[1]李丽萍,李宁凤,陈杉杉,等.大鼠视盘筛板星形胶质细胞的培养纯化及鉴定[J].眼科新进展,2018,38(1):023-26.[doi:10.13389/j.cnki.rao.2018.0004]
　LI Li-Ping,LI Ning-Feng,CHEN Shan-Shan,et al.Purification and characterization of rat optic nerve head astrocytes[J].Recent Advances in Ophthalmology,2018,38(1):023-26.[doi:10.13389/j.cnki.rao.2018.0004]
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大鼠视盘筛板星形胶质细胞的培养纯化及鉴定/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
38卷
期数:
2018年1期
页码:
023-26
栏目:
实验研究
出版日期:
2018-01-05

文章信息/Info

Title:
Purification and characterization of rat optic nerve head astrocytes
作者:
李丽萍; 李宁凤; 陈杉杉; 郭翠菊; 张旭
330006　江西省南昌市，南昌大学附属眼科医院；江西省眼科学和视觉科学研究所；江西省眼科学重点实验室
Author(s):
LI Li-Ping; LI Ning-Feng; CHEN Shan-Shan; GUO Cui-Ju; ZHANG Xu
Affiliated Eye Hospital of Nanchang University，Jiangxi Research Institute of Ophthalmology & Visual Sciences，Key Laboratory of Ophthalmology of Jiangxi Province，Nanchang 330006，Jiangxi Province，China
关键词:
视盘; 筛板; 细胞培养; 星形胶质细胞; 青光眼
Keywords:
optic disc; lamina cribrosa; cell culture; astrocytes; glaucoma
分类号:
R774
DOI:
10.13389/j.cnki.rao.2018.0004
文献标志码:
A
摘要:
目的　探讨大鼠视盘筛板部位原代星形胶质细胞体外分离纯化培养方法。方法　在解剖显微镜下分离成年大鼠视盘及其后一小段视神经，切块后放入含DMEM/F-12培养瓶中进行原代培养。经2.5 g·L-1胰蛋白酶消化、星形胶质细胞选择性培养基纯化，传代2次后，用胶质细胞标志性抗体胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）、神经元细胞黏附分子（neural cell adhesion molecule，NCAM） 和波形蛋白（Vimentin）进行免疫荧光化学染色；证实为星形胶质细胞后，用表皮生长因子刺激星形胶质细胞24 h，观察其形态变化并用Western blot法检测培养细胞GAFP、NCAM和Vimentin蛋白的表达情况。结果　组织块贴壁后10 d左右，细胞开始爬出，且形态各异。经星形胶质细胞选择性培养基纯化后，细胞形态多呈星形或不规则形，胞质丰富；免疫荧光化学染色显示GFAP、NCAM、Vimentin蛋白染色阳性，阳性细胞率达90%以上；表皮生长因子刺激后，细胞增生，形状发生改变，且与空白对照组相比，刺激后GAFP、Vimentin和NCAM蛋白表达增加，差异均有统计学意义（均为P<0.05）。结论　本方法可在体外培养并获得纯度较高的大鼠视盘筛板部位的原代星形胶质细胞。
Abstract:
Objective　To develop a set of methods for the culture and purification of astrocytes grown from explanted rat optic disc.Methods　Rat optic disc and optic nerve were separated with anatomic microscopy and the blocks were cultivated primarily in culture flask with the medium containing DMEM/F-12.After digested by 2.5 g·L-1 trypsin，astrocytes were purified on the selective medium and passaged for two generations.Then cells were identified by immunofluorescent staining of glial fibrillary acidic protein (GFAP)，neural cell adhesion molecule (NCAM) and Vimentin.Next，epidermal growth factor (EGF) was used to stimulate the purified cells for 24 h to observe their morphologic alteration.Finally，Western blot was used to detect the expression of GAFP，NCAM and Vimentin in the cells.Results　When the tissue blocks were attached to the wall for 10 days，cells began to crawl out of the tissues and had different morphologies.After purified by the selective culture medium，satellite or anomalistic cells，possessing abundant cytoplasm，began to take a leading place.Immunofluorescent staining showed GFAP，NCAM and Vimentin were stained positively，and the purity of positive cells was above 90%.After stimulated by EGF，cells proliferated and the morphology changed；meanwhile，there was an increasing expression of GFAP，NCAM and Vimentin after stimulated by EGF when compared with the blank control group，and the difference was statistically significant (P<0.05).Conclusion　Highly-purified astrocytes can be achieved by the culture of explanted lamina cribiosa and optic disc of rats in vitro.

