Effect and mechanism of human adipose-derived stem cell exosomes on diabetic peripheral neuropathy
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摘要:
目的 探讨糖尿病周围神经病变（DPN）中神经鞘胚素蛋白表达的变化及人脂肪干细胞（ADSC）外泌体对神经鞘胚素蛋白表达变化的调节作用。 方法 该研究为前瞻性观察性临床研究联合实验研究。将空军军医大学第一附属医院（以下简称本院）2022年5月—2023年10月收治的13例符合入选标准的DPN患者（男9例、女4例，年龄32~68岁）作为DPN组，将本院该段时间收治的5例符合入选标准的非糖尿病患者（男4例、女1例，年龄29~61岁）作为对照组。采集2组患者清创或截肢后弃用组织中的趾神经或腓肠神经组织，行苏木精-伊红染色后观察神经组织的病理学变化，行免疫荧光染色后观察神经组织中S100β、神经鞘胚素的蛋白表达并对神经鞘胚素蛋白表达进行定量，分别采用蛋白质印迹法和实时荧光定量反转录PCR法检测神经鞘胚素的蛋白和mRNA表达（DPN组样本数均为13，对照组样本数均为5）。取12只雄性3~5 d龄C57BL/6小鼠，提取施万细胞，并将细胞分为常规培养组（常规培养）、单纯高糖组（仅用高浓度葡萄糖溶液培养）、高糖+外泌体组（用高浓度葡萄糖溶液和提取的人ADSC外泌体培养）。培养24 h后，采用细胞计数试剂盒8检测细胞增殖活力（样本数为6）；培养48 h后，采用蛋白质印迹法检测神经鞘胚素的蛋白表达（样本数为3）。 结果 相较于对照组，DPN组患者神经组织中神经支持细胞减少、炎症细胞增多，具有典型的神经损伤表现。免疫荧光染色检测显示，相较于对照组，DPN组患者神经组织中细胞核更多，S100β的蛋白表达更少；DPN组患者神经组织中神经鞘胚素蛋白表达为71±31，明显低于对照组的1 729±62（ t=76.92， P<0.05）。蛋白质印迹法检测显示，DPN组患者神经组织中的神经鞘胚素蛋白表达为0.74±0.08，明显低于对照组的0.97±0.06（ t=5.49， P<0.05）。DPN组患者神经组织中神经鞘胚素的mRNA表达明显低于对照组（ t=7.65， P<0.05）。培养24 h后，与常规培养组比较，单纯高糖组和高糖+外泌体组施万细胞增殖活力均明显降低（ P<0.05）；与单纯高糖组比较，高糖+外泌体组施万细胞增殖活力明显升高（ P<0.05）。培养48 h后，与常规培养组比较，单纯高糖组、高糖+外泌体组施万细胞神经鞘胚素蛋白表达均明显降低（ P<0.05）；与单纯高糖组比较，高糖+外泌体组施万细胞神经鞘胚素蛋白表达明显升高（ P<0.05）。 结论 DPN患者神经组织中神经鞘胚素的蛋白表达较正常神经组织降低，这可能与高糖降低了施万细胞增殖活力相关；人ADSC外泌体可能通过提高神经鞘胚素蛋白表达，改善施万细胞增殖活力，进而延缓DPN的进展。 Abstract:Objective To investigate the changes of artemin protein expression in diabetic peripheral neuropathy (DPN) and to explore the regulatory effect of human adipose-derived stem cell (ADSC) exosomes on the change of artemin protein expression. Methods This research was a prospective observational clinical research combined with experimental research. Thirteen DPN patients (9 males and 4 females, aged 32 to 68 years) who were admitted to the First Affiliated Hospital of Air Force Medical University (hereinafter referred to as our hospital) from May 2022 to October 2023 and met the inclusion criteria were selected as DPN group, and 5 non-diabetes patients (4 males and 1 female, aged 29 to 61 years) who were admitted to our hospital in the same period of time and met the inclusion criteria were selected as control group. The toe nerve or sural nerve tissue in the abandoned tissue after debridement or amputation of patients in the two groups was collected. The pathological changes of nerve tissue were observed after hematoxylin-eosin staining; the protein expressions of S100β and artemin in nerve tissue were observed after immunofluorescence staining, and the artemin protein expression was quantified; the protein and mRNA expressions of artemin were detected by Western blotting and real-time fluorescent quantitative reverse transcription polymerase chain reaction, respectively (the sample number in DPN group and control group was 13 and 5, respectively). Twelve male C57BL/6 mice aged 3 to 5 days were collected to isolate Schwann cells, and the cells were divided into conventional culture group cultured routinely, high glucose alone group (cultured with high concentration of glucose solution only), and high glucose+exosome group (cultured with high concentration of glucose solution and extracted human ADSC exosomes). After 24 hours of culture, the cell proliferation activity was detected by cell counting kit 8 ( n=6). After 48 hours of culture, the protein expression of artemin