SD大鼠星形胶质细胞的原代培养

目的 设计原代培养SD大鼠星形胶质细胞。方法 取新生第2-3dSD红皮鼠，无菌操作下取大脑皮质，剪碎、胰酶消化结合机械吹打使细胞分散，网筛过滤，差速粘附处理去除成纤维细胞，将未粘附的细胞悬液接种培养9～12d，置摇床．舍弃含脱落细胞的细胞悬液，以去除少突胶质细胞和小胶质细胞。细胞传代，GFAP免疫组化染色鉴定。结果 功分离培养了原代神经胶质细胞，并进行了星形胶质细胞的纯化，GFAP鉴定星形胶质细胞比例为95％以上。结论 建立星形胶质细胞体外培养方法，总结操作关键，以此为实验材料对于比较学研究是非常有帮助的。     

小鼠脊髓原代星形胶质细胞培养方法的建立

目的获得纯度较高的脊髓源性星形胶质细胞。方法采用小鼠脊髓神经胶质细胞的原代培养和传代培养。无菌条件下取生后1～3dICR(Institute of Cancer Research)小鼠的脊髓,剪碎后胰酶分次消化,结合机械吹打使细胞分散,制成单细胞悬液,接种于无底物黏附的玻璃培养瓶中,置37℃,5%CO2培养箱中培养。培养液为DMEM/F12混合培养液。培养9～12d待细胞达到80%～90%融合后进行摇床纯化,舍弃含脱落细胞的细胞悬液,以去除少突胶质和小胶质细胞,继续培养,待细胞铺满瓶底后传代,并对传代细胞进行形态观察和星形胶质细胞的免疫荧光鉴定,GFAP免疫细胞化学染色阳性细胞可高达98%。结果通过恒温振荡和传代后得到了形态典型,含量较高的脊髓源性星形胶质细胞。结论用无底物贴附,恒温振荡和传代方法可获得高纯度脊髓源性星形胶质细胞。     

体外血脑屏障模型的建立及功能测定

目的利用大鼠原代微血管内皮细胞及星形胶质细胞建立体外血脑屏障模型,并通过跨内皮细胞电阻(Trans-epithelium electrical resistant,TEER)方法对血脑屏障模型进行功能测定。方法原代分离纯化SD大鼠脑微血管内皮细胞和星形胶质细胞,用免疫荧光检测内皮细胞标志物VWF,紧密连接蛋白ZO-1,星形胶质细胞标志物GFAP;用微血管内皮细胞和星形胶质细胞在Transwell小室上建立体外血脑屏障模型,观察TEER值的动态变化。结果原代的微血管内皮细胞培养至融合后具有典型的梭形"铺路石"样外观,VWF鉴定细胞纯度达到95%以上,ZO-1免疫荧光鉴定证实细胞间形成紧密连接;原代培养的星形胶质细胞呈现具有多个突起的典型形态,GFAP鉴定细胞纯度达到95%以上;在第10 d,单独微血管内皮细胞血脑屏障模型的TEER值为(42±1.41)Ωcm2,内皮细胞和星形胶质细胞共培养血脑屏障模型的TEER值为(65±1.42)Ωcm2。结论建立了体外血脑屏障模型,通过TEER值测定证明共培养使模型更加完整,更加接近在体血脑屏障模型的特性。     

液压性脑损伤后室下区神经干细胞的分离培养与鉴定

目的分离液压性脑损伤室下区巢蛋白（nestin）和胶质酸性纤维蛋白（GFAP）阳性（nestin^＋／GFAP^＋）共存细胞进行培养和诱导分化，观察其分裂、增殖和分化能力，以阐明损伤反应性星形胶质细胞增生过程中nestin^＋／GFAP^＋共存细胞是否具有神经干细胞特性。方法用液压冲击法建立动物模型，分离损伤成年SD大鼠室下区nestin^＋／GFAP^＋共存细胞，制成单细胞悬液，培养和诱导分化，以免疫荧光化学方法对原代和传代培养形成的神经球以及原代和传代培养诱导分化的细胞进行鉴定。结果结果显示培养及传代的细胞不断分裂增殖，可以形成悬浮生长nestin阳性的神经球；神经球诱导分化后可以分化为少突胶质细胞、神经元和星形胶质细胞。结论成年大鼠液压性脑损伤后分离的室下区细胞具有自我更新能力和多向分化潜能，是中枢神经系统神经干细胞。     

