四种显微技术观测大鼠颌下腺细胞与丝素-壳聚糖体外共培养的形态学特点

Morphological Characteristics of Rat Submandibular Gland Cells Co-Cultured with Silk Fibroin-Chitosan Observed by Four Types of Microscopic Techniques

目的采用倒置显微镜、扫描电镜(scanning electron microscopy,SEM)、荧光显微镜和激光共聚焦显微镜((laser scanning confocal microscopy,LSCM))技术对大鼠颌下腺细胞(rat submandibular gland cells,RSMGs)与丝素-壳聚糖(silk fibroin-chitosan,SFCs)的体外复合培养进行形态学观察。为观测、评估种子细胞在三维支架的内部生长情况提供技术支持。方法取0～8 d龄SD大鼠的颌下腺,对大鼠颌下腺细胞进行原代培养、分离纯化并传代;用抗细胞角蛋白单克隆抗体(CK8)及淀粉酶抗体的免疫细胞化学染色鉴定细胞来源。选取传至第二代的对数生长期的RSMGs作为种子细胞,选取SFCs共混膜(5×5×2)mm作为支架材料构建组织工程化涎腺样结构。将种子细胞与支架材料复合培养并分别于倒置显微镜、SEM、荧光显微镜和LSCM下观察二者复合生长情况。结果倒置显微镜可以直接观察活细胞与支架复合生长情况,方法简单易行。SEM可以较精确的展示细胞支架复合生长的表面超微结构。经过荧光染料的着色,荧光显微镜和LSCM都可以观察到支架上锚定的种子细胞。荧光显微镜可见细胞核的荧光信号均匀的分布在支架孔隙内。LSCM通过层扫描及三维重建技术对较厚的标本获取图像;并可以通过旋转图像,从不同角度观察细胞支架复合物的三维剖面或整体结构,得到更为准确的定位信息。结论四种显微技术均可应用于RSMGs与SFCs体外共培养的形态学观测。LSCM的三维重建技术结合荧光染料标记可以较好地获得RSMGs与SFCs复合生长的情况,有着较广泛的应用价值。

Objective To observe the morphological characteristics of rat submandibular gland cells（RSMGs） co-cultured with silk fibroin-chitosan,（SFCs） by inverted microscopy,scanning electron microscopy（SEM）,fluorescence microscopy and laser scanning confocal microscopy（LSCM）,and provide technical support for observation and evaluation of cell growth on three-dimensional（3D） scaffolds.Methods The submandibular glands were excised from SD rats at age of 0 to 8-days and RSMGs were primarily cultured,purified and subcultured.The origin of cells were identified by immunocytochemistry（CK8 and amylase）.Then RSMGs were chosen for the further studies when cells reached 75% to 80% confluence.Meanwhile SFCs blend membranes（5 mm x 5 mm x 2 mm） were prepared.RSMGs cultured on the scaffolds were monitored for 3,5 to 7 days.The morphological appearance of the seeded scaffolds was observed by inverted microscopy,SEM,fluorescence microscopy and LSCM.Results Living cells that were seeded on SFCs medium of 3D scaffolds could be observed directly under the invert microscope.SEM showed the surface ultrastructure of the complex growth situation more clearly.After fluorescent staining,cells anchoring on scaffolds were observed by fluorescence microscopy and LSCM.The fluorescent signals of cell nuclei were evenly distributed in the pores of scaffolds.Through layer scanning and 3D LSCM reconstruction technique,images of thick specimens could be obtained.By rotating the images,3D profile or whole structure could be observed from different angles and more accurate information of positioning could be obtained.Conclusions All four kinds of microscopic techniques can be used in examining the co-cultivation of RSMGs and SFCs.Images can be observed more distinctly through 3D LSCM reconstruction technique combined with fluorescent staining technique.It has a wide application value in this research field.