Osegui-Barcenilla Nadia, Sendino Maria, Martín-González Sergio, González-Moro Itziar, Benito-Agustino Ainhoa, Torres-Conde Noemi, López-Martínez Andrea, Jiménez-Mallebrera Cecilia, López-Márquez Arístides, Arechavala-Gomeza Virginia
Nucleic Acid Therapeutics for Rare Disorders (NAT-RD), Biobizkaia Health Research Institute, Barakaldo, Spain.
Laboratory of Applied Research in Neuromuscular Diseases, Neuromuscular Pathology Unit, Neuropediatric Service, Institut de Recerca Sant Joan de Déu, Esplugues de Llobregat, Spain.
Neuropathol Appl Neurobiol. 2025 Jun;51(3):e70020. doi: 10.1111/nan.70020.
This study aims to develop a quantitative method for assessing collagen VI expression in cell cultures, which is crucial for the diagnosis and treatment of collagen VI-related dystrophies.
We developed a combined in-cell western (ICW) and on-cell western (OCW) assay, which we have called 'collablot', to quantify collagen VI and its organisation in the extracellular matrix of cell cultures from patients and healthy controls. To optimise it, we optimised cell density and the protocols to induce collagen expression in cultures, as well as the cell fixation and permeabilisation methods. This was completed with a thorough selection of collagen antibodies and a collagen-hybridising peptide (CHP). We then used collablots to compare cultures from patients and controls and evaluate therapeutic interventions in the cultures.
Collablots enabled the quantification of collagen VI expression in both control and patient cells, aligning with immunocytochemistry findings and detecting variations in collagen VI expression following treatment of the cultures. Additionally, CHP analysis revealed a marked increase in collagen network disruption in patients compared to the controls.
The collablot assay represents a suitable method for quantifying collagen VI expression and its organisation in culture and assessing the effect of therapies.
本研究旨在开发一种定量方法，用于评估细胞培养物中胶原蛋白VI的表达，这对于胶原蛋白VI相关营养不良症的诊断和治疗至关重要。
我们开发了一种结合细胞内western（ICW）和细胞上western（OCW）的检测方法，我们称之为“胶原印迹法”，以量化患者和健康对照者细胞培养物细胞外基质中胶原蛋白VI及其组织结构。为了对其进行优化，我们优化了细胞密度、诱导培养物中胶原蛋白表达的方案，以及细胞固定和通透化方法。这一过程还包括对胶原蛋白抗体和胶原蛋白杂交肽（CHP）的全面筛选。然后，我们使用胶原印迹法比较患者和对照的培养物，并评估培养物中的治疗干预效果。
胶原印迹法能够对对照细胞和患者细胞中的胶原蛋白VI表达进行定量，与免疫细胞化学结果一致，并能检测培养物处理后胶原蛋白VI表达的变化。此外，CHP分析显示，与对照组相比，患者的胶原网络破坏明显增加。
胶原印迹检测法是一种适用于量化培养物中胶原蛋白VI表达及其组织结构并评估治疗效果的方法。