免疫磁珠法分离、培养人脑胶质瘤干细胞

目的建立免疫磁珠法分离，并培养人脑胶质瘤干细胞的方法。方法将术中取得的脑胶质瘤标本，通过剪切、消化和吹打成单细胞悬液，筛网过滤，免疫磁珠分选试剂盒分选出CD133^＋细胞，用神经干细胞无血清培养法培养出具有单细胞克隆能力的细胞球，取第3代进行诱导分化，分化前后用免疫细胞荧光化学方法鉴定肿瘤干细胞及分化后细胞。结果免疫磁珠分选出的CD133^＋细胞，可悬浮生长并形成神经干细胞样细胞球，有较强的增殖能力，干细胞标志物巢蛋白（nestin）阳性，分化后细胞表达神经元小管相关蛋白β-3（β-tubulin3）和星形胶质细胞胶质纤维酸性蛋白质（GFAP）特异性抗原，而巢蛋白、CD133^＋阴性，并具有肿瘤的核型。结论免疫磁珠分选法可避免原代培养中众多细胞混杂生长的发生，能够从大量肿瘤细胞中分离出只占极少比例的肿瘤干细胞，细胞结合磁珠后在体外可以长期培养和传代，进一步证实了肿瘤干细胞的存在，并为胶质瘤干细胞的研究奠定基础。     

TREK-1激动剂亚麻酸对星形胶质细胞谷氨酸诱导电流和摄取谷氨酸的影响

目的观察双孔钾通道TREK-1激动剂亚麻酸对星形胶质细胞谷氨酸诱导电流和摄取谷氨酸的影响。方法应用大鼠海马脑片膜片钳技术研究TREK-1激动剂亚麻酸对星形胶质细胞谷氨酸诱导电流的影响;原代培养大鼠皮层星形胶质细胞,采用免疫荧光双标法检测TREK-1在星形胶质细胞的表达,采用谷氨酸浓度检测法观察TREK-1激动剂亚麻酸对星形胶质细胞摄取谷氨酸的影响。结果 TREK-1在星形胶质细胞上表达丰富,谷氨酸可诱导星形胶质细胞产生内向电流,与对照组相比,给予TREK-1激动剂亚麻酸干预后星形胶质细胞谷氨酸诱导电流显著增加(P0.05),并显著增强原代培养的星形胶质细胞摄取谷氨酸能力(P0.05)。结论 TREK-1在星形胶质细胞上表达,TREK-1激动剂亚麻酸显著增加星形胶质细胞谷氨酸诱导电流,增强原代培养的星形胶质细胞摄取谷氨酸能力,提示TREK-1活性改变与星形胶质细胞平衡缓冲功能密切相关。     

丹参酮ⅡA对星形胶质细胞细胞周期和增殖的影响

目的探讨丹参酮ⅡA对体外培养星形胶质细胞细胞周期和增殖的影响。方法本研究采用体外细胞培养方法获得新生SD小鼠大脑皮质星形胶质细胞,然后在培养液中分别加入不同浓度的丹参酮ⅡA,作用8 h及24 h后,通过流式细胞仪检测丹参酮ⅡA对星形胶质细胞细胞周期的影响。结果丹参酮ⅡA作用于星形胶质细胞后,可引起细胞周期的显著变化,表现为G0/G1期细胞数减少,S期、G2/M期细胞数增多。结论丹参酮ⅡA可促进体外培养的星形胶质细胞增殖。     

NPPB对星形胶质细胞增殖的抑制作用

目的观察体积调节性阴离子通道阻滞剂NPPB对体外培养星形胶质细胞增殖的影响。方法采用原代培养的大鼠大脑皮层星形胶质细胞,实验分为对照组(正常培养基培养)与NPPB干预组;在不同时间点(0、6、12、24、48h),应用流式细胞技术以及免疫细胞荧光双标法(Brdu/DAPI)检测各实验组星形胶质细胞增殖及细胞周期进展的情况。结果与对照组相比,NPPB干预组在12、24h时星形胶质细胞增殖率较正常对照组降低(P<0.05),同时处于S期细胞的百分率亦相对正常对照组明显下降(P<0.01)。结论体积调节性氯离子通道阻滞剂NPPB可以显著抑制体外培养星形胶质细胞增殖及细胞周期进展,提示VRAC通道参与了体外培养星形胶质细胞的增殖。     

