优化人胃癌细胞SGC-7901细胞骨架图像的激光共聚焦显微技术  

Optimization of cytoskeleton observation of human gastric cancer cell line SGC-7901 by laser scanning confocal

作　　者：高建[1] 郑倩倩[2] 张四洋[1] 

机构地区：[1]中国医科大学科学实验中心三部,辽宁沈阳110014 [2]中国医科大学基础医学院细胞病理生物学研究室,辽宁沈阳110014

出　　处：《解剖科学进展》2015年第3期291-293,共3页Progress of Anatomical Sciences

基　　金：国家自然科学基金资助项目(No.81101599)

摘　　要：目的利用激光共聚焦技术,对比三种固定液不同固定时间对胃癌细胞系SGC-7901的细胞骨架荧光染色的影响。方法选择三种常用固定液:2.5%戊二醛,4%多聚甲醛及95%乙醇,对接种24h后的SGC-7901细胞分别进行10min,20min及30min等不同时间点固定,5%BSA(含0.2%Triton X-100)封闭透膜1h,荧光抗体FITC标记的鬼笔环肽37℃避光孵育2h,DAPI室温染核15min,激光共聚焦扫描显微镜扫描,对比观察其染色结果。结果三种固定液及不同固定时间对SGC-7901细胞骨架染色效果不尽相同。95%乙醇固定后微丝之间区分不明,排列混乱,无网状结构,染色效果与固定时长无关。2.5%戊二醛固定后微丝结构完整,但层次不清晰。4%多聚甲醛固定后微丝结构完整,层次清晰,固定20min及30min微丝荧光染色清晰。结论 4%多聚甲醛固定20min对细胞骨架固定效果最好,荧光染色标记结果为后续细胞骨架功能研究优化实验方法并提供理论依据。Objective To observe the results of F-actin fluorescein stain in SGC-7901 cells treated by different fixatives and different fixed time by laser scanning confocal technology. Methods SGC-7901 cells were fixed by 2.5% Glutaraldehyde（2.5% GA）, 4% Paraformaldehyde（4% PFA） and 95% ethanol at room temperature for 10 min, 20 min and 30 min respectively. Antigen was blocked by 5% BSA（containing 0.2% Triton X-100） for 1h. The samples were incubated with first antibody F-actin at 37℃ for 2h, avoiding of light. The nuclei were stained by DAPI at room temperature for 15 min. Results Different fixed time showed different effects of F-actin positive immunofluorescein staining. The constructure of microfilament（MF） fixed by 2.5% GA was complete but unclear, incomplete and unclear for 95% ethanol fixation, however, complete and clear for 4% PFA fixation for 20 min. Conclusion The fixation of SGC-7901 cells skeleton by 4% PFA for 20 min was the best.

关 键 词：固定液 细胞骨架 胃癌 激光共聚焦 

分 类 号：Q366[生物学—遗传学]