线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究
Summary
在这里, 我们提出了一个详细的协议, 应用罗丹明123识别线粒体膜电位 (基质) 和研究 CLIC4 击倒诱导 HN4 细胞凋亡的体外。在普通荧光显微镜和共聚焦激光扫描荧光显微镜下, 记录了基质金属蛋白酶的实时变化。
Abstract
线粒体膜电位的耗竭 (基质金属蛋白酶, ΔΨm) 被认为是凋亡级联的最早事件。它甚至发生在核凋亡特征之前, 包括染色质凝结和 DNA 断裂。一旦基质细胞崩溃, 癌细胞的凋亡就会不可逆转地启动。一系列的亲脂阳离子染料可以通过细胞膜和聚集在线粒体基质内, 作为荧光标记来评价基质金属蛋白酶的变化。CLIC4 作为 Cl 胞内通道 (点击)家族的六名成员之一, 主要通过线粒体通路参与细胞凋亡过程。在这里, 我们描述了一个详细的协议, 通过监测罗丹明 123 (Rh123) 的荧光涨落来测量基质, 通过它, 我们研究 CLIC4 的凋亡诱导。本文详细讨论了共焦激光扫描和正常荧光显微镜的优点和局限性, 并与其它方法进行了比较。
Introduction
Rh123 是一种阳离子荧光染料, 可作为跨膜电位的指标。Rh123 能够穿透细胞膜, 进入线粒体基质, 这取决于细胞膜内外电位的差异1。细胞凋亡导致线粒体膜完整性受损。线粒体通透性过渡孔隙 (MPTP) 将打开并导致基质细胞的崩溃, 从而使 Rh123 释放到线粒体的外部。最后, 在荧光显微镜下检测出更强的绿色荧光信号。有充分的证据表明, 细胞凋亡和高膜通透性的减少是2的早期迹象。因此, Rh123 可用于检测基质金属蛋白酶的变化和细胞凋亡的发生。
头颈癌作为世界上第六大常见的癌症, 严重恶化了一个人的健康3。虽然近年来发展了许多方法, 但治疗头颈部鳞状细胞癌 (HNSCC) 患者的临床疗效仍不理想4。探索新的治疗方法可以提高 HNSCC5的治疗效果。涉及许多生物过程的离子通道在不同的癌症的发展中显示了重要的作用6。氯离子通道的部分或全部参与高度参与了肿瘤转化的各种性质, 包括主动迁移、高增殖率和侵袭性。有鉴于此, 新的蛋白质家族点击已被列为癌症治疗6、7治疗靶点。最近的研究表明, 点击家族的成员, 包括 CLIC1, CLIC4 和 CLIC5, 定位到心脏线粒体和活性氧 (ROS) 水平上调的 CLIC5, 表明线粒体定位 Cl的功能作用-在凋亡反应中的通道8。CLIC4, 点击家族的一个成员 (也称为 mtCLIC, P64H1 和 RS43), 已被广泛研究, 其凋亡调节性质的癌细胞和亚细胞位置, 包括高尔基, 内质网和线粒体的人角质细胞7,9,10。CLIC4 的表达谱由肿瘤坏死因子α (TNF α)、P53 和外部刺激调节。CLIC4 的过度表达和下调引起的凋亡反应主要是通过线粒体通路伴随 Bcl-2 家族成员的不平衡, 活化 caspase 叶栅和释放细胞色素 C11,12,13. 因此, 基质金属蛋白酶的测量对探讨 CLIC4-related 细胞凋亡至关重要, Rh123 是理想的荧光指标。
本研究描述了一个详细的协议, 以检测基质干细胞研究 CLIC4 击倒诱导凋亡的 HN4 细胞。Rh123 用作荧光探针观察基质金属蛋白酶的变化。在普通荧光显微镜和共聚焦激光扫描荧光显微镜下, 可解决该基质金属蛋白酶的实时波动。本文详细讨论了共焦激光扫描荧光显微镜的优点和局限性, 并与其他方法进行了比较。该协议也可应用于其他与凋亡相关的研究。
Protocol
Representative Results
Discussion
有充分的文献表明, Cl−通道是必不可少的保持止血的内部环境, 并发挥重要作用, 细胞增殖和凋亡15,16。因此, 了解离子通道靶向干预与细胞凋亡之间的关系, 对于寻找一种更好的治疗方法对17种癌症有很大的必要性和意义。线粒体维持细胞正常的生物状态, 其功能与细胞膜通透性和跨膜电位密切相关。除了癌症, 积累病理调查记?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢赵先生的细胞培养。这项工作得到了中国自然科学基金的资助 (81570403、81371284号赠款);安徽省自然科学基金 (批准号: 1408085MH158);安徽医科大学优秀青年研究员;安徽省高校优秀青年人才支持方案。
Materials
| HNSCC cells | ATCC | CRL-3241 | |
| Polylysine | Thermo Fisher Scientific | P4981 | |
| Specific siRNA for human CLIC4 | Biomics | NM_013943 | (accession numbers, NM_013943; corresponding to the cDNA sequence 5-GCTGAAGGAGGAGGACAAAGA-3) and scrambled siRNA (5 ACGCGUAACGCGGGAAUUU-3) were designed and obtained from Biomics Company |
| Lipofectamine 2000 Transfection Reagent | Thermo Fisher Scientific | L3000-015 | |
| Opti-MEM I Reduced Serum Medium, GlutaMAX | Thermo Fisher Scientific | 51985-042 | |
| Rhodamine 123, FluoroPure grede | Thermo Fisher Scientific | R22420 | |
| Dulbecco’smodified Eagle medium (DMEM, 4.5 g/L glucose) | Gibco | 11965-084 | |
| Fetal Bovine Serum, Qualified, Australia Origin | Gibco | 10099141 | |
| Trypsin-EDTA Solution | Beyotime | C0201 | |
| Antibiotic-Antimycotic, 100X | Gibco | 15240062 | |
| Laser Scanning Confocal Microscopy | Leica Microsystems GmbH | LEICA.SP5-DMI6000-DIC | |
| Nikon Eclipse TE300 Inverted Microscope | Nikon | N/A | |
| Metaflour, V7.5.0.0 | Universal Imaging Corporation | N/A | |
| Leica application suite, v2.6.0.7266 | Leica Microsystems GmbH | N/A | |
| Microsoft office Excel 2007 | Microsoft | N/A | |
| Sigma Plot 12.5 | Systat Software | N/A | |
| Attofluor Cell Chamber | Thermo Fisher Scientific | A7816 |
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