Nogo-66受体分子在体外培养的星形胶质细胞中的表达

目的检测Nogo-66受体（NgR）在体外培养的大鼠星形胶质细胞中的表达。方法利用RT—PCR、WesteiTl Blot和间接免疫荧光染色技术，检测原代培养并纯化的星形胶质细胞中NgR mRNA和蛋白分子的表达。结果利用RT—PCR技术，从星形胶质细胞总RNA中扩增出特异的NgR条带；利用Western Blot技术，从星形胶质细胞抽提物中检测到64kD特异性的NgR反应条带；间接免疫荧光染色和Cotffocal显微镜进一步显示，星形胶质细胞中的NgR免疫反应物主要位于细胞内。同时，来源于大鼠的C6胶质瘤细胞也证明表达NgR蛋白。结论体外培养的星形胶质细胞中表达NgR分子，这为证明NgR在体内胶质细胞中的表达提供了更直接的证据。     

缺氧/复氧对体外培养星形胶质细胞脑源性神经营养因子合成及释放的影响

目的观察缺氧/复氧条件下体外培养星形胶质细胞活力变化及脑源性神经营养因子(BDNF)释放和表达的变化。方法采用原代培养的大鼠皮质星形胶质细胞,实验分为正常组(N)及缺氧/复氧组(H/R)。在缺氧/复氧各个时间点,应用MTT法检测缺氧/复氧时培养星形胶质细胞的活力变化,应用Western blot检测BDNF的表达水平;ELISA检测星形胶质细胞条件培养液上清中BDNF的含量。结果与对照组相比,在缺氧6 h、复氧72 h以内体外培养星形胶质细胞,细胞活力不会发生明显改变,BDNF的释放量无明显变化,但是缺氧/复氧可诱导体外培养星形胶质细胞BDNF表达量的增加。结论单纯的缺氧/复氧条件可影响体外培养星形胶质细胞BDNF的合成,但不足以引起BNDF的释放改变以及细胞的活力变化。     

Astrogliosis in culture. IV. Effects of basic fibroblast growth factor

Previous studies have shown that the mechanical wounding of 3-week-old cultured rat astrocytes results in cell proliferation and hypertrophy resembling astrocyte responses to a brain injury in vivo. We now report the effects of basic fibroblast growth factor (bFGF) and an anti-bFGF antibody on astrocyte morphology, proliferation, and migration following in vitro wounding of confluent secondary cultures. Addition of bFGF (20 ng/ml) to wounded cultures induced morphological changes characteristic of differentiation in wounded and nonwounded areas of the culture. Combined treatment with bFGF and an anti-bFGF antibody (100 μg/ml) prevented this effect. Astrocyte proliferation along the edges of a scratch wound was at maximum 24 hr after wounding in cells growing in Eagle's minimum essential medium (EMEM) containing 10% serum. Low serum concentration and treatment with dibutyryl cyclic adenosine monophosphate (dbc-AMP) reduced injury-associated astrocyte proliferation. Addition of bFGF to cultures in EMEM with serum increased astrocyte proliferation at 18 and 24 hr after wounding. This effect was reduced considerably by treatment of cultures with bFGF in combination with an anti-bFGF antibody. The combined treatment and the antibody alone reduced cell division to a level lower than in control cultures. Twenty-four hr following wounding, astrocytes along the edges of the wound exhibited extension of thick, flat processes into the wound area. At 3 and 5 days after wounding, a bodily migration of astrocytes into the wounded area was observed. Addition of bFGF significantly increased astrocyte migration 1 day after wounding, with maximum effect on day 3 and no subsequent increase on day 5. A combination of bFGF and anti-bFGF antibody as well as the antibody alone reduced astrocyte migration to a level lower than in controls. Immunohistochemical localization and isoform pattern of bFGF in astrocytes did not change with dbc-AMP treatment or wounding. We conclude that mechanically wounded confluent astrocytes respond to bFGF added to the culture medium by enhancing cell division, differentiation, and migration. In addition, the results of the antibody treatment also suggest a role for endogenous bFGF in astrocyte proliferation and migration elicited by wounding in vitro. These results support the notion that in vivo, both bFGF released by injury and endogenous bFGF synthesized by astrocytes, contribute to the cellular responses that lead to astrogliosis. © 1995 Wiley-Liss, Inc.     

