Name: the use of primary human fibroblasts for monitoring mitochondrial phenotypes in the field of parkinson's disease
Uploaded: 2012-10-03T13:00:00
Description: Watch this Scientific Journal Video about The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease at JoVE.com

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。

1。皮肤活检和培养的人类成纤维细胞<ol><li>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。 </li><li>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。 </li><li>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...

<ol>
	<li>de Rijk, M. C., Launer, L. J., Berger, K., Breteler, M. M., Dartigues, J. F., Baldereschi, M., Fratiglioni, L., Lobo, A., Martinez-Lage, J., Trenkwalder, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+Parkinson's+disease+in+Europe:+A+collaborative+study+of+population-based+cohorts.">Prevalence of Parkinson's disease in Europe: A collaborative study of population-based cohorts.</a> Neurologic Diseases in the Elderly Research Group. Neurology. 54, 21-23 (2000).</li><li>Dorsey, E. R., Constantinescu, R., Thompson, J. P., Biglan, K. M., Holloway, R. G., Kieburtz, K., Marshall, F. J., Ravina, B. M., Schifitto, G., Siderowf, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projected+number+of+people+with+Parkinson+disease+in+the+most+populous+nations.">Projected number of people with Parkinson disease in the most populous nations.</a> Neurology. 68, 384-386 (2005).</li><li>Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R., Goedert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+in+Lewy+bodies.">Alpha-synuclein in Lewy bodies.</a> Nature. 388, 839-840 (1997).</li><li>Chung, K. K., Zhang, Y., Lim, K. L., Tanaka, Y., Huang, H., Gao, J., Ross, C. A., Dawson, V. L., Dawson, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin+ubiquitinates+the+alpha-synuclein-interacting+protein,+synphilin-1:+implications+for+Lewy-body+formation+in+Parkinson+disease.">Parkin ubiquitinates the alpha-synuclein-interacting protein, synphilin-1: implications for Lewy-body formation in Parkinson disease.</a> Nature. 7, 1144-1150 (2001).</li><li>Kruger, R., Eberhardt, O., Riess, O., Schulz, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease:+one+biochemical+pathway+to+fit+all+genes.">Parkinson's disease: one biochemical pathway to fit all genes.</a> Trends Mol. Med. 8, 236-240 (2002).</li><li>Krebiehl, G., Ruckerbauer, S., Burbulla, L. F., Kieper, N., Maurer, B., Waak, J., Wolburg, H., Gizatullina, Z., Gellerich, F. N., Woitalla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+basal+autophagy+and+impaired+mitochondrial+dynamics+due+to+loss+of+Parkinson's+disease-associated+protein+DJ-1.">Reduced basal autophagy and impaired mitochondrial dynamics due to loss of Parkinson's disease-associated protein DJ-1.</a> PLoS One. 5, e9367 (2010).</li><li>Exner, N., Treske, B., Paquet, D., Holmstrom, K., Schiesling, C., Gispert, S., Carballo-Carbajal, I., Berg, D., Hoepken, H. H., Gasser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss-of-function+of+human+PINK1+results+in+mitochondrial+pathology+and+can+be+rescued+by+parkin.">Loss-of-function of human PINK1 results in mitochondrial pathology and can be rescued by parkin.</a> J. Neurosci. 27, 12413-12418 (2007).</li><li>Burbulla, L. F., Krebiehl, G., Kruger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balance+is+the+challenge--the+impact+of+mitochondrial+dynamics+in+Parkinson's+disease.">Balance is the challenge--the impact of mitochondrial dynamics in Parkinson's disease.</a> European journal of clinical investigation. 40, 1048-1060 (2010).</li><li>Strauss, K. M., Martins, L. M., Plun-Favreau, H., Marx, F. P., Kautzmann, S., Berg, D., Gasser, T., Wszolek, Z., Muller, T., Bornemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+function+mutations+in+the+gene+encoding+Omi/HtrA2+in+Parkinson's+disease.">Loss of function mutations in the gene encoding Omi/HtrA2 in Parkinson's disease.</a> Human molecular genetics. 14, 2099-2111 (2005).</li><li>Narendra, D., Tanaka, A., Suen, D. F., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin+is+recruited+selectively+to+impaired+mitochondria+and+promotes+their+autophagy.">Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.</a> The Journal of cell biology. 183, 795-803 (2008).</li><li>Dagda, R. K., Cherra, S. J., Kulich, S. M., Tandon, A., Park, D., Chu, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+PINK1+function+promotes+mitophagy+through+effects+on+oxidative+stress+and+mitochondrial+fission.">Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission.</a> The Journal of biological chemistry. , 284-13843 (2009).</li><li>Kieper, N., Holmstrom, K. M., Ciceri, D., Fiesel, F. C., Wolburg, H., Ziviani, E., Whitworth, A. J., Martins, L. M., Kahle, P. J., Kruger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+mitochondrial+function+and+morphology+by+interaction+of+Omi/HtrA2+with+the+mitochondrial+fusion+factor+OPA1.">Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1.</a> Experimental cell research. 316, 1213-1224 (2010).</li><li>Burbulla, L. F., Schelling, C., Kato, H., Rapaport, D., Woitalla, D., Schiesling, C., Schulte, C., Sharma, M., Illig, T., Bauer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+role+of+the+mitochondrial+chaperone+mortalin+in+Parkinson's+disease:+functional+impact+of+disease-related+variants+on+mitochondrial+homeostasis.">Dissecting the role of the mitochondrial chaperone mortalin in Parkinson's disease: functional impact of disease-related variants on mitochondrial homeostasis.</a> Human molecular genetics. 19, 4437-4452 (2010).</li><li>Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Nguyen, H. N., Byers, B., Cord, B., Shcheglovitov, A., Byrne, J., Gujar, P., Kee, K., Schule, B., Dolmetsch, R. E., Langston, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+mutant+iPSC-derived+DA+neurons+demonstrate+increased+susceptibility+to+oxidative+stress.">LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress.</a> Cell Stem Cell. 8, 267-280 (2011).</li><li>Seibler, P., Graziotto, J., Jeong, H., Simunovic, F., Klein, C., Krainc, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Parkin+recruitment+is+impaired+in+neurons+derived+from+mutant+PINK1+induced+pluripotent+stem+cells.">Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced pluripotent stem cells.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 31, 5970-5976 (2011).</li></ol>

