差速贴壁技术对大鼠脑皮质星形胶质细胞纯化率的影响

1、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 ratsAbstractAIM: To observe the proportion of rat cerebral astrocyte cultured by differential velocity adherent te

2、chnique,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

3、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

4、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 37and shook at 250 r/min for 18 hours. Superna

5、tant was added, and then the cells were washed with D-Hank's 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

6、 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 identi

7、fy purification of the astrocytes. At the same time,integrated optical density(IOD)was used to measure growth state of astrocytes.RESULTS: Purity of astrocytes could be elevated markedly by differential velocity adherent techniqueroutine culture group: (82±3)%, differential velocity adherent gr

8、oup of 15 minutes: (94±2)%, differential velocity adherent group of 30 minutes: (95±2)%, P<0.01. Differential velocity adherent technique needed enough time. There was no significant difference in elevating cell purity in the 15 minutes group and 30 minutes group. IOD of astrocytes in t

9、he differential velocity adherent group was higher than that in the routine culture group(routine culture group: 528±25, differential velocity adherent group of 15 minutes: 972±17, differential velocity adherent group of 30 minutes: 996±35, P<0.05. CONCLUSION:The differential veloc

10、ity adherent technique can remarkably elevate the purity of astrocytes, and the growth is distinctly better than that by routine culture method.The best differential velocity adherent time is 15 minutes, and there is no significant effect of longer adherent time on elevating purity. Zhou X, Ming XY,

11、 Kang SJ, Bai B ,Duan YK. Influence of differential velocity adherent technique on the purified rate of cerebral astrocytes in rats. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2007; 11(15): 2829-2831(China) 摘要目的: 观察差速贴壁技术对星形胶质细胞纯化率的影响,旨在建立一套可靠的大鼠脑皮质星形胶质细胞的取材分离、纯化培养技术。方法: 实验于2006-06/08在泰山医学院

12、生命科学研究所完成。实验材料：出生23 d的Wistar大鼠，雌雄不拘，由泰山医学院生命科学研究所实验动物中心提供。实验方法：选用出生二三天的Wistar大鼠进行脑皮质星形胶质细胞原代培养。实验分两组培养：常规培养组和差速贴壁培养组。差速贴壁培养组分别于15，30 min取出，轻轻翻转培养瓶，将上清液移至另一培养瓶中，放入培养箱中继续培养。710 d后传代，待细胞分层生长后，置于37摇床中250 r/min振荡18 h，倒掉上清液，D-Hanks液洗3次后，加入0.25%胰酶消化，倒置显微镜下观察，待细胞突起回缩后加入含血清的培养基终止消化，用吸管反复吹打使细胞从瓶壁上脱落，细胞悬液1 000

13、 r/min离心5 min后，弃上清液，加入含体积分数为0.2血清的DMEM培养基混悬沉淀，接种入预先涂有L-多聚赖氨酸的培养瓶中继续培养。采用双重免疫荧光法鉴定星形胶质细胞纯度，测定积分吸光度值判断星形胶质细胞的生长状况。结果：应用差速贴壁技术培养星形胶质细胞可明显提高星形胶质细胞纯度常规培养组：(82±3)%,差速贴壁培养组15 min：(94±2)%,差速贴壁培养组30 min：(95±2)%，P<0.01。差速贴壁需要充分的时间，15 min组和30 min组在提高星形胶质细胞纯度方面无明显差别。差速贴壁培养组星形胶质细胞积分吸光度值高于常规培养组(

14、常规培养组：528±25，差速贴壁培养组15 min：972±17，差速贴壁培养组30 min：996±35，P<0.05)。结论： 差速贴壁技术可明显提高星形胶质细胞纯化度，并且星形胶质细胞生长状态明显优于常规培养方法。 最佳差速贴壁时间为15 min，过长差速贴壁时间对提高星形胶质细胞纯度无明显影响。关键词：差速贴壁技术；细胞培养；星形胶质细胞周欣, 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J. 中国组织工程研究与临床康复, 2007, 11(15):2829-2831. 1 材料与方法星形胶质细胞原代培养1

15、-3: 每组取新生二三天的Wistar大鼠三四只,超净台上体积分数为0.75的乙醇消毒后置于无菌培养皿中,放血处死后,取脑,解剖显微镜下仔细剥离脑膜,取两侧大脑皮质,D-Hanks液漂洗、剪碎后,置于0.25%的胰酶中37消化5 min,用含有血清的培养基终止消化后,轻轻吹打,200目尼龙滤网过滤后,1 000 r/min离心5 min,加入含体积分数为0.2血清的DMEM培养基,轻轻吹打后接种入50 mL培养瓶中,置于CO2培养箱中培养。差速贴壁培养组分别于15,30 min后取出,轻轻翻转培养瓶,将上清液移至另一培养瓶中,放入培养箱中继续培养。3 d换液1次。星形胶质细胞纯化培养4-6:培

16、养710 d,待细胞分层生长后,置于37摇床中250 r/min振荡18 h,倒掉上清液,D-Hanks液洗3次后,加入0.25%胰酶消化,倒置显微镜下观察,待细胞突起回缩后加入含血清培养基终止消化,用吸管反复吹打使细胞从瓶壁上脱落,细胞悬液1 000 r/min离心5 min后,弃上清液,加入含体积分数为0.2血清的DMEM培养基混悬沉淀,接种入预先涂有L-多聚赖氨酸的培养瓶中继续培养。为提高星形胶质细胞的纯度,待细胞融合后,可再放入摇床振荡,重复上述操作。星形胶质细胞的鉴定:取传代二三次的细胞,接种至置有盖玻片(预先用L-多聚赖氨酸包被)的6孔培养板,放入培养箱中培养二三天后,吸去培养基,

