损害，从而在创面修复过程中发挥积极调节作用。目前的一些研究也支持这一结论，在糖尿病心肌病和糖尿病肾病等糖尿病并发症中，某些miR在局部微环境中能通过外泌体的形式传递保护信号或损伤信号，从而影响靶细胞的存活、分化与功能[19-24]。

本研究中蛋白质印迹法实验结果进一步印证了BMSC-Exo中miR-140-3p在抑制焦亡相关通路、保护高糖环境下Fb方面的重要作用。高糖状态常伴随着糖毒性物质显著增加和氧化应激水平显著升高，这种微环境可促使炎症信号通路持续激活。本研究显示，转染miR-140-3p模拟物的大鼠BMSC-Exo能够明显下调高糖环境下大鼠Fb中焦亡相关蛋白的表达，说明经miR-140-3p修饰后外泌体对焦亡信号通路具有抑制作用。miR-140-3p抑制剂组大鼠Fb则呈现相反的趋势，证实了可通过改变外泌体中miR-140-3p表达水平来调控高糖引发的焦亡。NLRP3炎症小体是由NLRP3蛋白、凋亡相关斑点样蛋白以及caspase-1前体等分子组成的多聚体，被认为在多种代谢性和炎症疾病中发挥核心作用。当细胞感受到代谢紊乱与氧化应激等危险信号时，NLRP3蛋白被激活，与凋亡相关斑点样蛋白形成寡聚体复合物，激活caspase-1。活化的caspase-1不仅能够催化IL-1β和IL-18从无活性形式转变为成熟形态，还会切割消皮素D，产生能够在细胞膜上打孔的N端片段，从而诱发焦亡[25-26]。焦亡既能通过向细胞外释放炎症因子加剧组织损伤，又会导致Fb功能的进一步衰退，给创面修复带来双重阻碍[27-31]。本研究显示，miR-140-3p模拟物可以在大鼠BMSC-Exo中被显著富集，通过与靶基因结合或干扰其关键蛋白翻译，减少NLRP3炎症小体等炎症级联因子的异常激活。随着NLRP3炎症小体形成减少，caspase-1的活化程度降低，IL-1β、IL-18的成熟和消皮素D的裂解也相应减弱，焦亡过程得到抑制。miR-140-3p抑制剂则会促进这条焦亡信号通路的活化，加剧炎症反应与焦亡，形成“高糖—炎症—焦亡—损伤加深”的恶性循环。蛋白质印迹法的检测结果为本研究提供了有力的蛋白质分子证据，表明miR-140-3p过表达的大鼠BMSC-Exo可通过阻断NLRP3炎症小体及其下游级联反应来实现对焦亡的抑制，从而在高糖环境下加快大鼠Fb的增殖和迁移。

焦亡与其他细胞死亡方式（如凋亡、坏死）相比，具有鲜明的形态学特征，即细胞膜上产生明显的泡状或气球样突起，并最终导致细胞裂解以释放大量炎症内容物，对周围组织造成极大的刺激和损伤[32-34]。本研究显示，当大鼠BMSC-Exo中miR-140-3p水平提高时，会抑制NLRP3炎症小体及其下游级联反应的激活，从而减少对大鼠Fb细胞膜结构的破坏，也就减少了气球样突起的数量；而当miR-140-3p被抑制时，焦亡信号被进一步放大，大鼠Fb细胞膜表面出现更多明显的气球样突起并伴随炎症物质的大量释放。同时，扫描电子显微镜观察到的大鼠Fb的这些变化与蛋白质印迹法检测的定量数据相互佐证，进一步从形态学层面揭示了miR-140-3p调控焦亡的事实。

值得注意的是，本研究得出的大鼠BMSC-Exo中miR-140-3p对高糖环境诱导的大鼠Fb焦亡的抑制作用，与近年来其他研究团队在糖尿病相关病变中的结论形成了有益的呼应。有研究者报道了人间充质干细胞来源外泌体通过递送miR-125b保护人糖尿病肾病中的足细胞，主要通过沉默信息调节因子2相关酶7/核因子κB信号通路发挥作用[35]，而本研究则聚焦于miR-140-3p通过NLRP3炎症小体信号通路的调控作用，提示不同miR可能通过不同的分子机制在糖尿病并发症中发挥保护作用。有研究显示，大鼠BMSC-Exo中的TNF-α诱导蛋白6能够直接抑制糖尿病心肌病中的炎症反应，这提示外泌体的治疗效应可能是多种活性成分协同作用的结果[36]。然而，本研究通过特异性调控miR-140-3p的表达水平，证明了miR成分也可以显著影响细胞焦亡过程，这为开发更精准的外泌体工程化策略提供了理论依据。

综上所述，大鼠BMSC-Exo中miR-140-3p对高糖环境下大鼠Fb具有重要保护作用，可促进Fb的增殖和迁移并降低焦亡相关蛋白的表达，能减少细胞气球样突起的数量，降低细胞焦亡水平。随着研究的深入，miR-140-3p及其相关分子信号通路有望成为糖尿病及其并发症治疗的重要策略之一。

Funding Statement

甘肃省自然科学基金（22JR5RA692）；甘肃省卫生健康委员会骨干和青年人才项目（GSWSQN2025-20）；甘肃省烧伤与创面修复临床医学研究中心（21JR7RA674）；兰州市青年科技人才创新项目（2024-QN-38）

Gansu Provincial Natural Science Foundation (22JR5RA692); Backbone and Young Talent Project of Gansu Provincial Health Commission (GSWSQN2025-20); Gansu Provincial Clinical Medical Research Center for Burns and Wound Repair (21JR7RA674); Lanzhou Youth Science and Technology Talent Innovation Project (2024-QN-38)

Footnotes

利益冲突  所有作者声明不存在利益冲突

作者贡献声明  武岳：研究设计、实施研究、采集数据、统计学分析、撰写论文初稿；尹文辉、于淼、王达：采集数据、统计学分析；吴健：研究指导、经费支持、论文审核

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