Visualization of apoptosis in three-dimensional cell aggregates based on molecular beacon imaging
Visualization of apoptosis in three-dimensional cell aggregates based on molecular beacon imaging
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基于分子信标成像的三维细胞聚集体凋亡可视化
DOI：
10.1089/ten.tec.2020.0338
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Tabata Yasuhiko
中科院分区：
文献类型：
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作者：
Murata Yuki;Jo Jun-ichiro;Tabata Yasuhiko
本研究的目的是可视化细胞凋亡的三维（3D）细胞聚集体的基础上分子信标（MB）。使用两种类型的信使RNA的MB：半胱天冬酶-3 MB作为细胞凋亡的靶标，甘油醛-3-磷酸脱氢酶（GAPDH）MB作为细胞中稳定荧光的对照。为了增强MB内化到细胞中，将胱天蛋白酶-3和GAPDH MB分别掺入阳离子化明胶纳米球（cGNS）中（cGNScasp 3 MB和cGNSGAP MB）。此外，制备共掺入半胱天冬酶-3和GAPDH MB的cGNS（cGNSDual MB），以对相同的细胞聚集体进行双色成像。将cGNSMB与小鼠骨髓间充质干细胞共孵育，在二维培养中用MB标记。通过cGNSdual MB以与单一MB相同的程度以比率计量法检测到通过添加死亡受体Fas的抗体介导的细胞凋亡。从MB标记的细胞制备细胞聚集体，并且即使在3D聚集体中也从几乎所有细胞检测到MB荧光，以显示均匀分布。除了Fas介导的细胞凋亡外，还用低分子量细胞凋亡诱导剂喜树碱处理聚集体。caspase-3 MB的荧光主要分布在Fas介导的凋亡聚集体的表面周围部位而不是中心部位，而GAPDH MB的荧光甚至在内部部位也被检测到。另一方面，在喜树碱诱导的凋亡聚集体中，从聚集体的内部位点以及周围位点检测到case 3和GAPDH MB荧光。MB荧光可能反映了由不同分子大小的凋亡诱导剂引起的凋亡位置的定位以及随后渗透到聚集体中。结论cGMSMB是一个很有前途的系统，在三维细胞聚集体中显示细胞凋亡，而不破坏聚集体。
The objective of this study is to visualize cell apoptosis in three-dimensional (3D) cell aggregates based on molecular beacons (MB). Two types of MB for messenger RNA were used: caspase-3 MB as a target for apoptosis and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MB as a control of stable fluorescence in cells. To enhance the MB internalization into cells, caspase-3 and GAPDH MB were incorporated in cationized gelatin nanospheres (cGNS), respectively (cGNScasp3 MBand cGNSGAP MB). In addition, cGNS co-incorporating caspase-3 and GAPDH MB (cGNSdual MB) were prepared to perform the dual-color imaging for the same cell aggregate. The cGNSMBwere incubated with mouse mesenchymal stem cells to label with MB in the two-dimensional culture. The cell apoptosis mediated by the addition of antibody for a death receptor Fas was ratiometrically detected by the cGNSdual MBto the same extent as single MB. The cell aggregates were prepared from MB-labeled cells, and the MB fluorescence was detected from almost all the cells even in the 3D aggregates to show the homogenous distribution. In addition to the Fas-mediated apoptosis, the aggregates were treated with camptothecin of a low-molecular weight apoptosis inducer. The fluorescence of caspase-3 MB was mainly distributed at the surface surrounding site of Fas-mediated apoptotic aggregates rather than the center site, while that of GAPDH MB was detected even in the interior site. On the other hand, in the camptothecin-induced apoptotic aggregates, both caspae-3 and GAPDH MB fluorescence were detected from the interior site of aggregates as well as the surrounding site. It is likely that the MB fluorescence reflected the localization of apoptotic position caused by the different molecular sizes of apoptosis inducer and the consequent penetration into the aggregates. It is concluded that the cGMSMBare a promising system to visualize cell apoptosis in 3D cell aggregates without the destruction of aggregates.