大鼠主动脉内皮细胞和平滑肌细胞的原代培养及生物学特性比较

DIFFERENCES IN THE PRIMARY CULTURE AND BIOLOGICAL CHARACTERISTICS BETWEEN ENDOTHELIAL CELLS AND SMOOTH MUSCLE CELLS FROM RAT AORTA

目的培养大鼠主动脉平滑肌细胞和内皮细胞,细胞纯化与鉴定,比较生物学特性的差异。方法采用血管环贴壁法培养动脉内皮细胞,组织块贴壁法培养动脉平滑肌细胞,并采用有限稀释法挑选内皮细胞单克隆,免疫细胞荧光鉴定二者的特异性标志,相差显微镜观察二者单个细胞及细胞群体在形态上的差异性,CCK-8试剂盒检测细胞的增殖,比较二者对胰酶消化,粘附,冻存后复苏的情况。结果血管环贴壁法成功培养血管内皮细胞,组织块培养法成功培养出血管平滑肌细胞,内皮细胞能够形成单克隆集落,培养的细胞均表达相应的特异性标志,内皮细胞增殖速度和平滑肌细胞有差异,内皮细胞对胰酶的耐受性较差,内皮细胞粘附所需时间短,对冻存后的耐受性较好。结论组织块贴壁法适合内皮细胞和平滑肌细胞的培养,有限稀释法能够纯化原代培养的内皮细胞,大鼠主动脉平滑肌细胞和内皮细胞在细胞形态、增殖、粘附、对胰酶的反应、冻存后复苏均存在差异。

Objective To investigate the cell culture, purification and identification, and differences in biological characteristics between endothelial ceils and smooth muscle ceils from rat aorta. Methods Endothelial cells were obtained via vascular ring adherence, while smooth muscle cells were separated from tissue pieces of rat aorta. Monoclonal endothelial cells were selected by limiting dilution assay. Both cells were identified by the specific cell markers with immunofluorescence; phase contrast microscopy was used to observe the morphological differences between endothelial and smooth muscle cells both as a single cell and as a colony. Cell proliferation was determined by Cell Counting Kit-8. Differences in trypsin digestion time, adherence time and resuscitation after frozen preservation between endothelial and smooth muscle cells were also evaluated. Results Endothelial cells and smooth muscle cells were cultured successfully with the methods of vascular ring adherence and tissue pieces respectively. Endothelial cells formed monoclones. The two cells expressed specific markers, and the proliferation time of endothelial cells different from that of smooth muscle cells. Endothelial cells were more sensitive to trypsin digestion and had a shorter adherence time and better survival after frozen preservation than smooth muscle cells. Conclusion Vascular ring adherence and tissue pieces are suitable for the culture of endothelial cells and smooth muscle cells from rat aorta. Endothelial cells and smooth muscle cells have different characteristics in cell morphology, proliferation, adherence, trypsin digestion and resuscitation after frozen preservation.