用荧光线粒体分离条件永生化的小鼠肾小球内皮细胞
Summary
本文描述了从表达热不稳定猿病毒40和光激活线粒体PhAM切除的转基因小鼠的肾脏中分离条件永生化肾小球内皮细胞的方法。我们描述了使用磁珠、消化步骤、播种和培养 GEC-CD31 阳性从整个肾脏中分离肾小球的程序。
Abstract
肾小球内皮细胞（GEC）功能障碍可引发并导致肾小球滤过屏障破坏。线粒体氧化应激增加被认为是导致某些肾小球疾病发病机制中GEC功能障碍的机制。从历史上看，由于难以从肾小球中分离纯培养物，因此从 体内 模型中分离GECs一直具有挑战性。GEC在 体外 具有复杂的生长要求，并且寿命非常有限。在这里，我们描述了用荧光线粒体分离和培养有条件永生化GEC的程序，从而能够跟踪线粒体裂变和融合事件。GECs从表达不稳定性的SV40 TAg（来自Immortomouse）的双转基因小鼠的肾脏中分离，有条件地促进增殖和抑制细胞分化，以及所有线粒体中的光转换荧光蛋白（Dendra2）（来自光激活线粒体[PhAM切除]小鼠）。产生的稳定细胞系允许在永生化SV40 TAg基因失活和线粒体亚群的光活化后进行细胞分化，从而导致荧光从绿色切换到红色。使用mitoDendra2-GEC可以对荧光线粒体的分布，融合和裂变事件进行实时成像，而不会对细胞进行染色。
Introduction
肾小球通过限制大分子通过肾小球滤过屏障而对血液滤过至关重要1，2。肾小球包含四种细胞类型：壁上皮细胞、足细胞（内脏上皮细胞）、肾小球内皮细胞 （GEC） 和系膜细胞3。肾小球内皮的特征在于独特的血管结构，根据大过滤量所需的Fenestrae的存在4。肾小球内皮的顶端表面覆盖着带负电荷的糖萼层和称为内皮表层的涂层，该涂层在内皮和血液之间形成空间。这种结构提供了高电荷选择性，限制了带负电荷的分子（如白蛋白）的通过，并防止了白细胞和血小板粘附5。
GEC对代谢变化非常敏感，例如与糖尿病环境相关的高血糖症。事实上，糖尿病会导致有害物质循环增加，葡萄糖代谢途径饱和，并扰乱细胞氧化还原平衡3，6。此外，活性氧的增加诱发线粒体功能障碍，从而影响内皮功能7。
当前方案的总体目标是分离具有荧光线粒体特征的永生化肾小球内皮细胞。事实上，原代GEC的细胞培养具有有限的增殖周期和早期衰老8。此外，荧光线粒体的存在有助于检查高血糖或任何其他治疗的裂变和融合事件。作为一种替代方法，其他实验室使用h-TERT在体外使细胞永生化9。
这里描述的方法允许从4-6周龄的动物中分离有条件永生化的mitoDendra2肾小球内皮细胞（图1）。该详细协议描述了使用携带猿病毒40大肿瘤抗原（SV40 TAg）基因10，11的转基因小鼠（H-2K b-tsA58）来产生热不稳定的条件永生化细胞。tsA58 TAg基因产物在小鼠H-2Kb基因的诱导5’侧翼启动子的控制下在33°C的允许温度下起作用，该启动子在暴露于干扰素γ（IFNγ）时升高到基础水平以上，因此维持条件增殖表型12。在没有IFNγ的情况下，H-2Kb在37°C的非允许温度下迅速降解，消除了细胞中tsA58Tag的永生化功能，并允许细胞发展出更分化的表型13，14，15。H-2Kb-tsA58转基因小鼠与PhAM小鼠的选择性杂交，PhAM小鼠表达线粒体特异性（细胞色素c氧化酶亚基VIII）Dendra2-green，允许活检测荧光线粒体16。Dendra2 绿色荧光在暴露于 405 nm 激光16 后切换到红色荧光。当线粒体在光开关后融合时，它们形成细长的形状，由于绿色和黄色物质的交换而呈现黄色，或者在发生裂变时呈现红色7，17。mitoDendra2-GEC是研究GEC线粒体对不同刺激的细胞反应的好工具。
Protocol
Representative Results
Discussion
线粒体对细胞代谢、稳态和应激反应至关重要，其功能障碍与许多疾病有关，包括肾脏疾病。线粒体在过量活性氧（ROS）的病理生成，细胞内钙水平，细胞死亡途径和细胞骨架动力学的调节中起作用21，22，23。
小鼠GEC的分离具有挑战性，因为它们的数量，大小，基质因子很少，并且与其他肾小球细胞的相?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
作者感谢何慈江教授和傅佳博士在小鼠内皮细胞分离方面的见解，感谢Mone Zaidi教授为PhAM切除 小鼠提供有价值的讨论。作者还要感谢西奈山伊坎医学院的显微镜CORE和工作人员为我们提供的指导。这项工作得到了美国国立卫生研究院拨款R01DK097253和国防部CDMRP拨款E01 W81XWH2010836对I.S.D.的支持。
Materials
| 100 µm cell strainer | Fisher | 22-363-549 | |
| 1ml Insulin Syringes | BD | 329424 | |
| 25G butterfly | BD | 367298 | |
| 3 mm cutting edge scissors | F.S.T | 15000-00 | |
| 30ml syringe | BD Biooscience | 309650 | |
| 40 µm cell strainer | Fisher | 22-363-547 | |
| 40 µm nylon mesh | |||
| Bonn Scissors | F.S.T | 14184-09 | |
| Bovine serum albumin | Fisher | BP1600-100 | |
| CD31 | abcam | ab7388 | |
| Collagenase type I | Corning | 354236 | |
| Collagenase type II | SIGMA | C6885 | 125CDU/mg |
| Collagene type IV | SIGMA | C5533-5M | |
| Dnase-I | Qiagen | 79254 | |
| Dynabeads 450 | Thermofisher Scientific | 14013 | |
| endothelial cells growth medium | Lonza | cc-3156 | |
| Extra fine graefe forceps | F.S.T | 11150-10 | |
| FBS | Gemini | 100-106 | Heat inactivated |
| Fibronectin | Thermofisher | 33016015 | |
| Fine forceps | F.S.T Dumont | E6511 | |
| HBSS | GIBCO | 14065-056 | |
| IFNg | Cell Science | CRI001B | |
| Immortomouse | Jackson laboratory | 32619 | Tg(H2-K1-tsA58)6Kio/LicrmJ |
| L-Glutamine 100x | Thermofisher Scientific | 25030081 | |
| Magnetic particle concentrator | Thermofisher Scientific | 12320D | |
| mitotracker | Thermofisher Scientific | M7512 | |
| PBS 1X | Corning | 46-013-CM | |
| penecillin streptomycin 100x | Thermofisher Scientific | 10378016 | |
| PhaM mice | Jackson laboratory | 18397 | B6;129S-Gt(ROSA)26Sortm1.1(CAG-COX8A/Dendra2)Dcc/J |
| Protease (10 mg/ml) | SIGMA | P6911 | |
| RPMI | GIBCO | 3945 | |
| Sodium Pyruvate 100mM | Thermofisher Scientific | 11360070 | |
| Standard pattern forceps | F.S.T | 11000-12 | |
| Surgical Scissors – Sharp-Blunt | F.S.T | 14008-14 | |
| synaptopodin | Santa Cruz | sc-515842 | |
| Trypsin 0.05% | Thermofisher Scientific | 25300054 |
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