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?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录没有。 </td></tr><tr><td>罗斯韦尔公园纪念研究所1640培养基（RPMI） </td><td> Invitrogen公司</td><td> 52400-025 </td></tr><tr><td> RPMI 1640培养基，无酚红</td><td> Invitrogen公司</td><td> 11835-063 </td></tr><tr><td> Dulbecco氏磷酸盐缓冲盐水（DPBS） </td><td> Invitrogen公司</td><td> 14190-094 </td></tr><tr><td>碱性成纤维细胞生长因子2（FGF2） </td><td> PeproTech公司</td><td> 100-18B </td></tr><tr><td> ACCUMAX（支队的解决方案） </td><td> PAA </td><td> L11-008 </td></tr><tr><td>的实验室，TekTMII腔coverglasses </td><td> Nalge Nunc公司国际</td><td> 115382 </td></tr><tr><td> tetramethylrodamine乙酯（TMRE） </td><td> Invitrogen公司</td><td> T-669 </td></tr><tr><td> MitotraCKER绿色FM </td><td> Invitrogen公司</td><td> M-7514 </td></tr><tr><td> Mitotracker有CM-H2XRos </td><td> Invitrogen公司</td><td> M-7513 </td></tr><tr><td> DND-99 Lyostracker红， </td><td> Invitrogen公司</td><td> L-7528 </td></tr><tr><td>赫斯特33342 </td><td> Invitrogen公司</td><td> H-3570 </td></tr></tbody></table> 表1中。具体的试剂和设备。

the use of primary human fibroblasts for monitoring mitochondrial phenotypes in the field of parkinson's disease

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。

Watch this Scientific Journal Video about The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease at JoVE.com

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

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&gt;体外</em&gt;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。

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

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Research

JoVE Journal

Medicine

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

科研

医学

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

1单侧损毁6  - 羟基多巴胺帕金森病小鼠模型的发展

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI成像帕金森病的神经肽质谱

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

推导人类纤维原细胞的皮肤穿刺活检植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

在正常人脑分离的可溶性和不溶性朊蛋白低聚物

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

原位主动脉瓣移植在小鼠血管疾病的研究

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

基因-环境相互作用模型揭开帕金森病的隐形易感机制

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

控制帕金森氏症与自适应深部脑刺激

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Developing Neuroimaging Phenotypes of the Default Mode Network in PTSD: Integrating the Resting State, Working Memory, and Structural Connectivity

发展中的默认模式网络的神经影像学表型在创伤后应激障碍：整合静息状态下，工作记忆和结构连接

Complementary structural and functional neuroimaging techniques used to examine the Default Mode Network (DMN) could potentially improve assessments of ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...