肌源性祖细胞衍生外质细胞的纯化与移植, 改善杜氏肌营养不良性小鼠的心功能
Summary
在这里, 我们提出了一个方案, 以过渡改善心脏功能的杜氏肌营养不良小鼠移植从正常的肌原细胞外周肌。
Abstract
杜琴肌肉营养不良 (DMD) 是一种由缺乏功能性营养不良蛋白引起的 x 连锁隐性遗传病。这种疾病无法治愈, 随着病情的发展, 患者会出现扩张型心肌病、心律失常和充血性心力衰竭的症状。DMD Mdx突变体小鼠不表达营养不良蛋白, 常被用作 DMD 的小鼠模型。在我们最近的研究中, 我们观察到大类型 (WT)-肌原体细胞衍生外显子 (MPC-Exo) 的心肌内注射短暂地恢复了 DMDMdx突变体小鼠心肌中营养不良蛋白的表达, 这与之相关心脏功能的短暂改善表明 WT-MPC-Exo 可以提供一个选项来缓解 DMD 的心脏症状。本文介绍了 MPC-Exo 纯化并移植到dmd Mdx 突变体小鼠心脏中的技术。
Introduction
杜氏肌营养不良症 (DMD) 是一种 x 连锁隐性、进行性神经肌肉疾病, 由 DMD 基因突变和功能性营养不良蛋白 1的丧失引起。肌营养蛋白主要表现在骨骼肌和心肌中, 在平滑肌、内分泌腺体和神经元2,3中表达较少。Dmd 是最常见的肌肉营养不良症, 发病率为每3, 500 至 5,000名新生男孩1人。个人通常会出现进行性肌肉坏死, 在青春期早期失去独立行走, 以及在生命的第二至第三年死于心力衰竭和呼吸衰竭.
扩张型心肌病、心律失常和充血性心力衰竭是 dmd7,8的常见心血管表现。这种疾病无法治愈, 支持性治疗可能会改善症状或延缓心力衰竭的进展, 但很难改善心脏功能9,10.
与 DMD 患者类似, x 连锁肌营养不良症 (MDX) 小鼠缺乏营养不良蛋白, 并出现心肌病11的症状, 因此被广泛用于 DMD 相关心肌病的研究。为了恢复受影响肌肉中的营养不良蛋白, 异基因干细胞治疗已被证明是治疗 dmd12、13、14的有效方法。外体, 30-150nm 膜囊泡分泌的各种细胞类型, 发挥关键作用, 通过遗传物质运输细胞间的通信, 如信使 rna (mrna) 和非编码 rna15,16,17 ,18,19,20,21。
我们以前的研究表明, 从肌源性祖细胞 (MPC) 中提取的外显子, 如 C2C12 细胞系, 可以转移营养不良素 mRNA 到宿主心肌细胞后, 直接心脏注射22, 这表明同种异体传递Mpc 衍生外显子 (MPC-Exo) 可以短暂恢复 MDX 小鼠的 DMD 基因表达。本文的重点是 MPC-Exo 纯化和移植技术。
Protocol
动物是根据奥古斯塔大学佐治亚医学院动物护理和使用机构委员会批准的协议和动物福利条例处理的。 1. mpc 衍生的外显子的分离和纯化 种子 5 x10 6 c2c12 细胞在一个15厘米的细胞培养盘与20毫升完成 dulbecco 的改性鹰的培养基 (dmem) 含有10% 的胎儿牛血清 (fbs), 100 Uml 青霉素 g 和100μgml 链霉素。在37°c 和 5% co 2 下孵化. 利用摆动的桶式转子, 在 100, 000 <…
Representative Results
图 1a 显示了从 c2c12 细胞中分离和纯化外显体的流程图。为了确认外显子的存在, 我们进行了透射电子显微镜分析。透射电子显微镜图像 (图 1b) 显示了 c2c12 衍生外显子的明亮和圆形囊泡的形态。西方印迹分析证实存在外显子标记, 包括 CD63 和 TSG101 (图 1c)。 我们观察到心?…
Discussion
分离纯外质体的方法对于研究外显子的功能是必不可少的。外显子体分离的常用技术之一是聚乙二醇介导的沉淀17,18,25。外显子可以沉淀在 Peg 中, 并通过低速离心颗粒。Peg 介导的纯化非常方便, 成本低, 它不需要任何先进的设备, 但人们担心外显子的纯度, 因为其他脂蛋白可能沉淀在一起, 很难去除。超滤是一种常规的外显子分?…
Declarações
The authors have nothing to disclose.
Acknowledgements
唐得到了美国心脏协会的部分支持: GRNT31430008、NIH-AR070029、NIH-HL086555、NIH-HL134354。
Materials
| 0.22-μm Filter | Fisherbrand | 09-720-004 | |
| 15-cm Cell Culture Dish | Thermo Fisher Scientific | 157150 | |
| 24-gauge catheter | TERUMO | SR-OX2419CA | |
| 31-gauge insulin needle | BD | 328291 | |
| 4% paraformaldehyde | Affymetrix | AAJ19943K2 | |
| 50 mL Centrifuge Tubes | Thermo Fisher Scientific | 339652 | |
| 6-0 suture | Pro Advantage by NDC | P420697 | |
| Alexa Fluor 488 goat anti-rabbit IgG | Thermo Fisher Scientific | A-11008 | |
| Antibiotic Antimycotic Solution | Corning | 30-004-CI | |
| Anti-Dystrophin antibody | Abcam | ab15277 | |
| Antigen retriever | Aptum Biologics | R2100-US | Antigen recovery |
| Autofluorescence Quenching Kit | Vector Laboratories | SP-8400 | |
| C2C12 cell line | ATCC | CRL-1772 | |
| Centrifuge | Unico | C8606 | |
| Change-A-Tip High Temp Cauteries | Bovie Medical Corporation | HIT | |
| Confocal microscopy | Zeiss | Zeiss 780 Upright Confocal | |
| DBA/2J-mdx mice | The Jackson Laboratory | 013141 | |
| DMEM | Corning | 10-013-CM | |
| Fetal Bovine Serum (FBS) | Corning | 35-011-CV | |
| Goat serum | MP Biomedicals, LLC | 191356 | |
| Isoflurane | Patterson Veterinary | 07-893-1389 | |
| Ketamine | Henry Schein | 056344 | |
| Mounting Medium with DAPI | Vector Laboratories | H-1500 | |
| Mouse Retractor Set | Kent Scientific | SURGI-5001 | |
| Polyethylene glycol tert-octylphenyl ether | Fisher Scientific | BP151-100 | |
| Rodent ventilator | Harvard Apparatus | 55-7066 | |
| SW-28 Ti rotor | Beckman | 342207 | |
| The Vevo 2100 Imaging Platform | FUJIFILM VisualSonics | Vevo 2100 | Ultrasound System |
| Ultracentrifuge | Beckman | 365672 | |
| Ultra-Clear Tubes | Beckman | 344058 | |
| Xylazine (XylaMed) | Bimeda-MTC Animal Health Inc. | 1XYL003 8XYL006 |
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Citar este artigo
Su, X., Shen, Y., Jin, Y., Jiang, M., Weintraub, N., Tang, Y. Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice. J. Vis. Exp. (146), e59320, doi:10.3791/59320 (2019).