17、PBS冲洗后,加入4%甲醛,4固定6 h,去固定液,用含0.1%的PBS洗3遍,每次3 min,吸尽液体。5%BSA封闭60 min,去封闭液,滴加一抗GFAP抗体(1100),以PBS代替一抗作为阴性对照,4过夜。去除一抗,PBS洗涤3次,每次5 min。加11 000稀释的cy3标记的二抗作用60 min。PBS洗涤3次,每次5 min,加1100稀释的DAPI作用30 min后,洗去DAPI,滴一滴抗荧光淬灭封片液于载玻片上,盖上贴有细胞的盖玻片,尽量避免气泡。使细胞接触封片液,相差荧光显微镜下观察并拍照。观察指标:胶质纤维酸性蛋白(GFAP)是星形胶质细胞的标志性蛋白7-9,cy3阳性

18、者呈现鲜艳的红色荧光,即为星形胶质细胞。4,6-二脒基-2-苯基吲哚(DAPI)标记的细胞核呈现蓝色。纯化率=GFAP(+)/DAPI(+)×100%。每组取6张爬片,每张爬片取3个视野,计数红色荧光细胞占所有细胞(蓝色荧光)的比例,即为星形胶质细胞纯化率,取平均值。照片经Image Pro Plus软件处理后计算星形胶质细胞的积分吸光度(A),样本采集同上。主要观察指标:差速贴壁技术对大鼠脑皮质星形胶质细胞纯化率的影响。统计学分析:由第一作者采用SPSS 10.0处理统计学数据,数据以x±s表示,组间、组内比较进行t检验。2 结果星形胶质细胞在接种培养710 d后,细胞已

19、融合并可见分层生长,星形胶质细胞在底层,在倒置显微镜下观察时,细胞轮廓清晰,胞核一般不可见。经差速贴壁后,星形胶质细胞的纯化度明显提高,且生长状态明显优于常规培养组,具体表现为胞体较大、铺展明显、胞突丰富以及积分吸光度明显增高等。t检验显示差速贴壁时间过长对提高星形胶质细胞纯化度无明显作用。3 讨论星形胶质细胞是中枢神经系统内数量最多、分布最广的胶质细胞。对其生物学作用的认识日益受到学者们的重视10-12。由于星形胶质细胞是与其它细胞成分混杂存在, 对其生物学功能研究的关键在于获得纯化星形胶质细胞。星形胶质细胞的分裂增殖高峰发生在动物胚胎晚期及出生以后, 故一般选择出生后 1 周内的新生大鼠1

20、3,14, 特别是二三天的新生大鼠。此期的大鼠脑膜较易完整剥离, 可以尽可能地避免成纤维细胞的污染。1 周后的大鼠脑沟回已较明显, 脑膜很难剥离干净, 因此成纤维细胞的污染难以避免。且脂质成分增多, 不利于细胞的生长。成纤维细胞是星形胶质细胞原代培养中的主要污染细胞15, 其贴壁速度明显快于星形胶质细胞, 可以起到纯化星形胶质细胞的目的。本实验使用的是 50 mL 玻璃培养瓶, 底面积大, 净化细胞的能力明显强于 25 mL 培养瓶。另外差速贴壁时间对于净化效果至关重要。时间太短起不到净化目的, 太长则会损失大量星形胶质细胞。由于培养瓶的材质、L- 多聚赖氨酸的浓度、培养条件、取材手法等差异的

21、存在, 差速贴壁的时间很难统一。在本实验中差速贴壁 15 min 与 30 min 相比, 纯化率无明显差别, 考虑到时间过长会损失部分星形胶质细胞, 故作者认为对于普通玻璃培养瓶来讲, 差速贴壁的时间以 15 min 左右为宜, 不宜过长。Cy3 是近年新开发的一种红色荧光探针16,17。它比绝大部分其它的红色荧光探针更加明亮, 更加不容易淬灭, 而且背景更低。胶原纤维酸性蛋白(GFAP)是星形胶质细胞的标志蛋白, 通过间接法被 Cy3 标记后, 在荧光显微镜下可发出明亮的红色。DAPI 是一种可以穿透细胞膜的蓝色荧光染料18-20, 与双链 DNA结合后可以产生比 DAPI 自身强 20

22、多倍的荧光, 常用于普通的细胞核染色。通过计算二者的比例来获得星形胶质细胞的纯化率, 本实验证实了此方法的可靠性。积分吸光度(A 值)反映了星形胶质细胞中胶原纤维酸性蛋白的数量, 由于通过差速贴壁去除了绝大部分的成纤维细胞, 保证了星形胶质细胞生长中的营养供应, 故其积分吸光度明显高于未差速贴壁组星形胶质细胞。综合上述, 作者认为差速贴壁技术是一种简便易行、效果可靠的提高星形胶质细胞纯化率的方法4 参考文献1 Olsen C, Rustad A, Fonnum F, et al. 3-Nitropropionic acid: an astrocyte-sparing neurotoxin in

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