摘要背景 角膜细胞的形态学参数及其密度对角膜结构的完整性及其正常功能的维持具有重要意义.目前,BALB/c小鼠已成为角膜研究中广泛应用的实验动物,了解正常角膜细胞的大小、形态及密度可为相应的实验研究提供参考依据. 目的 应用双光子激光扫描显微镜对BALB/c小鼠的不同角膜细胞进行活体扫描,对细胞密度、细胞大小参数进行活体三维测量,活体观察角膜不同细胞的形态学特征.方法 收集6只8 ～13周龄BALB/c小鼠,麻醉后荧光解剖显微镜下于角膜基质内注射20 μg/ml的胞质膜染料1μl,注射后1.5h于注射部位的对侧注射500 μg/ml Hoechst 33342 1 μl,30 min后使用双光子激光扫描荧光显微镜对角膜中央视野(XY轴)的角膜内皮层、基质层和上皮层进行Z轴逐层扫描,每层扫描厚度为2μm,图像像素为512×512,图像位数为12 bit.应用Imaris软件对扫描的图像进行细胞密度以及细胞大小参数的测量,细胞密度通过计算3维空间内细胞核数目获得;通过软件自动测量角膜上皮基底层、中基质层和内皮层细胞和细胞核的表面积和体积,并计算细胞和细胞核的表面积/体积值和细胞的核质比. 结果 BALB/c小鼠角膜上皮基底层和内皮层均为单层细胞,而中基质层包括2层细胞.小鼠角膜上皮基底层细胞密度为(108.00±18.97) ×104/mm3,明显大于中基质层的(9.27±0.48)×104/mm3和内皮层的(22.30±2.28)×104/mm3,3个组间总体比较差异有统计学意义(F=141.592,P=0.000);以中基质层的细胞表面积和体积最大,其次为内皮层,上皮基底层的细胞表面积和体积最小,3层细胞间的表面积和体积的总体比较差异均有统计学意义(F=2 222.000、598.504,均P=0.000).小鼠角膜上皮基底层、中基质层和内皮层细胞的表面积/体积值分别为(0.80±0.13)、(0.51±0.07)和(0.40±0.04)/μm,以上皮基底层细胞最大,3层间总体比较差异有统计学意义(F=127.075,P=0.000).小鼠角膜以内皮层的细胞核表面积和体积最大,其次为中基质层,以上皮基底层细胞的细胞核表面积和体积最小,但以上皮基底层细胞核的表面积/体积值最大,以内皮层细胞核的表面积/体积值最小;小鼠角膜内皮层细胞的核质比明显大于上皮基底层和中基质层细胞. 结论 双光子激光扫描显微镜可对BALB/c小鼠中央角膜3层细胞参数,包括细胞和细胞核表面积及体积进行活体定量测定,为BALB/c小鼠角膜生理、病理和疾病的相关研究提供参考依据.

abstractsBackground Corneal cell size and density are among the primary parameters that determine the structural integrity and normal physiological function of the cornea.Recently,BALB/c mice have become the most widely used laboratory animal in corneal researches and studies.An understanding of normal corneal cell size,morphology and cell density is desirable for future comparisons when mouse models are used in corneal research.Objective The aim of this study was to measure the cell size and cell density in three layers of the central cornea in the widely used inbred BALB/c mice strain based on in vivo three-dimensional (3D) two-photon (2PH) imaging.Methods Corneal endothelium layer,corneal stromal layer and corneal epithelium layer were sequentially scanned in six BALB/c mice by using a 2PH laser scanning microscope after staining with plasma membrane stain and Hoechst 33342 by intrastromal injection with the scanning thickness 2 μm for each layer,pixels 512 ×512 and 12 bit.Good quality 3D images were selected for the cell density and cell size analyses.Cell density was determined by counting the cell nuclei in a predefined cube of 3 D images.Cell size measurements including cell surface area,cell volume,nuclear surface area,and nuclear volume were automatically quantified using the Imaris software.The cell and nuclear surfacearea-to-volume ratio and the cell nuclear-to-cytoplasmic ratio were calculated.Results Corneal basal epithelium layer and endothelium layer appeared the monolayer cells,while middle stromal layer showed the double-layer cells under the 2PH laser scanning microscope.The cell density was (108.00± 18.97) × 104/mm3 in the basal epithelium layer,which was significantly higher than (9.27±0.48) ×104/mm3 in the middle stromal layer and (22.30± 2.28) ×104/mm3 in the endothelium layer (F=141.592,P=0.000).The cell surface area and volume were highest in the middle stromal layer and lowest in the basal epithelium layer,with significant differences among the three layers (F=2 222.000,598.504,both at P =0.000).The area/volume ratios of cells were (0.80±0.13),(0.51 ±0.07) and (0.40±0.04)/μm in the basal epithelium layer,middle stromal layer and endothelium layer,with the highest ratio in the basal epithelium layer and lowest ratio in the endothelium layer (F =127.075,P =0.000).The cell nuclear surface area and volume were highest in the endothelium and lowest in the basal epithelium.The cell nuclear surface area/volume ratio was highest in the basal epithelial cell and lowest in the endothelial cell.In addition,the nucleus/cytoplasm ratio was highest in the endothelial cells and lowest in the middle keratocytes.Conclusions 2PH laser scanning microscope can quantitatively measure the parameters of corneal cells in vivo in BALB/c mice,including cell surface area and volume,cell nuclear surface area and volume.These data provide important cell morphology features for the study of corneal physiology,pathology and disease in mice,particularly in BALB/c mice.