Proteomic analysis and bioluminescent reporter gene assays to investigate effects of simulated microgravity on Caco-2 cells
Proteomic analysis and bioluminescent reporter gene assays to investigate effects of simulated microgravity on Caco-2 cells
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DOI：
10.1002/pmic.201700081
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发表时间：
2017-08-01
期刊：
影响因子：
3.4
通讯作者：
Lagana, Aldo
中科院分区：
文献类型：
--
作者：
La Barbera, Giorgia;Capriotti, Anna Laura;Lagana, Aldo
微重力是空间飞行中最重要的特征之一。以前的体外研究证据表明，在模拟微重力下会发生重大变化。为此，选择人结肠腺癌Caco-2细胞作为肠上皮屏障的细胞模型，并研究它们对改变的重力条件的响应，特别是在蛋白质水平上。在这项研究中，我们结合无标记鸟枪蛋白质组学和生物发光报告基因检测，以确定在参考和微重力条件下的Caco-2细胞的反应中涉及的关键蛋白质和途径。用3D打印的适配器修改了二维回转器，以容纳传统的T25培养瓶。比较蛋白质组分析导致识别38和26蛋白质不同的调节模拟微重力后48和72小时，分别。这些蛋白质的大部分参与调节、细胞和代谢过程以及定位。采用生物发光报告基因检测法研究了微囊化诱导的NF-κ B通路和CYP 27 A1等关键靶点转录调控的改变。虽然在基础转录中没有发现显著差异，但据报告，模拟微重力条件下NF-κ B基础活化较低，证实了微重力条件下免疫力降低的假设。
Microgravity is one of the most important features in spaceflight. Previous evidence from invitro studies has shown that significant changes occur under simulated microgravity. For this reason, human colon adenocarcinoma Caco-2 cells were selected as cell model of intestinal epithelial barrier and their response to altered gravity conditions was investigated, especially on the protein level. In this study, we combined label-free shotgun proteomics and bioluminescent reporter gene assays to identify key proteins and pathways involved in the response of Caco-2 cells under reference and microgravity conditions. A two-dimensional clinostat was modified with 3D-printed adaptors to hold conventional T25 culture flasks. The comparative proteome analysis led to identify 38 and 26 proteins differently regulated by simulated microgravity after 48 and 72 h, respectively. Substantial fractions of these proteins are involved in regulation, cellular and metabolic processes and localization. Bioluminescent reporter gene assays were carried out to investigate microgavity-induced alterations on the transcriptional regulation of key targets, such as NF-kB pathway and CYP27A1. While no significant difference was found in the basal transcription, a lower NF-kB basal activation in simulated microgravity conditions was reported, corroborating the hypothesis of reduced immunity in microgravity conditions.