摘要
目的探索来源于脐带间充质干细胞(huc-MSCs)的含有腺病毒-TGF-β1 shRNA的外泌体对单侧输尿管梗阻(UUO)大鼠纤维化肾组织的靶向性和缓解肾间质纤维化的疗效。方法构建单侧输尿管梗阻(UUO)大鼠模型;腺病毒-TGF-β1 shRNA转导脐带间充质干细胞,收集其外泌体;采用纳米粒子追踪分析、透射电子显微镜、Western blot法和RT-qPCR法对外泌体及其对TGF-β1的干扰效率进行鉴定;IVIS Lumina系统荧光成像技术分析外泌体在体(in vivo)分布情况;HE染色、Masson染色、TGF-β1免疫组织化学染色(IHC)分析外泌体对UUO大鼠肾间质纤维化的疗效;Western blot法检测大鼠肾组织Collagen I、Fibronectin、α-SMA以及TGF-β1、p-Smad 3和Smad 3的表达情况。结果含有腺病毒TGF-β1 shRNA的外泌体在体外对TGF-β1具有显著的敲低效率(P<0.01)。此外泌体经尾静脉注射至大鼠体内后,大部分聚集于左侧损伤肾脏,具有定向迁移能力。相对于UUO组大鼠而言,经含有腺病毒-TGF-β1 shRNA的外泌体治疗后,能够明显缓解UUO大鼠肾间质纤维化的程度,HE染色和Masson染色显示的肾间质组织胶原纤维沉积显著减少;IHC染色显示TGF-β1表达明显被抑制(P<0.01)。Western blot结果显示,相较于UUO组,外泌体治疗组大鼠纤维化肾组织的Collagen I、Fibronectin和α-SMA表达显著降低(P<0.01);TGF-β1和磷酸化-Smad 3(P<0.01)被显著抑制。结论将huc-MSCs来源的含有腺病毒-TGF-β1 shRNA的外泌体尾静脉注射至UUO大鼠体内,外泌体能够特异性靶向纤维化的肾组织并通过抑制TGF-β1/Smad 3通路修复肾间质纤维化。
Objective To explore the effects of exosomes containing adenovirus-TGF-β1 shRNA derived from umbilical cord mesenchymal stem cells(huc-MSCs)on the targeting of fibrotic renal tissues and the therapeutic effects on renal interstitial fibrosis in unilateral ureteral obstruction(UUO)rat models.Methods UUO rat models were constructed.The huc-MSCs were transducted with adenovirus-TGF-β1 shRNA and exosomes were collected.The exosomes and knock-down efficiency of TGF-β1 were determined with nanoparticle tracking analysis,transmission electron microscopy,Western blot and RT-qPCR.The distribution of exosomes in vivo was analyzed with IVIS Lumina system.The therapeutic effect of exosomes on renal interstitial fibrosis in UUO rats were determined with HE staining,Masson staining and TGF-β1 immunohistochemistry(IHC).The expressions of collagen I,fibronectin,α-SMA,TGF-β1,p-Smad 3 and Smad 3 in renal tissue were detected with Western blot.Results Exosomes containing adenovirus-TGF-β1 shRNA had a significant knock-down efficiency on TGF-β1(P<0.01).After the exosomes were injected into rats through the tail vein,most of them gathered in the left kidney,with a directional migration behavior.The exosomes containing adenovirus-TGF-β1 shRNA could significantly alleviate the renal interstitial fibrosis in UUO rats.HE staining and Masson staining showed the collagen deposition in fibrotic renal tissues was significantly reduced.IHC showed the expression of TGF-β1 was significantly inhibited(P<0.01).Western blot showed the expressions of collagen I,fibronectin andα-SMA(P<0.01)were significantly reduced,and TGF-β1 and phosphorylated-Smad 3(P<0.01)were significantly inhibited.Conclusion Exosomes containing adenovirus-TGF-β1 shRNA derived from huc-MSCs injected into UUO rats through the tail vein can specifically target and repair renal interstitial fibrosis by inhibiting TGF-β1/Smad 3 pathway.
作者
俞静
戴世荣
单荣梅
刘香
YU Jing;DAI Shirong;SHAN Rongmei;LIU Xiang(Department of Laboratory Medicine,Nantong Second People s Hospital,Nantong 226002,China)
出处
《现代泌尿外科杂志》
CAS
2022年第7期593-599,共7页
Journal of Modern Urology
基金
南通市卫生和计划生育委员会科研课题(No.WKZL2018053)。
