摘要
目的探讨一种简单易行的大鼠胰岛β细胞的原代分离及培养方法,并探索近细胞膜钙离子的测定技术。方法雄性SD大鼠胰腺用V型胶原酶消化分离得到胰岛,胰酶逐级消化胰岛,分离出β细胞,从而建立大鼠胰岛β细胞分离、原代培养方法。用近膜钙离子荧光探针FFP-18/AM孵育、负载β细胞,利用激光共聚焦技术测定β细胞近膜钙离子的分布和变化。结果原代分离获得的胰岛具有良好的活性,β细胞经鉴定纯度较好。激光共聚焦测定FFP-18/AM负载胰岛β细胞,近膜钙离子呈蓝色荧光,不均匀分布于近细胞膜附近。结论建立了大鼠胰岛β细胞的原代分离、培养方法,以及β细胞近膜钙离子测定技术。
Objective To explore a convenient and reliable method to isolate rat pancreatic β-cells for primary culture,and to develop the technique to measure sub-plasma-membrane calcium in the cells.Methods Male SD rat islets were isolated using digestion by ductal injection of collagenase V and treated with trypsin to obtain isolated cells.After β-cells were loaded with fluorescent probe FFP-18-AM,fluorescent images were collected using laser scanning confocal microscope with an excitation of 340 nm to monitor changes of sub-plasma-membrane calcium of β-cells. Results Bile duct collagenase V injection and digestion can reproducibly yield functional and pure β-cells.After β-cells were loaded with FFP-18-AM,fluorescent images show that Ca2＋ located near-plasma lemma presented nonuniformity blue fluorescence.Conclusions A reliable method to isolate and primary culture rat pancreatic β-cell is obtained. Using fluorescent probe FFP-18 could observe the changes of sub-plasma-membrane calcium of β-cells.
出处
《临床儿科杂志》
CAS
CSCD
北大核心
2011年第8期769-772,共4页
Journal of Clinical Pediatrics
关键词
胰岛
近膜钙离子
大鼠
pancreatic islet
sub-plasma-membrane Ca2＋
rat