was detected by Western blotting ( n=3). Results Compared with those in control group, the neural supporting cells decreased and the inflammatory cells increased in the nerve tissue of patients in DPN group, showing typical manifestations of nerve injury. Immunofluorescence staining showed that compared with those in control group, the nuclei was more, and the protein expression of S100β was lower in nerve tissue of patients in DPN group. The protein expression of artemin in nerve tissue of patients in DPN group was 71±31, which was significantly lower than 1 729±62 in control group ( t=76.92, P<0.05). Western blotting detection showed that the protein expression of artemin in nerve tissue of patients in DPN group was 0.74±0.08, which was significantly lower than 0.97±0.06 in control group ( t=5.49, P<0.05). The artemin mRNA expression in nerve tissue of patients in DPN group was significantly lower than that in control group ( t=7.65, P<0.05). After 24 hours of culture, compared with that in conventional culture group, the proliferation activities of Schwann cells in high glucose alone group and high glucose+exosome group were significantly decreased ( P<0.05); compared with that in high glucose alone group, the proliferation activity of Schwann cells in high glucose+exosome group was significantly increased ( P<0.05). After 48 hours of culture, compared with those in conventional culture group, the protein expressions of artemin of Schwann cells in high glucose alone group and high glucose+exosome group were significantly decreased ( P<0.05); compared with that in high glucose alone group, the protein expression of artemin of Schwann cells in high glucose+exosome group was significantly increased ( P<0.05). Conclusions The protein expression of artemin in nerve tissue of DPN patients is lower than that in normal nerve tissue, which may be related to the reduction of proliferation activity of Schwann cells by high glucose. Human ADSC exosomes may improve the proliferation activity of Schwann cells by increasing artemin protein expression, thereby delaying the progression of DPN. -
Key words:
- Diabetic neuropathies /
- Exosomes /
- Cell proliferation /
- Schwann cells /
- Artemin /
- Adipose-derived stem cells
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曹涛：实施研究，采集数据，统计分析，撰写论文；郝彤、肖丹：实施研究，采集数据；张伟锋、计鹏、贾艳慧、王婧：起草文章，修改文章；王许杰：实施研究，采集数据；官浩：设计研究，修改文章；陶克：酝酿和设计研究，分析数据，修改文章，获取研究经费所有作者均声明不存在利益冲突
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参考文献
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1 DPN组DPN患者及对照组非糖尿病患者趾神经组织病理学观察 苏木精-伊红×20。1A、1B.分别为对照组和DPN组，图1B较图1A神经支持细胞减少，炎症细胞增多
注：DPN为糖尿病周围神经病变；红色箭头指示神经支持细胞，蓝色箭头指示炎症细胞
2 DPN组DPN患者及对照组非糖尿病患者趾神经组织中神经鞘胚素和S100β的蛋白表达。2A、2B、2C、2D、2E.分别为对照组细胞核与神经鞘胚素和S100β染色重叠、细胞核染色、S100β染色、神经鞘胚素染色、图2D方框中图形放大10倍的图片；2F、2G、2H、2I、2J.分别为DPN组细胞核与神经鞘胚素和S100β染色重叠、细胞核染色、S100β染色、神经鞘胚素染色、图2I方框中图形放大10倍的图片，可见DPN组神经组织中细胞核数量较对照组明显增多，S100β和神经鞘胚素蛋白表达明显减少
注：进行异硫氰酸荧光素-花青素3-4′，6-二脒基-2-苯基吲哚染色；图2A、2B、2C、2D及2F、2G、2H、2I放大倍数为20倍；DPN为糖尿病周围神经病变；细胞核染色为蓝色，神经鞘胚素染色为红色，S100β染色为绿色
3 蛋白质印迹法检测DPN组DPN患者及对照组非糖尿病患者趾神经组织中的神经鞘胚素蛋白表达
注：DPN为糖尿病周围神经病变；条带上方的1表示对照组1例患者，2、3表示DPN组2例患者
5 小鼠原代施万细胞的鉴定。5A.加入博来霉素48 h后，原代细胞大部分呈双极、梭形或三角形 倒置相差显微镜×200；5B.加入博来霉素培养3 d，细胞中有S100β表达 花青素3-4′，6-二脒基-2-苯基吲哚×200
注：S100β阳性染色为红色
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