Influence and mechanism of exosomes derived from rat bone marrow mesenchymal stem cells on rat Fbs under high glucose conditions
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摘要:
目的 探讨大鼠骨髓间充质干细胞（BMSC）来源外泌体（BMSC-Exo）对高糖环境下大鼠成纤维细胞（Fb）的影响及其机制,为糖尿病创面的治疗探索潜在的新途径。 方法 该研究为成组设计实验研究。取大鼠原代BMSC提取BMSC-Exo,并成功鉴定。取BMSC-Exo,分为对照组和高糖组,其中对对照组BMSC-Exo行常规培养,对高糖组BMSC-Exo采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）培养。对2组BMSC-Exo行真核mRNA测序,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。取第1~3代大鼠BMSC,采用随机数字表法分为微小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,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BMSC-Exo,用实时荧光定量PCR法检测BMSC-Exo中miR-140-3p的表达。取对数生长期大鼠Fb,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,采用高糖培养基培养24 h后分别加入转染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抑制剂后的BMSC分泌的外泌体（分组及处理后同）,于转染24 h后,采用细胞计数试剂盒-8测量细胞吸光度值,代表细胞增殖活力。取对数生长期大鼠Fb,分组及处理后行划痕试验检测划痕后24 h的细胞迁移率；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白细胞介素-1β（IL-1β）、IL-18、含pyrin结构域的NOD样受体蛋白3（NLRP3）、胱天蛋白酶-1（caspase-1）和消皮素D的蛋白表达。样本数均为3。 结果 与对照组相比,高糖组BMSC-Exo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为miR-140-3p。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（P<0.05）,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<0.05）。转染24 h后,miR-140-3p模拟物组Fb吸光度值为0.940±0.031,明显高于miR-140-3p模拟物对照组的0.781±0.020（P<0.05）；miR-140-3p抑制剂组Fb吸光度值为0.510±0.041,明显低于miR-140-3p抑制剂对照组的0.822±0.061（P<0.05）。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<0.05）,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<0.05）。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）。 结论 大鼠BMSC-Exo通过递送miR-140-3p,促进高糖环境下大鼠Fb的增殖、迁移并抑制其焦亡相关蛋白的表达,减少细胞焦亡,为糖尿病创面修复提供了潜在治疗靶点。 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 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 microRNAs (miRs) that strongly interacted with the classical pyroptosis signaling pathway were screened and identified. The rat BMSCs at passages 1 to 3 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 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 exosomes 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 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 Fbs in the logarithmic growth phase were divided 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 markers, including interleukin-1β (IL-1β), IL-18, NOD-like receptor pyrin domain-containing protein 3 (NLRP3), 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 expression levels of miR-140-3p and miR-542-5p were significantly upregulated in BMSC-Exo of high-glucose group. MiR-140-3p was identified as the differential miRNA with strong interaction 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), 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). 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); 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 expression levels 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 expression levels 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 proteins and alleviate cell pyroptosis. This study provides a promising therapeutic target for diabetic wound repair. -
Key words:
- Mesenchymal stem cells /
- Exosomes /
- Diabetes mellitus /
- Pyroptosis /
- Fibroblasts /
- MicroRNAs
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参考文献
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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
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 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
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 3 4组大鼠Fb划痕后各时间点迁移情况 荧光显微镜 ×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
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 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
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 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
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
Table 1. 4组大鼠Fb转染24 h后焦亡相关蛋白的蛋白表达比较（
组别 样本数 消皮素D caspase-1 IL-1β IL-18 NLRP3 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 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 
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