人牙周膜成纤维细胞的培养及牵张应变诱导凋亡研究

Study on Human Periodontal Ligament Fibroblasts Culture and Apoptosis by Stretch Strain

目的:探讨人牙周膜成纤维细胞的分离、培养及在牵张应变作用下发生早期凋亡的情况。方法:刮取健康人前磨牙牙周膜,进行牙周膜成纤维细胞的分离、培养及生长曲线描绘。经3～4代传代培养后,对细胞加载1%～20%的牵张应变,加载时间分别为30min,1h、6h、12h然后通过流式细胞仪检测Annexin V-FITC(异硫氰酸荧光素)及激光扫描共聚焦显微镜进行细胞的早期凋亡鉴定。结果:人牙周膜成纤维细胞传代到12代以后,细胞逐渐呈衰老生状,胞体变大,细胞生长缓慢。人牙周膜成纤维细胞的凋亡率在30min组低于对照组,但两者间无统计学差异。在6h内,人牙周膜成纤维细胞的凋亡情况随加载时间和加载应力的增加而增加(P<0.05)。在12h时所有组的细胞凋亡均减少。结论:人牙周膜成纤维细胞的使用最好在12代以内,尤其是3～4代细胞的活力、增殖能力较好。牵张应变可以诱导细胞发生早期凋亡。

Objective： To evaluate human periodontal ligament fibroblasts （HPLFs） isolation and culture and early apoptosis by loading stretch strain in vitro. Methods： Scraping periodontal ligament from healthy teeth, and isolating HPLFs to primary culture then depicting the growth curve. The HPLFs through 3 to 4 passages were stretched by strain 1% to 200/00 for 30min, 1 h, 6 h and 12h, whereafter examing Annexin V-FITC binding by flow cytometry and confocal laser scanning microscope to detect the early apoptosis. Results： HPLFs can be obtained from periodontal ligment and became aged after 12 passages distinctly. The rate of early apoptosis increased along with the time and strain within 6h （P〈0.05）, however, apoptosis decreased at 12h. Conclusion： The experiment indicated that HPLFs were used before 12 passages,especially the vitality of cells in 3-4 passages is better. Early apoptosis in HPLFs were found apprently when stretching strain.