胰蛋白酶消化强度优化获得高纯度体外培养的星形胶质细胞  

Optimization of trypsin digestion intensity to obtain high-purity in vitro cultured astrocytes

作　　者：靳辉[1] 杨蓬勃[1] 冯改丰[1] 贾宁[1] 杨维娜[1] 王唯析[1] 

机构地区：[1]西安交通大学医学院人体解剖与组织胚胎学系,西安710061

出　　处：《生理学报》2015年第1期103-109,共7页Acta Physiologica Sinica

基　　金：supported by the Natural Science Basic Research Plan in Shaanxi Province;China(No.2012JQ4011;2014JM4096)

摘　　要：本文旨在观察胰蛋白酶消化对体外培养的星形胶质细胞纯度的影响,优化星形胶质细胞培养方法。常规分离新生Sprague Dawley(SD)大鼠大脑皮质,分别用0.25%胰蛋白酶消化20、30和40 min制备单细胞悬液并接种细胞。细胞长满瓶底时进行恒温摇床振荡,前两个不同消化时间组再分为常规消化的对照组和二次胰蛋白酶消化组进行传代纯化。倒置相差显微镜观察细胞生长情况,MTT法检测细胞增殖,GFAP免疫荧光分析星形胶质细胞纯度并观察其形态,流式细胞术分析细胞凋亡。结果显示,胰蛋白酶消化20 min的细胞在原代培养9 d长满瓶底。而延长胰蛋白酶消化时间至30 min,细胞增殖更快,培养7 d即可铺满瓶底,且星形胶质细胞形态正常,纯度达(70.2±4.0)%,较20 min组有显著性提高(P<0.05)。40 min组虽然星形胶质细胞纯度也较20 min组有所提高,但细胞增殖缓慢且损伤明显。在恒温摇床振荡结束后,进行二次胰蛋白酶消化可减少传代后杂细胞数量。二次胰蛋白酶消化组第一代(P1)的GFAP阳性率普遍高于各自对照组,其中30 min+二次胰蛋白酶消化组GFAP阳性率为(98.1±1.7)%,相当于20 min+对照组P3水平,且二者的凋亡率无显著性差异。以上结果表明,胰蛋白酶消化30 min+二次消化能有效提高星形胶质细胞纯度,缩短原代培养及纯化时间,是体外快速获得高纯度星形胶质细胞的有效方法。The aim of the present study was to observe the effect of trypsin digestion on the purity of in vitro cultured astrocytes and optimize the culture methods. The cerebral cortical tissue from newborn Sprague Dawley （SD） rats was isolated and digested with 0.25% trypsin for 20, 30, or 40 min. The obtained single cell suspension was then cultured. Once reaching confluence, the cells were shaken at a constant temperature. Then, each of 20 and 30 min groups was subdivided into two groups, the control group with normal digestion and two-time-digestion group, and the cells were passaged and purified. Through inverted phase contrast microscope and MTT assay, cell growth and proliferation were observed, respectively. Immunofluorescence for glial fibrillary acidic protein （GFAP） was used to observe the morphology of astrocytes and to assess their purity in different stages. Flow cytometric analysis was used to detect the apoptotic rates of purified astrocytes. The results showed that, the cells being digested for 20 min usually reached conflu- ence at 9 d after seeding. When the digestion time was extended to 30 min, the cells grew faster and reached confluence at 7 d after seeding, meanwhile the morphology of astrocytes was normal, GFAP positive rate （70.2 ± 4.0）% being much higher than that of the 20 min group （P 〈 0.05）. Compared with 20 min group, 40 min group showed higher GFAP positive rate, whereas the cell prolifera- tion was slower, and cell injury was more obvious. After shaking at constant temperature, two times of trypsin digestion could decrease the number of contaminated cells after passage. The GFAP positive rates of two-time-digestion groups in passage 1 （P1） were higher than those of corresponding control groups, and the GFAP positive rate of 30 min ＋ two-time-digestion group in P1 reached （98.1 ± 1.7）%, which was equivalent to that of the 20 min ＋ control group in P3. However, the apoptotic rate showed no significant difference between these two groups. Based on above mentio

关 键 词：星形胶质细胞 细胞培养 胰蛋白酶 

分 类 号：Q421[生物学—神经生物学]