参考文献/References:

［1］　KELLIHER C，KENNY D，O’BRIEN C.Trends in blind registration in the adult population of the Republic of Ireland 1996-2003［J］.Br J Ophthalmol，2006，90（3）：367-371.
［2］　QUIGLEY HA，BROMAN AT.The number of people with glaucoma worldwide in 2010 and 2020［J］.Br J Ophthalmol，2006，90（3）：262-267.
［3］　QUIGLEY HA.Number of people with glaucoma worldwide［J］.Br J Ophthalmol，1996，80（5）：389-393.
［4］　WEINREB RN，AUNG T，MEDEIROS FA.The pathophysiology and treatment of glaucoma：a review［J］.JAMA，2014，311（18）：1901-1911.
［5］　ABE RY，GRACITELLI CPB，DINIZ-FILHO A，TATHAM AJ，MEDEIROS FA.Lamina cribrosa in glaucoma：diagnosis and Monitoring［J］.Curr Ophthalmol Rep，2015，3（2）：74-84.
［6］　YAN DB，COLOMA FM，METHEETRAIRUT A，TROPE GE，HEATHCOTE JG，ETHIER CR.Deformation of the lamina cribrosa by elevated intraocular pressure［J］.Br J Ophthalmol，1994，78（8）：643-648.
［7］　CRAWFORD DOWNS J，ROBERTS MD，SIGAL IA.Glaucomatous cupping of the lamina cribrosa：a review of the evidence for active progressive remodeling as a mechanism［J］.Exp Eye Res，2011，93（2）：133-140.
［8］　MORCOS Y，CHAN-LING T.Concentration of astrocytic filaments at the retinal optic nerve junction is coincident with the absence of intra-retinal myelination：comparative and developmental evidence［J］.J Neurocytol，2000，29（9）：665-678.
［9］　DING L，YAMADA K，TAKAYAMA C，INOUE Y.Development of astrocytes in the lamina cribrosa sclerae of the mouse optic nerve，with special reference to myelin formation［J］.Okajimas Folia Anat Jpn，2002，79（5）：143-157.
［10］　SCHNEIDER M，FUCHSHOFER R.The role of astrocytes in optic nerve head fibrosis in glaucoma［J］.Exp Eye Res，2016，142（1）：49-55.
［11］　HERNANDEZ MR.The optic nerve head in glaucoma：role of astrocytes in tissue remodeling［J］.Prog Retin Eye Res，2000，19（3）：297-321.
［12］　HERNANDEZ MR，MIAO H，LUKAS T.Astrocytes in glaucomatous optic neuropathy［J］.Prog Brain Res，2008，173（3）：353-373.
［13］　SOFRONIEW MV，VINTERS HV.Astrocytes：biology and pathology［J］.Acta Neuropathol，2010，119（1）：7-35.
［14］　JOHNSON EC，MORRISON JC.Friend or foe?Resolving the impact of glial responses in glaucoma［J］.J Glaucoma，2009，18（5）：341-353.
［15］　DOWNS JC，SUH JK，THOMAS KA，BELLEZZA AJ，HART RT，BURGOYNE CF.Viscoelastic material properties of the peripapillary sclera in normal and early-glaucoma monkey eyes［J］.Invest Ophthalmol Vis Sci，2005，46（2）：540-546.
［16］　BURGOYNE CF，MORRISON JC.The anatomy and pathophysiology of the optic nerve head in glaucoma［J］.J Glaucoma，2001，10（5 Suppl 1）：S16-18.
［17］　GARCA DM，KOKE JR.Astrocytes as gate-keepers in optic nerve regeneration-a mini-review［J］.Comp Biochem Physiol A Mol Integr Physiol，2009，152（2）：135-138.
