Gavin Herbert Eye Institute, University of California, Irvine, Irvine, California 92697, USA.
Invest Ophthalmol Vis Sci. 2012 Feb 16;53(2):770-8. doi: 10.1167/iovs.11-9092.
The purpose of this study was to determine whether myofibroblast differentiation altered keratocyte crystallin protein concentration and increased cellular light scattering.
Serum-free cultured rabbit corneal keratocytes and TGFβ (5 ng/mL) induced myofibroblasts were harvested and counted and protein/RNA extracted. Expression of myofibroblast and keratocyte markers was determined by real-time PCR and Western blot analysis. The cell volume of calcein AM-loaded keratocytes and myofibroblasts was determined by using nonlinear optical microscopy. Cellular light scattering of transformed myofibroblasts expressing human keratocyte crystallins was measured by reflectance confocal microscopy.
Differentiated myofibroblasts showed a significant decrease in RNA levels for the keratocyte markers ALDH1A1, lumican, and keratocan and a significant increase in the myofibroblast marker α-smooth muscle actin. Volumetric and protein measurements showed that myofibroblast differentiation significantly increased cytoplasmic volume (293%; P < 0.001) and water-soluble and -insoluble protein content per cell (respectively, 442% and 431%; P < 0.002) compared to keratocytes. Western blot analysis showed that the level of ALDH1A1 protein per cell was similar between myofibroblasts and keratocytes, but was substantially reduced as a percentage of total water-soluble protein. Light scattering measurements showed that induced expression of corneal crystallins significantly decreased light scattering.
These data suggest that myofibroblast differentiation leads to a marked increase in cell volume and dilution of corneal crystallins associated with an increase in cellular light scattering.
本研究旨在确定成肌纤维细胞分化是否会改变角膜基质细胞晶体蛋白浓度并增加细胞光散射。
从无血清培养的兔角膜基质细胞和 TGFβ（5ng/ml）诱导的成肌纤维细胞中收获和计数细胞，并提取蛋白质/RNA。通过实时 PCR 和 Western blot 分析确定成肌纤维细胞和角膜基质细胞标志物的表达。使用非线性光学显微镜测定 calcein AM 加载的角膜基质细胞和成肌纤维细胞的细胞体积。通过反射共焦显微镜测量表达人角膜基质细胞晶体蛋白的转化成肌纤维细胞的细胞光散射。
分化的成肌纤维细胞显示角膜基质细胞标志物 ALDH1A1、luman 和角膜蛋白的 RNA 水平显著降低，而肌成纤维细胞标志物α-平滑肌肌动蛋白的水平显著增加。体积和蛋白质测量表明，与角膜基质细胞相比，成肌纤维细胞分化显著增加了细胞质体积（293%；P<0.001）和细胞内水溶性和不溶性蛋白质含量（分别为 442%和 431%；P<0.002）。Western blot 分析显示，成肌纤维细胞和角膜基质细胞中每个细胞的 ALDH1A1 蛋白水平相似，但作为总水溶性蛋白的百分比显著降低。光散射测量表明，角膜晶体蛋白的诱导表达显著降低了光散射。
这些数据表明，成肌纤维细胞分化导致细胞体积显著增加，并与细胞光散射增加相关的角膜晶体蛋白稀释。
