差速貼壁技術(shù)對大鼠腦皮質(zhì)星形膠質(zhì)細胞純化率的影響

1、周欣,明曉云,康頌建,白波,段耀奎. 差速貼壁技術(shù)對大鼠腦皮質(zhì)星形膠質(zhì)細胞純化率的影響. 中國組織工程研究與臨床康復, 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: 觀察差速貼壁技術(shù)對星形膠質(zhì)細胞純化率的影響,旨在建立一套可靠的大鼠腦皮質(zhì)星形膠質(zhì)細胞的取材分離、純化培養(yǎng)技術(shù)。方法: 實驗于2006-06/08在泰山醫(yī)學院

12、生命科學研究所完成。實驗材料：出生23 d的Wistar大鼠，雌雄不拘，由泰山醫(yī)學院生命科學研究所實驗動物中心提供。實驗方法：選用出生二三天的Wistar大鼠進行腦皮質(zhì)星形膠質(zhì)細胞原代培養(yǎng)。實驗分兩組培養(yǎng)：常規(guī)培養(yǎng)組和差速貼壁培養(yǎng)組。差速貼壁培養(yǎng)組分別于15，30 min取出，輕輕翻轉(zhuǎn)培養(yǎng)瓶，將上清液移至另一培養(yǎng)瓶中，放入培養(yǎng)箱中繼續(xù)培養(yǎng)。710 d后傳代，待細胞分層生長后，置于37搖床中250 r/min振蕩18 h，倒掉上清液，D-Hanks液洗3次后，加入0.25%胰酶消化，倒置顯微鏡下觀察，待細胞突起回縮后加入含血清的培養(yǎng)基終止消化，用吸管反復吹打使細胞從瓶壁上脫落，細胞懸液1 000

13、 r/min離心5 min后，棄上清液，加入含體積分數(shù)為0.2血清的DMEM培養(yǎng)基混懸沉淀，接種入預先涂有L-多聚賴氨酸的培養(yǎng)瓶中繼續(xù)培養(yǎng)。采用雙重免疫熒光法鑒定星形膠質(zhì)細胞純度，測定積分吸光度值判斷星形膠質(zhì)細胞的生長狀況。結(jié)果：應(yīng)用差速貼壁技術(shù)培養(yǎng)星形膠質(zhì)細胞可明顯提高星形膠質(zhì)細胞純度常規(guī)培養(yǎng)組：(82±3)%,差速貼壁培養(yǎng)組15 min：(94±2)%,差速貼壁培養(yǎng)組30 min：(95±2)%，P<0.01。差速貼壁需要充分的時間，15 min組和30 min組在提高星形膠質(zhì)細胞純度方面無明顯差別。差速貼壁培養(yǎng)組星形膠質(zhì)細胞積分吸光度值高于常規(guī)培養(yǎng)組(

14、常規(guī)培養(yǎng)組：528±25，差速貼壁培養(yǎng)組15 min：972±17，差速貼壁培養(yǎng)組30 min：996±35，P<0.05)。結(jié)論： 差速貼壁技術(shù)可明顯提高星形膠質(zhì)細胞純化度，并且星形膠質(zhì)細胞生長狀態(tài)明顯優(yōu)于常規(guī)培養(yǎng)方法。 最佳差速貼壁時間為15 min，過長差速貼壁時間對提高星形膠質(zhì)細胞純度無明顯影響。關(guān)鍵詞：差速貼壁技術(shù)；細胞培養(yǎng)；星形膠質(zhì)細胞周欣, 明曉云, 康頌建, 白波, 段耀奎. 差速貼壁技術(shù)對大鼠腦皮質(zhì)星形膠質(zhì)細胞純化率的影響J. 中國組織工程研究與臨床康復, 2007, 11(15):2829-2831. 1 材料與方法星形膠質(zhì)細胞原代培養(yǎng)1

15、-3: 每組取新生二三天的Wistar大鼠三四只,超凈臺上體積分數(shù)為0.75的乙醇消毒后置于無菌培養(yǎng)皿中,放血處死后,取腦,解剖顯微鏡下仔細剝離腦膜,取兩側(cè)大腦皮質(zhì),D-Hanks液漂洗、剪碎后,置于0.25%的胰酶中37消化5 min,用含有血清的培養(yǎng)基終止消化后,輕輕吹打,200目尼龍濾網(wǎng)過濾后,1 000 r/min離心5 min,加入含體積分數(shù)為0.2血清的DMEM培養(yǎng)基,輕輕吹打后接種入50 mL培養(yǎng)瓶中,置于CO2培養(yǎng)箱中培養(yǎng)。差速貼壁培養(yǎng)組分別于15,30 min后取出,輕輕翻轉(zhuǎn)培養(yǎng)瓶,將上清液移至另一培養(yǎng)瓶中,放入培養(yǎng)箱中繼續(xù)培養(yǎng)。3 d換液1次。星形膠質(zhì)細胞純化培養(yǎng)4-6:培