［18］　GALOU M，GAO J，HUMBERT J，MERICSKAY M，LI Z，PAULIN D，et al.The importance of intermediate filaments in the adaptation of tissues to mechanical stress：evidencefrom gene knockout studies［J］.Biol Cell，1997，89（2）：85-97.
［19］　CALVO JL，CARBONELL AL，BOYA J.Coexpression of vimentin and glial fibrillary acidic protein in astrocytes of the adult rat optic nerve［J］.Brain Res，1990，532（1-2）：355-357.
［20］　VARELA HJ，HERNANDEZ MR.Astrocyte responses in human optic nerve head with primary open-angle glaucoma［J］.J Glaucoma，1997，6（5）：303-313.
［21］　YANG P，HERNANDEZ MR.Purification of astrocytes from adult human optic nerve heads by immunopanning［J］.Brain Res Brain Res Prot，2003，12（2）：67-76.
［22］　WU N，XU JJ，ZHANG SH，BO Q，LE QH，WU JH，et al.Purification and characterization of adult optic nerve head astrocytes［J］.Chin J Ophthalmol，2012，48（7）：615-618.
武娜，徐建江，张圣海，伯齐，乐琦华，吴继红，等.成人视盘星形胶质细胞的纯化培养及鉴定［J］.中华眼科杂志，2012，48（7）：615-618.
［23］　MI H，BARRES BA.Purification and characterization of astrocyte precursor cells in the developing rat optic nerve［J］.J Neurosci，1999，19（3）：1049-1061.
［24］　SCHEEF E，WANG S，SORENSON CM，SHEIBANI N.Isolation and characterization of murine retinal astrocytes［J］.Mol Vis，2005，11：613-624.
［25］　KERSTETTER AE，MILLER RH.Isolation and culture of spinal cord astrocytes［J］.Methods Mol Biol，2012，814（1）：93-104.
［26］　CHOI HJ，SUN D，JAKOBS TC.Isolation of intact astrocytes from the optic nerve head of adult mice［J］.Exp Eye Res，2015，137（2）：103-110.
［27］　RICARD CS，KOBAYASHI S，PENA JD，SALVADOR-SILVA M，AGAPOVA O，HERNANDEZ MR.Selective expression of neural cell adhesion molecule（NCAM）-180 in optic nerve head astrocytes exposed to elevated hydrostatic pressure in vitro［J］.Brain Res Mol Brain Res，2000，81（1-2）：62-79.
［28］　MENET V，GIMNEZ Y RIBOTTA M，CHAUVET N，DRIAN MJ，LANNOY J，COLUCCI-GUYON E，et al.Inactivation of the glial fibrillary acidic protein gene，but not that of vimentin，improves neuronal survival and neurite growth by modifying adhesion molecule expression［J］.J Neurosci，2001，21（16）：6147-6158.
［29］　LIU B，CHEN H，JOHNS TG，NEUFELD AH.Epidermal growth factor receptor activation：an upstream signal for transition of quiescent astrocytes into reactive astrocytes after neural injury［J］.J Neurosci，2006，26（28）：7532-7540.
［30］　ZHANG X，NEUFELD AH.Activation of the epidermal growth factor receptor in optic nerve astrocytes leads to early and transient induction of cyclooxygenase-2［J］.Invest Ophthalmol Vis Sci，2005，46（6）：2035-2041.

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备注/Memo

备注/Memo:
国家自然科学基金资助（编号：81260148）
更新日期/Last Update: 2018-01-22