利用人类纤维原细胞线粒体表型的监测领域的帕金森氏病
Summary
成纤维细胞在帕金森氏症的致病基因携带突变的患者代表一个方便<em>体外</em>模型来研究疾病相关的表型。活细胞成像提供了学习的机会，在活细胞的形态和功能参数。在这里，我们描述了准备的人成纤维细胞的粒线体的表型及随后的监测。
Abstract
帕金森氏病（PD）是第二个最常见的运动障碍，并影响到1％的人年龄超过60 1。由于老龄化是最重要的危险因素，PD的情况下，将增加在未来几十年里2。病态的蛋白质折叠和受损的蛋白质降解途径，线粒体功能和形态的改变，并指出了3-11 PD的神经退行性疾病中的又一标志。
经过多年的研究，在小鼠和人类肿瘤细胞的体外模型解剖帕金森病的分子途径，利用人成纤维细胞的体外模型从患者和适当的控制已成为一个有价值的研究工具，如果被认为是潜在的警告。除永生，而人工细胞模型，主要的成纤维细胞从患者进行疾病相关的基因突变显然反映了重要的病理特点of的人类疾病。
在这里，我们描绘出的皮肤活组织切片检查，培养人成纤维细胞和必要的显微技术，利用详细的协议定义线粒体的表型的过程。这些被用来研究不同的功能与PD相关的线粒体功能和动态。 体外 ，线粒体可以分析方面的功能，形态，共存与溶酶体（细胞器有辱人格的不正常的线粒体）和降解通过溶酶体途径。这些表型是高度相关的识别PD的早期迹象，并在人类疾病的基因携带者可能先于临床的运动症状。因此，这里提出的分析可以利用的宝贵工具，以确定病理特征的神经退行性疾病，并有助于确定新的治疗策略，PD。
Protocol
1。皮肤活检和培养的人类成纤维细胞皮肤活检，必须采取由有经验的医师。这个过程需要在无菌条件下，需要局部麻醉。用于活检的典型网站的内侧上臂，肩或腰部。 您可以采取的4x4mm或6x6mm的直径冲活检标本培养人成纤维细胞，以获得足够的组织。 切开皮肤活检分成相等的小块，在无菌条件下将它们区分成2-4 T25瓶中。每个烧瓶的表皮应该包含2-3个。培养基中含有15％FCS，1…
Discussion
病人的皮肤成纤维细胞体外模型的特点与疾病相关的遗传缺陷的一个重要工具。此外，皮肤成纤维细胞的方便和可扩展后的培养。因此，初级细胞从携带PD相关基因突变的患者得到的肿瘤细胞系的使用是优选的，因为它们包含不仅内源性致病基因，但整个受影响的个体的遗传背景。此外，已经示出的成纤维细胞，以反映在神经退行性疾病的重要病理特征的线粒体功能障碍。在本协议中所描?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是支持的补助弗里茨·蒂森基金会（10.11.2.153向RK），德国研究理事会（DFG，KR2119/3-2和KR2119/8-1至RK），联邦教育和研究部（BMBF NGFNplus; 01GS08134 RK）[LFB的慈善Hertie基金会的博士奖学金。我们感谢他们的支持在视频拍摄过程的Carolin奥伯迈尔和Julia Westermeier。
Materials
| Name of reagent | Company | Catalogue no. |
| Roswell Park Memorial Institute (RPMI) 1640 medium | Invitrogen | 52400-025 |
| RPMI 1640 medium, no Phenol Red | Invitrogen | 11835-063 |
| Dulbecco’s Phosphate-Buffered Saline (DPBS) | Invitrogen | 14190-094 |
| Fibroblast growth factor 2 (FGF2) | PeproTech | 100-18B |
| AccuMax (detachment solution) | PAA | L11-008 |
| Lab-TekTMII chambered coverglasses | Nalge Nunc International | 115382 |
| Tetramethylrodamine ethyl ester (TMRE) | Invitrogen | T-669 |
| Mitotracker Green FM | Invitrogen | M-7514 |
| Mitotracker CM-H2XRos | Invitrogen | M-7513 |
| Lyostracker Red DND-99 | Invitrogen | L-7528 |
| Hoechst 33342 | Invitrogen | H-3570 |
Table 1. Specific reagents and equipment.
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Cite This Article
Burbulla, L. F., Krüger, R. The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson’s Disease. J. Vis. Exp. (68), e4228, doi:10.3791/4228 (2012).