[1]郭柏华,王建峰,张轶男,等.骨髓间充质干细胞源性外泌体在大鼠巨结肠模型中的干预性研究及相关机制初探[J].临床小儿外科杂志,2025,(08):786-792.[doi:10.3760/cma.j.cn101785-202502059-015]
　Guo Bohua,Wang Jianfeng,Zhang Yinan,et al.Therapeutic effect and related mechanisms of bone marrow mesenchymal stem cell-derived exosomes on giant colon model rats[J].Journal of Clinical Pediatric Surgery,2025,(08):786-792.[doi:10.3760/cma.j.cn101785-202502059-015]
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骨髓间充质干细胞源性外泌体在大鼠巨结肠模型中的干预性研究及相关机制初探

《临床小儿外科杂志》[ISSN:1671-6353/CN:43-1380/R] 卷: 期数: 2025年08 页码: 786-792 栏目: 实验研究 出版日期: 2025-08-28
Title:
Therapeutic effect and related mechanisms of bone marrow mesenchymal stem cell-derived exosomes on giant colon model rats
作者:
郭柏华1;  王建峰2;  张轶男1;  张剑峰1;  樊长河1;  黄婵1;  童国煜1;  曾祥勇3
1. 上海交通大学医学院附属上海儿童医学中心海南医院儿外科, 三亚 572013;
2. 嘉兴市妇幼保健院儿外科, 嘉兴 314000;
3. 海南医科大学第二附属医院普通外科, 海口 570100
Author(s):
Guo Bohua1;  Wang Jianfeng2;  Zhang Yinan1;  Zhang Jianfeng1;  Fan Changhe1;  Huang Chan1;  Tong Guoyu1;  Zeng Xiangyong3
1. Department of Pediatrics, Hainan Branch, Shanghai Children’s Medical Center, Shanghai Jiao Tong University, Sanya 572013, China;
2. Department of Pediatrics, Jiaxing Maternal & Children’s Healthcare Hospital, Jiaxing 314000, China;
3. Department of General Surgery, Second Affiliated Hospital, Hainan Medical University, Haikou 570100, China
关键词:
外泌体; 骨髓间充质干细胞; Hirschsprung病; 信号通路; 大鼠; 模型;  动物; 动物;  实验
Keywords:
Exosomes; Bone Marrow Mesenchymal Stem Cell; Hirschsprung Disease; Rats; Signal Pathway; Models; Animal; Animals; Laboratory
DOI:
10.3760/cma.j.cn101785-202502059-015
摘要:
目的 分离骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)源性外泌体(exosomes)并作用于巨结肠模型大鼠与人神经母细胞瘤细胞(SH-SY5Y),观察其治疗作用并探讨相关机制。方法 通过全骨髓粘附法体外分离、培养和纯化斯泼累格·多雷(Sprague-Dawley,SD)大鼠骨髓组织中的BMSCs。采用ExoQuick法从BMSCs培养上清液中分离Exosomes。将30只SD大鼠称重后随机等额分为巨结肠大鼠模型组、对照组和干预组(干预组自尾静脉注射100 μg/mL的Exosomes稀释液1 mL干预巨结肠大鼠模型,每日1次,连续7 d)。培养的SH-SY5Y细胞分为Exosomes干预组和对照组。HE染色观察结肠病理学结构改变;实时荧光定量PCR测定结肠组织NOTCH(为一组单次跨膜受体)、胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)和性别决定取Y框蛋白10基因(SRY-related HMG-box 10,SOX-10) mRNA的表达;蛋白免疫印迹(Western blot)检测结肠组织NOTCH、GDNF和Sox10蛋白的表达。四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)和Transwell小室检测细胞增殖和迁移能力。结果 采用ExoQuick法分离出的BMSCs源性Exosomes在大小上存在异质性,介于50~120 nm之间,且表达Exosomes特异性标记CD9和CD63。HE染色显示,干预组大鼠结肠组织病理结构改变较模型组轻微。模型组大鼠结肠组织中NOTCH、GDNF和Sox10 的mRNA(F=17.420、19.740和25.820,P均<0.001)及蛋白表达(F=7.690、8.470和11.780,P均<0.001)均明显低于对照组,而在干预组大鼠结肠组织较模型组大鼠而言明显升高(F=18.120,P<0.001)。干预组SH-SY5Y细胞NOTCH、GDNF和Sox10较对照组明显升高(t=3.130、2.420和3.340,P<0.05),干预组SH-SY5Y细胞增殖率和划痕后24 h迁移距离均高于对照组(P<0.05)。结论 BMSCs源性Exosomes能够改善巨结肠模型大鼠结肠的病理变化,其作用可能是通过调控NOTCH、GDNF和Sox10相关信号通路实现的。
Abstract:
Objective To explore the therapeutic effect of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) on the model of megacolon in rats and elucidate its related mechanisms. Methods BMSCs from bone marrow of normal Sprague-Dawley (SD) rats were isolated,cultured and purified in vitro by total bone marrow adhesion.Exosomes were isolated from BMSCs supernatant by ExoQuick reagent method.Thirty SD rats were randomized into model group (megacolon rat model without any intervention),control group (same operation as model group,normal saline used instead of 0.1% BAC solution); intervention group (rat model of megacolon was interfered with by an injection of 100 μg/mL exosomes dilution 1ml into tail vein,once a day for 7 days).Cultivated SH-SY5Y cells were divided into intervention groups (adding 50 ng to conventional culture medium)/μL BMSCs derived exosomes and control group (conventional culture without any intervention).Hematoxylin-eosin (HE) stain was utilized for observing the pathological structural changes of colon; Real-time fluorescent quantitative polymerase chain reaction (PCR) and Western blot were employed for detecting the expressions of NOTCH,GDNF and Sox10 in colonic tissue.Tetramethylazolamide (MTT) and Transwell cell assays were performed for cell proliferation and migration capability. Results BMSCs of rats were isolated and cultured and exosomes derived from BMSCs successfully extracted.HE stain indicated that the pathological changes in colonic tissue of intervention groups were milder than those of model group.The expressions of NOTCH,GDNF and Sox10 mRNA and protein in colonic tissue of model group were significantly lower than those of control group.The expressions of NOTCH,GDNF and Sox10 was significantly higher in colonic tissue of intervention group than those of model group (F=18.120,P<0.001); The levels of NOTCH,GDNF and Sox10 levels of SH-SY5Y cells were significantly higher in intervention group than those in control group (t=3.130,2.420,and 3.340,P<0.05).The proliferation rate of SH-SY5Y cells at various time points and the number of cells passing through artificial basement membrane in intervention group were higher than those in control group (P<0.05). Conclusions BMSCs derived exosomes may improve the pathological changes of colon in rats with Hirschsprung‘s disease.And their effects are achieved through regulating NOTCH/GDNF/Sox10-related signaling pathways.

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备注/Memo

收稿日期:2025-2-28。
基金项目:浙江省卫生健康科技计划(临床研究应用项目2022KY390)
通讯作者:曾祥勇,Email:278409751@qq.com

更新日期/Last Update: 2025-08-28