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，1.1％，丙酮酸钠和1％青霉素/链霉素的在罗斯威尔公园Memorial研究所（RPMI）1640培养基中。 </li><li>孵育细胞在37℃和5％CO 2的 。直到第一次成纤维细胞生长的表皮样品的预期的传播时间是1-2周。如果经济增长遇到问题，你可以利用成纤维细胞生长发构造函数（5-10 ng / ml的FGF2），以提高细胞的生长。 </li><li>一旦细胞融合达到50％-70％，人类纤维原细胞可以冷冻。达到50-70％汇合时为止的时间，细胞系之间的不同。冻结在90％FCS中加10％的二甲亚砜（DMSO）中的成纤维细胞。 </li></ol> 2。活细胞显微成像技术的人成纤维细胞的制备在一般情况下，人成纤维细胞的实验应低于10通道与衰老相关的改变可以改变属性，多次传代后的细胞。在疑问的情况下，β-半乳糖苷酶的测定可以用来评估的老化过程，在各自的细胞诱导。一般情况下，不使用衰老成纤维细胞（见上文）。此外，用来比较的几个细胞系，每个细胞系具有相同的通路数工作。比较患者与健康对照组相比，不同的基因同质的建议。然而，有时患者进行特定的基因突变是罕见的，这可以说具有一定的通道数以及不同的控制线只有一个病人的皮肤活检必须包括 - 这是必要的，以确保相同的通道数包含所有行。如果一个细胞融合达到50％-70％，但没有实验计划，成纤维细胞被冻结。 <ol><li>在实验的第一天，洗一次附加的成纤维细胞，用PBS。然后松开他们的瓶底使用细胞分离蛋白水解DNA酶酶（ 即 ACCUMAX）的解决方案的。 </li><li>在4室盖玻片（1.8厘米2培养面积每孔）的每个孔中的细胞和种子50,000-70,000细胞计数。因此，一半的细胞数或少8腔coverglas，是适当的（0.8厘米2培养面积每孔）。这是重要的种子，而一个小的细胞数，以执行真正的单细胞分析。没有涂层的分庭，我小号需要。 </li><li> 24-48小时后，活细胞成像实验可以执行。确保活细胞成像显微镜配备的孵化器，其控制的温度和CO 2水平。在图像采集过程中，气氛中37℃和5％CO 2应该保持不变。 </li><li>每一个活细胞成像测量应进行至少在三个独立的实验。总体而言，最低的每50个细胞的细胞系进行成像。活细胞成像实验，以及离线分析下进行蒙蔽的条件，以保证没有偏见的数据采集。 </li></ol> 3。 “活的人类成纤维细胞线粒体功能的测量对于常规测量线粒体功能通过线粒体膜电位（MMP） - 依赖的染料，荧光激活细胞分选（FACS）是主要方法。在人成纤维细胞的情况下，蜂窝autofluore刘哲民，经常观察和，与实际染色信号的干扰可能会导致假阳性结果。自体荧光通常变得更加显着更高的通道数目的成纤维细胞。因此，，我们宁愿评估人成纤维细胞线粒体功能的活细胞显微成像技术。 <ol><li> 24-48小时后播种，培育成纤维细胞MMP-依赖的染料tetramethylrodamine乙基酯（TMRE），在RPMI培养基在50海里。这RPMI培养基中缺乏酚红，因为这会产生负面影响图像质量。 1.6μM的Hoechst染色应添加到可视化晶核。 </li><li>孵育细胞15分钟在37℃和5％CO 2的保护，光在制备的染色溶液。 </li><li>更换用RPMI培养基（无酚红）染色溶液。没有进行洗涤步骤，并立即图像单元格。使用63X的放大倍率和激光扫描共聚焦显微镜技术（或者荧光显微镜apotome技术）来定义的细胞的单层。适用于37℃和5％CO 2的细胞，在成像过程中的过程。 </li><li>当拍摄成纤维细胞，给予最大的关注荧光灯TMRE染色漂白剂快速的曝光时间。 200和300 ms之间定义适当的曝光时间，不超过这个时间间隔。在标准设置的图像采集是非常重要的，将无法进行比较的基础上强度，如果设置不同的细胞系之间的。 </li><li>作为定义的半径周围的成像单元被漂白，满分曝光后，保持充分远离光致漂白的部分的下一个视场。 </li><li>进行离线分析使用，ImageJ软件（韦恩·罗斯本;美国国立卫生研究院，美国）。 </li><li>使用选择工具，选择您感兴趣的细胞。 </li><li>将图像转换成8位（图像类型&lt;8位）。 </li><li>减少非特异性噪声BŸ使用“散斑”功能（过程噪声&lt;去斑）。 </li><li>选择“查找表 - 火”状态（图片&lt;查找表&lt;火）。 </li><li>在阈值函数切换到“上/下”功能和调节阈值（图像调整&lt;阈值）。 </li><li>选择“分析粒子”的选项，这将给你每个检测线粒体颗粒（分析分析颗粒）的平均灰度值。给定的结果列表保存为Excel电子表格中。 </li><li>在Excel程序打开结果文件，并计算线粒体结构的平均灰度值的平均值（表示为“平均”在给定的卓越结果列表）。 </li><li>要创建曲面图，选择“插件”交互式三维曲面图“中的”3D“插件部分。在这里，你可以进一步调整的情节使用不同的显示选项。改变原来的颜色“，”频谱LUT“提供适当的地积强度的比例尺。 </li><li>另外到使用TMRE用于分析的人成纤维细胞的线粒体功能通过活细胞成像，MMP独立染料Mitotracker有绿色FM组合，和MMP依赖性染料Mitotracker有可以使用CM-H 2 XRos的。同样地，人成纤维细胞的生活细胞成像显微镜是优选的，但在一般这种替代也被用来使用FACS分析单元12在其他种。当使用这种替代方法来衡量人的成纤维细胞，细胞线粒体功能的步骤3-7的协议第5条“测量”生活人类成纤维细胞溶酶体的共定位在mitochondrio应适用。舌头，信号的覆盖，无论是从线粒体积极FM和Mitotracker有CM-H 2 XRos Mitotracker有绿色计算和表示比所有线粒体现在的线粒体的功能。 </li></ol> 4。 “活的人类成纤维细胞的线粒体形态测量<ol><li> 24-48小时后播种，在200 nM的Mitotracker有绿色FM在RPMI培养基中（无酚红）孵育细胞避光15分钟，在37℃和5％CO 2的 。应该使用1.6μM的Hoechst染色以突出显示细胞核。 Mitotracker有绿色FM是一个独立于MMP-染料，污渍线粒体结构呈现。 </li><li>轻轻PBS洗细胞一次。 </li><li>加入新鲜RPMI培养基（无酚红）细胞，并立即图像细胞。使用63X的放大倍率和激光扫描共聚焦显微镜技术（或荧光显微镜技术的使用Apotome）定义单细胞层。 </li><li>离线分析取出由使用ImageJ的软件。 </li><li>使用选择工具，选择您感兴趣的细胞。 </li><li>将图像转换成8位（图像类型&lt;8位）。 </li><li>减少使用的“去散斑”功能（处理过程&lt;噪音&lt;去斑）的非特异的噪声。 </li><li>使用卷积滤镜突出线粒体drial结构（过程&lt;过滤器&lt;卷积）。 </li><li>调整的信号 - 噪声比是适当的阈值，使（图片&lt;调整&lt;阈值）。 </li><li>通过使用“分析颗粒”选项，启动线粒体的结构的自动识别的基础上选择的像素尺寸（分析&lt;分析颗粒）。随后，一些线粒体参数计算，如面积，周长，短轴和长轴或圆。这些测量结果可以单独设置（&lt;设定测量分析）。给定的结果列表保存为Excel电子表格中。 </li><li>在Excel程序中打开结果文件。随着使用这样的参数，可以进行分析线粒体形态。这包括线粒体网络分支[公式表示程度的形状系数的定义：（周长2）/（4 * PI（）*面积）]，以及纵横比线粒体（式定义的长度：主要轴/短轴）。 </li></ol> 5。活的人类成纤维细胞的Mitochondrio溶酶体的共定位测量<ol><li>播种后24-48小时，孵育细胞在200 nM的Mitotracker有绿色FM和100nM的Lyostracker红色DND-99在RPMI培养基（无酚红），15分钟，在37℃和5％CO 2的保护，光。 1.6微米的Hoechst染色是用来突出核。 </li><li>更换新鲜的RMPI介质（无酚红）含100纳米Lyostracker红DND-99培养基中。这种染料必须在整个成像会议。不要洗细胞后，潜伏期。立即图像的细胞。使用63X的放大倍率和共聚焦技术（或者Apotome技术），以确定单层细胞。 </li><li>离线分析取出由使用ImageJ的软件。 </li><li>打开相应的图像，你想分析的共存状态。 </li><li>将图像转换成8位（图像类型&lt;8位）。 </lI&gt; <li>减少非特异的噪声，通过使用“去散斑”功能在两个图像（处理过程&lt;噪声&lt;去斑）。 </li><li>选择：共存分析工具插件“JACoP”，它给你的各通道的值，并计算Pearson的系数，重叠系数大以及“曼德斯系数（插件&lt;JACoP）。 </li></ol> 6。