Immunodepressive mechanical of exosomes excreted by rat bone mesenchymal stem cells with overexpressed indoleamine 2,3-dioxygenase
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摘要: 目的:探讨过表达吲哚胺2, 3双加氧酶 (IDO) 大鼠骨髓间充质干细胞 (BMSCs) 通过分泌外排体 (exosome) 抑制免疫排异反应机制研究。方法:通过慢病毒载体GV308携带IDO转染干细胞构建过表达IDOBMSCs体系并加入基因开启剂强力霉素 (DOX) 后采用SBI公司的ExoQuick-TC提取分泌的exosome。同时建立大鼠腹腔异位移植心脏模型, 经尾静脉给予相应细胞分泌的exosome, 利用超声心动图检测经注射过表达IDOBMSCs exosome (过表达IDO-BMSCs exosome组, A组)、空载体-BMSCs exosome (空载体-BMSCs exosome组, B组)、BMSCs exosome (BMSCs exosome组, C组) 48h后移植心脏心功能变化, 将移植未处理组、给予吗替麦考组作为对照。完成心脏彩超后取A组、B组、C组大鼠注射48h后的脾脏, 采用流式细胞技术检测其CD40、CD86、CD80、MHCII、CD274、CD45RA、CD45RA+CD45RB、Treg细胞的表达情况, 将移植未处理组、给予吗替麦考组及正常大鼠 (正常组) 脾脏细胞作为对照。进一步采用TMT标记定量蛋白质组学分析A组有关调节免疫排异的蛋白。结果:A组的LVEF、FS较其他各组有提高;采用流式细胞技术证实其CD40、CD86、CD80、MHCII、CD45RA、CD45RA+CD45RB表达降低, 而CD274、Treg细胞的表达增高。蛋白质谱示:Four and a half LIM domains 1蛋白为调节免疫排异反应的关键蛋白。结论:采用TMT标记过表达IDO-BMSCs分泌exosome内蛋白, Four and a half LIM domains 1蛋白为调节免疫排异反应的关键蛋白。
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关键词:
- 大鼠骨髓间充质干细胞 /
- 吲哚胺2 /
- 3双加氧酶 /
- 外泌体 /
- FourandahalfLIMdomains1蛋白
Abstract: Objective:This study aimed to assess how indoleamine 2, 3-dioxygenase (IDO) overexpression in rat bone marrow mesenchymal stem cells (BMSCs) inhibits immune rejection mechanism by secreting exosomes.Method:Using added doxycycline as gene initiator, rat BMSCs overexpressing IDO were constructed by transfecting stem cells with the lentiviral vector GV308 carrying IDO gene.ExoQuick-TC from SBI was adopted to extract secreted exosomes.Concurrently, a rat heart model was established with abdominal heterotopic transplantation.Exosomes secreted by corresponding cells were administered through the caudal vein.Echocardiogram was used for two days to detect overexpression IDO-BMSC exosomes by injection, empty vector-BMSC exosomes, BMSC exosome, and changes in cardiac function after transplantation.Transplant-untreated group and mycophenolate mofetil group were used as control groups.Spleens of recipient rats in three exosome groups were harvested to detect expressions of cluster of differentiation CD 40, CD86, CD80, major histocompatibility complex (MHC) II, CD274, CD45RA, CD45RA+CD45RB, and Treg cell by flow cytometry.An untreated transplant group, a mycophenolate mofetil group, and a normal rat spleen cell group were adopted as controls.Tandem mass tag (TMT) quantitative proteomics was used to further analyze proteins regulating immune rejection in exosome secreted by overexpression IDO-BMSCs.Result:Corresponding cell treatment was performed after establishment of heterotopic heart transplant rat model.The group with overexpression IDO-BMSCs secreting exosome for two days showedhigher LVEF and FS of transplanted heart than other groups.Flow cytometry revealed that spleen cells of rats with IDO-BMSC exosomes presented downregulated expression levels of CD40, CD86, CD80, MHCII, CD45 RA, and CD45RA+CD45RB and upregulated expression levels of CD274 and Treg cell.Protein spectrum:Four and a half LIM domains 1 protein was the key protein for regulating immune rejection reaction.Conclusion:TMT was used to tag proteins in exosomes secreted from rat BMSCs with IDO overexpression.Four and a half LIM domains 1 protein is the key protein for regulating immune rejection reaction. -
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