16、養(yǎng)710 d,待細胞分層生長后,置于37搖床中250 r/min振蕩18 h,倒掉上清液,D-Hanks液洗3次后,加入0.25%胰酶消化,倒置顯微鏡下觀察,待細胞突起回縮后加入含血清培養(yǎng)基終止消化,用吸管反復吹打使細胞從瓶壁上脫落,細胞懸液1 000 r/min離心5 min后,棄上清液,加入含體積分數(shù)為0.2血清的DMEM培養(yǎng)基混懸沉淀,接種入預先涂有L-多聚賴氨酸的培養(yǎng)瓶中繼續(xù)培養(yǎng)。為提高星形膠質(zhì)細胞的純度,待細胞融合后,可再放入搖床振蕩,重復上述操作。星形膠質(zhì)細胞的鑒定:取傳代二三次的細胞,接種至置有蓋玻片(預先用L-多聚賴氨酸包被)的6孔培養(yǎng)板,放入培養(yǎng)箱中培養(yǎng)二三天后,吸去培養(yǎng)基,

17、PBS沖洗后,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,吸盡液體。5%BSA封閉60 min,去封閉液,滴加一抗GFAP抗體(1100),以PBS代替一抗作為陰性對照,4過夜。去除一抗,PBS洗滌3次,每次5 min。加11 000稀釋的cy3標記的二抗作用60 min。PBS洗滌3次,每次5 min,加1100稀釋的DAPI作用30 min后,洗去DAPI,滴一滴抗熒光淬滅封片液于載玻片上,蓋上貼有細胞的蓋玻片,盡量避免氣泡。使細胞接觸封片液,相差熒光顯微鏡下觀察并拍照。觀察指標:膠質(zhì)纖維酸性蛋白(GFAP)是星形膠質(zhì)細胞的標志性蛋白7-9,cy3陽性

18、者呈現(xiàn)鮮艷的紅色熒光,即為星形膠質(zhì)細胞。4,6-二脒基-2-苯基吲哚(DAPI)標記的細胞核呈現(xiàn)藍色。純化率=GFAP(+)/DAPI(+)×100%。每組取6張爬片,每張爬片取3個視野,計數(shù)紅色熒光細胞占所有細胞(藍色熒光)的比例,即為星形膠質(zhì)細胞純化率,取平均值。照片經(jīng)Image Pro Plus軟件處理后計算星形膠質(zhì)細胞的積分吸光度(A),樣本采集同上。主要觀察指標:差速貼壁技術(shù)對大鼠腦皮質(zhì)星形膠質(zhì)細胞純化率的影響。統(tǒng)計學分析:由第一作者采用SPSS 10.0處理統(tǒng)計學數(shù)據(jù),數(shù)據(jù)以x±s表示,組間、組內(nèi)比較進行t檢驗。2 結(jié)果星形膠質(zhì)細胞在接種培養(yǎng)710 d后,細胞已

19、融合并可見分層生長,星形膠質(zhì)細胞在底層,在倒置顯微鏡下觀察時,細胞輪廓清晰,胞核一般不可見。經(jīng)差速貼壁后,星形膠質(zhì)細胞的純化度明顯提高,且生長狀態(tài)明顯優(yōu)于常規(guī)培養(yǎng)組,具體表現(xiàn)為胞體較大、鋪展明顯、胞突豐富以及積分吸光度明顯增高等。t檢驗顯示差速貼壁時間過長對提高星形膠質(zhì)細胞純化度無明顯作用。3 討論星形膠質(zhì)細胞是中樞神經(jīng)系統(tǒng)內(nèi)數(shù)量最多、分布最廣的膠質(zhì)細胞。對其生物學作用的認識日益受到學者們的重視10-12。由于星形膠質(zhì)細胞是與其它細胞成分混雜存在, 對其生物學功能研究的關(guān)鍵在于獲得純化星形膠質(zhì)細胞。星形膠質(zhì)細胞的分裂增殖高峰發(fā)生在動物胚胎晚期及出生以后, 故一般選擇出生后 1 周內(nèi)的新生大鼠1

20、3,14, 特別是二三天的新生大鼠。此期的大鼠腦膜較易完整剝離, 可以盡可能地避免成纖維細胞的污染。1 周后的大鼠腦溝回已較明顯, 腦膜很難剝離干凈, 因此成纖維細胞的污染難以避免。且脂質(zhì)成分增多, 不利于細胞的生長。成纖維細胞是星形膠質(zhì)細胞原代培養(yǎng)中的主要污染細胞15, 其貼壁速度明顯快于星形膠質(zhì)細胞, 可以起到純化星形膠質(zhì)細胞的目的。本實驗使用的是 50 mL 玻璃培養(yǎng)瓶, 底面積大, 凈化細胞的能力明顯強于 25 mL 培養(yǎng)瓶。另外差速貼壁時間對于凈化效果至關(guān)重要。時間太短起不到凈化目的, 太長則會損失大量星形膠質(zhì)細胞。由于培養(yǎng)瓶的材質(zhì)、L- 多聚賴氨酸的濃度、培養(yǎng)條件、取材手法等差異的