代表性的成果在活的人体成纤维细胞线粒体功能可以通过活细胞成像技术评估。功能分析，TMRE荧光信号与线粒体膜电位（MMP）。正常MMP条件下，TMRE荧光显着更高（ 图1A，上排）比其特征在于通过降低基质金属蛋白酶（ 图1A，下排）的病理条件下。此荧光强度测定通过计算每线粒体结构的平均灰度值的平均的（ <stronG&gt;图1B）。可测得的荧光强度，通过ImageJ软件。在这里，使用的选项“交互式表面情节”允许不同的峰的强度（ 图1A中 ，右侧）的图示。 人成纤维细胞线粒体功能的评估，活细胞显微成像技术可用于定义不同的参数线粒体形态。通过使用Mitotracker有绿色FM染料线粒体结构可视化独立的各自的基质金属蛋白酶（ 图2）。这是很重要的，以确保纳入到分析的所有线粒体结构。使用ImageJ软件，该形态的二进制数字的基础上进行分析，因为只有在这里线粒体形态学参数可以评估（ 图2，“二进制”）。线粒体形态分析中的形式因素，以及纵横比的例子是最近公布的6，13。另一项重要的功能，可以测量活的人体成纤维细胞线粒体结构的退化。在这一进展的第一步，可评估的mitochondrio溶酶体的共定位在活细胞显微成像技术。某些病理状态下导致线粒体与溶酶体（ 图3A）中的增加的共定位。同样，Mitotracker有绿色FM染色线粒体结构，独立于它们各自的MMP。此外，与DND-99 Lysotracker红，染色，可以可视化的溶酶体结构。这是至关重要的，本Lysotracker红染料是在成像过程中，冲洗步骤减少的信号，用于可视化变得过低。黄恩启表示由于重叠的绿色（Mitotracker格林FM）和红色（Lysotracker红DND-99）染色（白色箭头， 图3A）的澄清的黄色的荧光信号，并确定所计算的Pearson安科fficient（ 图3B）。 在一般情况下，用ImageJ软件离线分析，它是有帮助的，每一个细胞图像可视化，可避免使一个特定的感兴趣区域（ROI）的定义。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/4228/4228fig1highres.jpg" src="/files/ftp_upload/4228/4228fig1.jpg" /> 图1。TMRE荧光信号，表明活细胞成像技术在活的人体成纤维细胞的线粒体膜电位（MMP）的测量。 （A）TMRE是一个MMP-依赖的荧光染料，其荧光强度与基质金属蛋白酶。 Hoechst染料被用于染色细胞核（蓝色）。 TMRE荧光正常MMP条件下（上排），显着高于其特征在于通过降低基质金属蛋白酶（下排）的病理条件下。被附加示出荧光强度强度曲面图。比例尺表示10微米。 （B）（A）中所示的示例性成纤维细胞的统计分析。在病理情况下，TMRE荧光的线粒体下降到正常MMP条件的测量相比， <a href="http://www.jove.com/files/ftp_upload/4228/4228fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/4228/4228fig2.jpg" /> 图2。活细胞成像技术在活的人体成纤维细胞的线粒体形态的测量。 mitotracker格林FM（绿色），用于可视化线粒体的结构，它是独立的MMP。 Hoechst染料被用于染色细胞核（蓝色）。使用ImageJ的软件，可以分析的形态上的二进制数字。比例尺表示10微米。<html>这里查看大图。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/4228/4228fig3highres.jpg" src="/files/ftp_upload/4228/4228fig3.jpg" /> 图3。测量的线粒体，溶酶体中活的人体成纤维细胞，活细胞成像技术的共存。 （A）Mitotracker格林FM（绿色）被用于染色线粒体的结构，Lyostracker红色DND-99（红色）可视化溶酶体结构。 Hoechst染料被用于染色细胞核（蓝色）。成功共存病理条件下，由于，重叠的Lysotracker红色和Mitotracker有绿染色（白色箭头）黄色信号表示。比例尺表示10微米。 （B）（A）中所示的示例性成纤维细胞的统计分析。程度较高，mitochondrio溶酶体的共定位结果在一个较高的Pearson相关系数的值。/ 4228/4228fig3large.jpg“目标=”_blank“&gt;点击这里查看大图。

