聚乙二醇诱导鸡红细胞融合实验方案的优化  

Optimization of the Experimental Scheme for Chicken Red Blood Cell Fusion Induced by PEG

作　　者：李玲[1] 刘欢[1] 贺亚玲[1] 崔林[1] 唐慧[1] 张君[1] 张亮[1] LI Ling;LIU Huan;HE Ya-ling;CUI Lin;TANG Hui;ZHANG Jun;ZHANG Liang(Shihezi University Medical College,Shihezi,Xinjiang 832002)

机构地区：[1]石河子大学医学院,新疆石河子832002

出　　处：《农垦医学》2020年第2期164-169,共6页Journal of Nongken Medicine

基　　金：石河子大学教育教学改革项目(SJ-2018-15);国家自然基金项目(81600325)。

摘　　要：目的:聚乙二醇(PEG)介导的细胞融合法是最为经济、简单的细胞融合技术,但受多种因素影响,实际实验效果并不理想,为获得高效稳定的细胞融合效率,本研究拟对PEG诱导的鸡红细胞的融合实验进行优化。方法:调整鸡红细胞的采集与保存方式、细胞浓度、缓冲系统(GKN与1640)、pH、诱导与孵育时间,以及染液的选择等。结果:弱碱PEG环境(pH≈7.8)下诱导与孵育时间均采用5min时,细胞融合率最高(17.87%),显著高于未调节pH组及诱导与孵育时间20min组(P<0.01);pH>8时,细胞聚集但胞膜皱缩破裂,计算融合率困难;亚甲基蓝、中性红染色时间>20min时,细胞膜亦皱缩破裂。结论:宜选择肝素类抗凝管采集及保存鸡血,在GKN或1640缓冲系统中,以全血与试剂1:9比例洗涤及孵育,弱碱PEG环境(pH≈7.8)分别诱导、孵育5min,并在20 min内观察亚甲基蓝或中性红染色结果,该方案适宜应用于PEG诱导的鸡红细胞融合实验教学。Objective:PEG-mediated cell fusion is the most economical and simple cell fusion technique.However the experimental results are not ideal due to various factors.In order to obtain efficient and stable cell fusion efficiency,we optimized the experiment.Methods:Methods of the chicken red blood cell collection and preservation,cell concentration,buffer system(GKN and 1640),pH,induction time and incubation time were adjusted,as well as other methods such as selected different dyes.Results:In the weak alkali environment(PEG pH≈7.8),the cell fusion rate was the highest(17.87%)when both the induction and incubation time were 5 min,which was significantly higher than that of the group without adjusted pH and the group with induction and incubation time of20 min(P<0.01).At pH>8,cells gathered trend obvious,but it was difficult to calculate the fusion rate due to the cell membrane shrinkage and rupture.The cell membranes were also crumpled when the staining time was more than20 min with methylene blue or neutral red dye.Conclusion:It is recommended to collect and store chicken blood with heparin anticoagulant tubes,whole blood was washed and incubated with GKN or 1640 buffer at a ratio of 1:9 and then weakly basic PEG was used to induce and incubate the cells for 5 min respectively,and the cell staining results within 20 min were observed with methylene blue or neutral red dye,this scheme is suitable for practical experiment teaching.

关 键 词：聚乙二醇 细胞融合 鸡红细胞 缓冲体系 PH 时间 染色剂 

分 类 号：G642.423[文化科学—高等教育学] R394-33[文化科学—教育学]