Shin HyeRim, Choi Jun-Hyuk, Lee Ji Youn
Biometrology Group, Division of Chemical and Biological Metrology, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea.
Biochem Biophys Rep. 2021 Sep 20;28:101137. doi: 10.1016/j.bbrep.2021.101137. eCollection 2021 Dec.
Cytoskeletal proteins are essential in maintaining cell morphology, proliferation, and viability as well as internalizing molecules in phagocytic and non-phagocytic cells. Orderly aligned cytoskeletons are disturbed by a range of biological processes, such as the epithelial-mesenchymal transition, which is observed in cancer metastasis. Although many biological methods have been developed to detect cytoskeletal rearrangement, simple and quantitative approaches are still in great demand. Herein, we applied a flow cytometry-based nanoparticle uptake assay to measure the degree of cytoskeletal rearrangement induced by transforming growth factor β1 (TGF-β1). For the assay, silica nanoparticles, selected for their high biocompatibility, were fluorescent-labeled to facilitate quantification with flow cytometry. Human keratinocyte HaCaT cells were treated with different concentrations of TGF-β1 and then exposed to FITC-labeled silica nanoparticles. Increasing concentrations of TGF-β1 induced gradual changes in cytoskeletal rearrangement, as confirmed by conventional assays. The level of nanoparticle uptake increased by TGF-β1 treatment in a dose-dependent manner, indicating that our nanoparticle uptake assay can be used as a quick and non-destructive approach to measure cytoskeletal rearrangement.
细胞骨架蛋白对于维持细胞形态、增殖和活力，以及在吞噬细胞和非吞噬细胞中内化分子至关重要。一系列生物学过程会扰乱有序排列的细胞骨架，比如在癌症转移过程中观察到的上皮-间质转化。尽管已经开发出许多生物学方法来检测细胞骨架重排，但仍迫切需要简单且定量的方法。在此，我们应用基于流式细胞术的纳米颗粒摄取试验来测量转化生长因子β1（TGF-β1）诱导的细胞骨架重排程度。在该试验中，选择具有高生物相容性的二氧化硅纳米颗粒进行荧光标记，以便通过流式细胞术进行定量分析。用人角质形成细胞HaCaT细胞分别用不同浓度的TGF-β1处理，然后使其暴露于异硫氰酸荧光素（FITC）标记的二氧化硅纳米颗粒。如传统试验所证实的，TGF-β1浓度的增加会诱导细胞骨架重排逐渐发生变化。TGF-β1处理使纳米颗粒摄取水平呈剂量依赖性增加，这表明我们的纳米颗粒摄取试验可作为一种快速且非破坏性的方法来测量细胞骨架重排。