21、存在, 差速貼壁的時間很難統(tǒng)一。在本實驗中差速貼壁 15 min 與 30 min 相比, 純化率無明顯差別, 考慮到時間過長會損失部分星形膠質(zhì)細胞, 故作者認為對于普通玻璃培養(yǎng)瓶來講, 差速貼壁的時間以 15 min 左右為宜, 不宜過長。Cy3 是近年新開發(fā)的一種紅色熒光探針16,17。它比絕大部分其它的紅色熒光探針更加明亮, 更加不容易淬滅, 而且背景更低。膠原纖維酸性蛋白(GFAP)是星形膠質(zhì)細胞的標志蛋白, 通過間接法被 Cy3 標記后, 在熒光顯微鏡下可發(fā)出明亮的紅色。DAPI 是一種可以穿透細胞膜的藍色熒光染料18-20, 與雙鏈 DNA結(jié)合后可以產(chǎn)生比 DAPI 自身強 20

22、多倍的熒光, 常用于普通的細胞核染色。通過計算二者的比例來獲得星形膠質(zhì)細胞的純化率, 本實驗證實了此方法的可靠性。積分吸光度(A 值)反映了星形膠質(zhì)細胞中膠原纖維酸性蛋白的數(shù)量, 由于通過差速貼壁去除了絕大部分的成纖維細胞, 保證了星形膠質(zhì)細胞生長中的營養(yǎng)供應(yīng), 故其積分吸光度明顯高于未差速貼壁組星形膠質(zhì)細胞。綜合上述, 作者認為差速貼壁技術(shù)是一種簡便易行、效果可靠的提高星形膠質(zhì)細胞純化率的方法4 參考文獻1 Olsen C, Rustad A, Fonnum F, et al. 3-Nitropropionic acid: an astrocyte-sparing neurotoxin in

23、 vitro. Brain Res 1999; 850(1-2):144-149. 2 Young JK, Dreshaj IA, Wilson CG, et al. An astrocyte toxin influences the pattern of breathing and the ventilatory response to hypercapnia in neonatal rats. Respir Physiol Neurobiol 2005; 147(1):19-30. 3 Stone R, Stewart VC, Hurst RD, et al. Astrocyte nitr

24、ic oxide causes neuronal mitochondrial damage, but antioxidant release limits neuronal cell death. Ann N Y Acad Sci 1999; 893:400-403. 4 Braet K, Paemeleire K, D'Herde K, et al. Astrocyte-endothelial cell calcium signals conveyed by two signalling pathways. Eur J Neurosci 2001; 13(1):79-91. 5 Gr

25、iffin S, Clark JB, Canevari L. Astrocyte-neurone communication following oxygen-glucose deprivation. J Neurochem 2005; 95(4):1015-1022. 6 Ma SH, Lepak LA, Hussain RJ, et al. An endothelial and astrocyte co-culture model of the blood-brain barrier utilizing an ultra-thin, nanofabricated silicon nitri

26、de membrane. Lab Chip 2005; 5(1):74-85. 7 梁衛(wèi)蘭,單巍松,吳希如.大鼠( 小鼠) 大腦皮層星形膠質(zhì)細胞培養(yǎng)方法J.北京醫(yī)科大學學報,1994, 26 (4):320. 8 Nirmala C, Jasti SL, Sawaya R, et al. Effects of radiation on the levels of MMP-2, MMP-9 and TIMP-1 during morphogenic glial-endothelial cell interactions. Int J Cancer 2000; 88(5): 766-771. 9

27、Kamiryo T, Kassell NF, Thai QA, et al. Histological changes in the normal rat brain after gamma irradiation. Acta Neurochir (Wien) 1996; 138(4): 451-459. 10 王凱.星形膠質(zhì)細胞與神經(jīng)功能修復J.中國臨床康復,2006, 10(41):158-159. 11 Araque A,Carmignoto G,Haydon PG. Dynamic signaling between astrocytes and neurons. Annu Rev P

28、hysiol 2001;63:795-813. 12 吳永炎,姜勇.腦星形膠質(zhì)細胞生物學功能研究進展J.生理科學進展,2002,33(1):72. 13 McCarthy KD, de Vellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 1980; 85(3):890-902. 14 Gerhardt H, Golding M, Fruttiger M, et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 2004; 161(6):1163-1177. 15 Ullian Em, Sapperstein SK, Christopherson KS, et al. Control of synapse number by glia. Science 2001;291(5504):657-661. 16 Dunn SL, Young EA, Hall MD, et al. Activat