差速贴壁技术对大鼠脑皮质星形胶质细胞纯化率影响.docVIP

周欣,明晓云,康颂建,白波,段耀奎. 差速贴壁技术对大鼠脑皮质星形胶质细胞纯化率的影响. 中国组织工程研究与临床康复, 2007, 11(15): 2829-2831. Influence of differential velocity adherent technique on the purified rate of cerebral astrocytes in rats Abstract AIM: To observe the proportion of rat cerebral astrocyte cultured by differential velocity adherent technique,so as to establish a set of reliable technique for the isolation and purification of the astrocytes cerebral cortex. METHODS:The experiment was performed in the Institute of Life Science,Taishan Medical College from June to August 2006.Wistar rats aged 2-3 days after birth of either sex were provided by Experimental Animal Center, Institute of Life Science, Taishan Medical College,and selected to carry out primary culture of cerebral astrocytes. They were divided into two groups:routine culture group and differential velocity adherent group. Astrocytes were removed at minutes 15 and 30 in the latter group,and then culture flask was rotated to remove supernatant into another culture flask,and then kept in incubator for following culture. After 7-10 days,each group was passaged till cells grew into demixing, and then put in swing bed at 37℃and shook at 250 r/min for 18 hours. Supernatant was added, and then the cells were washed with D-Hanks liquor three times, and treated with 0.25%trypsinization, observed under inverted microscope. Medium containing serum was added to stop digestion when process of cells recovered.The cells were blown and hit by pipette for keeping cells from flask wall, and then centrifuged at 1 000 r/min for 5 minutes.Supernatant was removed before DMEM medium containing 0.2 volume fraction was suspended and precipitated. The cells were incubated in L-polylysine culture flask successively. Double immunofluorescence staining was selected to identify purification of the astrocytes. At the same time,integrated optical density(IOD)was used to measure growth state of astrocytes. RESULTS: ①Purity of astrocytes could be elevated markedly by differe