Liskova Jana, Hadraba Daniel, Filova Elena, Konarik Miroslav, Pirk Jan, Jelen Karel, Bacakova Lucie
Institute of Physiology, the Czech Academy of Sciences, Prague, 142 20, Czech Republic.
Faculty of Physical Education and Sport, Charles University, Prague, 162 00, Czech Republic.
Microsc Res Tech. 2017 Aug;80(8):936-942. doi: 10.1002/jemt.22886. Epub 2017 Apr 28.
Collagen often acts as an extracellular and intracellular marker for in vitro experiments, and its quality defines tissue constructs. To validate collagen detection techniques, cardiac valve interstitial cells were isolated from pigs and cultured under two different conditions; with and without ascorbic acid. The culture with ascorbic acid reached higher cell growth and collagen deposition, although the expression levels of collagen gene stayed similar to the culture without ascorbic acid. The fluorescent microscopy was positive for collagen fibers in both the cultures. Visualization of only extracellular collagen returned a higher correlation coefficient when comparing the immunolabeling and second harmonic generation microscopy images in the culture with ascorbic acid. Lastly, it was proved that the hydroxyproline strongly contributes to the second-order susceptibility tensor of collagen molecules, and therefore the second harmonic generation signal is impaired in the culture without ascorbic acid.
胶原蛋白常作为体外实验的细胞外和细胞内标志物，其质量决定了组织构建物。为验证胶原蛋白检测技术，从猪身上分离出心脏瓣膜间质细胞，并在两种不同条件下培养：添加和不添加抗坏血酸。添加抗坏血酸的培养物细胞生长和胶原蛋白沉积更高，尽管胶原蛋白基因的表达水平与不添加抗坏血酸的培养物相似。两种培养物中胶原蛋白纤维的荧光显微镜检查均为阳性。在比较添加抗坏血酸的培养物中的免疫标记和二次谐波产生显微镜图像时，仅细胞外胶原蛋白的可视化返回了更高的相关系数。最后，证明了羟脯氨酸对胶原蛋白分子的二阶极化率张量有很大贡献，因此在不添加抗坏血酸的培养物中二次谐波产生信号受损。