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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>

没有利益冲突的声明。

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&#039;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 ...

the-use-primary-human-fibroblasts-for-monitoring-mitochondrial

Research

JoVE Journal

Medicine

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

16.7K 次观看.  DZNE. 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体外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。

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

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

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

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

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

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

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

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 imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson&#039;s Disease

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

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

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

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

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

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

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

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

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson&#039;s Disease

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

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

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Controlling Parkinson&#039;s Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of 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

Complementary structural and functional neuroimaging techniques used to examine the Default Mode Network (DMN) could potentially improve assessments of psychiatric illness severity and provide added validity to the clinical diagnostic process. Recent neuroimaging research suggests that DMN processes may be disrupted in a number of stress-related psychiatric illnesses, such as posttraumatic stress disorder (PTSD).
Although specific DMN functions remain under investigation, it is generally thought to be involved in introspection and self-processing. In healthy individuals it exhibits greatest activity during periods of rest, with less activity, observed as deactivation, during cognitive tasks, e.g., working memory. This network consists of the medial prefrontal cortex, posterior cingulate cortex/precuneus, lateral parietal cortices and medial temporal regions.
Multiple functional and structural imaging approaches have been developed to study the DMN. These have unprecedented potential to further the understanding of the function and dysfunction of this network. Functional approaches, such as the evaluation of resting state connectivity and task-induced deactivation, have excellent potential to identify targeted neurocognitive and neuroaffective (functional) diagnostic markers and may indicate illness severity and prognosis with increased accuracy or specificity. Structural approaches, such as evaluation of morphometry and connectivity, may provide unique markers of etiology and long-term outcomes. Combined, functional and structural methods provide strong multimodal, complementary and synergistic approaches to develop valid DMN-based imaging phenotypes in stress-related psychiatric conditions. This protocol aims to integrate these methods to investigate DMN structure and function in PTSD, relating findings to illness severity and relevant clinical factors.

This protocol describes the complementary neuroimaging techniques of resting state structural connectivity, task-induced deactivation, and structural connectivity analyses to examine the default network in post-traumatic stress disorder. The use of synergistic methods could potentially lead to improved diagnostics and assessments of severity, outcome, and other relevant clinical factors.

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

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

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

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

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 ...