从骨髓间充质干细胞制备血浆膜囊泡用于潜在的细胞质替代治疗
Summary
与年龄相关的疾病与细胞质组分中的多种缺陷有关。在这里，我们提出了从骨髓间充质干细胞制备质膜囊泡的方案。这种技术可能被用作细胞质替代疗法的手段，以改善或甚至逆转年龄相关表型。
Abstract
我们以前报道了通过机械挤压哺乳动物细胞产生质膜囊泡（PMV）。 PMV与线粒体缺陷Rho0细胞的融合在正常培养条件下恢复有丝分裂活性。据报道，动脉粥样硬化，2型糖尿病，阿尔茨海默病和癌症是与各种细胞类型的细胞溶质和细胞器中的多种机械和功能缺陷相关的年龄相关疾病。骨髓间充质干细胞（BMSCs）代表骨髓中独特的细胞群，具有自我更新能力，同时保持其多能性。通过PMV融合补充来自自体骨髓基质干细胞的年轻细胞质的衰老细胞提供了改善或甚至逆转年龄相关表型的有希望的方法。该方案描述了如何通过使用3的聚碳酸酯膜挤出从BMSC制备PMV1; m孔，确定线粒体的存在，并使用共聚焦显微镜检查PMV内膜电位的维持，通过离心浓缩PMV，并进行体内注射PMV至小鼠腓肠肌。
Introduction
已经投入了大量的努力来建立基因，酶和细胞替代疗法的方法。这导致了巨大的突破，甚至临床应用1，2，3。最近，基于核转移技术的有争议的线粒体替代疗法被应用于老年妇女的体外受精或携带致死线粒体DNA突变4 。在年龄相关疾病中发现的缺陷，包括动脉粥样硬化，2型糖尿病，阿尔茨海默病和癌症，通常是多方面的。已经证明脂滴的积累;沉淀淀粉样蛋白;展开蛋白质在内质网中的保留;缺陷型蛋白酶体，自噬体和线粒体有助于这些疾病的发展或恶化“外套参照”> 5,6,7,8,9,10,11。目前没有可用于直接修复细胞质和细胞器故障的机制，这导致衰老和衰老表型。
我们以前通过机械挤压哺乳动物细胞报道了生物质膜囊泡（PMVs） 12 。除了细胞核外，膜或细胞溶质中的组分（包括蛋白质和RNA）以及细胞器如线粒体都发现在PMV中。基本上，PMV可以被认为是一个微型的去核细胞。更重要的是，PMV与线粒体缺乏的Rho0细胞的融合在正常培养条件下恢复有丝分裂活性。这是关于成立的第一份报告为细胞质替代疗法建立潜在的有效途径。
骨髓间充质干细胞（BMSCs）是通常从骨髓产生并且在培养物中容易扩增的多能祖细胞。胚胎干细胞标记Oct4，Nanog和SOX2在MSCs 13中检测到低水平。端粒酶活性也是可测量的。此外，不存在共刺激分子和人白细胞抗原（HLA）II类分子以及MSC上的低HLA I类表达使其成为同种异体或“现成”使用的理想细胞再生医学和免疫调节应用14 。
在这里，我们描述如何通过挤出通过具有3-μm孔隙的聚碳酸酯膜来制备来自小鼠BMSC的PMV，确定线粒体的存在并使用confoc检查PMV中膜电位的维持通过离心法制备浓缩但不聚集的PMV，并进行PMVs 体内注射到小鼠的腓肠肌中。
Protocol
Representative Results
Discussion
Cytoplasm replacement therapy as proposed in this manuscript has unique advantages over other reported approaches such as gene, molecular, and cell therapy. PMVs generated from BMSCs encapsulate not only the products of stemness genes but also intact cellular organelles, which are essential to remedy the ageing phenotypes associated with senescence. When young cytoplasm is delivered to senescent cells, the malfunctioning mechanisms may gain a brief relief; at the same time, the epigenome could be reprogrammed and invigor…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究得到了李嘉诚基金会，广东省高校“海洋工业绿色科技”，“中国自然科学基金”项目（http://www.nsfc.gov.cn/资助号：30971665， 81172894,81370925）和广东省教育厅（http://www.gdhed.edu.cn/ Grant No.cxzd1123）。
Materials
| IsoporeTM membranes | Millipore | TSTP04700 | 3 mm pore |
| Disposable filter unit | Xinya, Shanghai, China | 25 mm | Medical grade polypropylene |
| Insulin syringe | BD | 328446 | 1 ml |
| pN1-EGFP | Clontech | 6085-1 | |
| MitoTracker | Molecular Probes | M7514 | Green FM, 1 μM |
| JC-1 | Beyotime, Haimen, China | C2006 | 10 mg/ml |
| CM-DiI | Beyotime, Haimen, China | C1036 | 10 mM |
| PEI | Sigma | P3143 | Mn = 75000 |
| Fluorescence Microscope | Nikon | Eclipse TE 2000 | With CCD camera |
| Confocol Microscope | Carl Zeiss | LSM 510 Meta | |
| PolyJet | SigaGen | SL100688 | For cell transfection |
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