Primary culture of astrocytic glial cells from rainbow trout, Salmo gairdneri L., brain.

A method is described for the preparation and maintenance of rainbow trout brain astrocytes in primary culture. A dissociated cell suspension was prepared from brains from young fish by mechanical sieving through nylon guazes, and the cells cultured in polylysine-treated plastic tissue culture flasks at 22 degrees C. The resultant cultures were characterised by specific immunofluorescent staining using glial fibrillary acidic protein (GFAP) for astrocytes and Thy 1 for fibroblastic cells. The cultures were greater than 95% astrocytes with fibroblasts the only other cell type present. These highly enriched astrocyte cultures provide a unique system for various neurochemical, neurophysiological and biochemical studies of fish neural cells. Polyunsaturated fatty acid metabolism will be an area of particular interest.     

Insulin-like growth factor-I (IGF-I) production by astroglial cells: Regulation and importance for epidermal growth factor-induced cell replication

The insulin-like growth factor are postulated to play a role during brain development. Because they are believed to act in a paracrine/autocrine manner, the production of insulin-like growth factor-I (IGF-I) by cultured astroglial cells was examined. Quantities of IGF-I in conditioned media were determined by RIA after separation of IGFs from IGF-binding proteins by high-pressure liquid chromatography. Astrocytes from 1-day-old rats and the rat glioma cell line (C6) both secreted 7.5-kDa IGF-I. A peak of immunoreactivity with an apparent mol wt of 12,000 was additionally present in media conditioned by C6 cells. Exposure to epidermal growth factor (EGF) increased media content of immunocreactive IGF-I slightly (60%) in C6 cells but more than 2-fold in normal astrocytes. Fibroblast growth factor also increased the amount of IGF-I contained in media conditioned by normal astrocytes. To determine whether the secreted IGF-I was biologically active, media IGFs were immunoneutralized with a monoclonal antibody (Sm 1.25). In the presence of the antibody, EGF-stimulated astrocyte replication was blocked. These data indicate that IGF-I secretion by rodent astrocytes is stimulated by factors thought to be important for brain growth and development and that the IGFs are likely intimate participants in EGF-induced astrocyte growth. © 1993 Wiley-Liss, Inc.     

Isolation and characterization of living primary astroglial cells using the new GLAST-specific monoclonal antibody ACSA-1

Astrocytes show large morphological and functional heterogeneity and are involved in many aspects of neural function. Progress in defining astrocyte subpopulations has been hampered by the lack of a suitable antibody for their direct detection and isolation. Here, we describe a new monoclonal antibody, ACSA-1, which was generated by immunization of GLAST1 knockout mice. The antibody specifically detects an extracellular epitope of the astrocyte-specific L-glutamate/L-aspartate transporter GLAST (EAAT1, Slc1a3). As shown by immunohistochemistry, immunocytochemistry, and flow cytometry, ACSA-1 was cross-reactive for mouse, human, and rat. It labeled virtually all astrocytes positive for GFAP, GS, BLBP, RC2, and Nestin, including protoplastic, fibrous, and reactive astrocytes as well as Bergmann glia, Müller glia, and radial glia. Oligodendrocytes, microglia, neurons, and neuronal progenitors were negative for ACSA-1. Using an immunomagnetic approach, we established a method for the isolation of GLAST-positive cells with high purity. Binding of the antibody to GLAST and subsequent sorting of GLAST-positive cells neither interfered with cellular glutamate transport nor compromised astrocyte viability in vitro. The ACSA-1 antibody is not only a valuable tool to identify and track astrocytes by immunostaining, but also provides the possibility of separation and further analysis of pure astrocytes.     

Immunocytochemical localization of NaCh6 in cultured spinal cord astrocytes

The expression of the alpha-subunit of voltage-gated sodium channel 6 (NaCh6) was examined in cultures of astrocytes from E18 rat spinal cord by using an antibody specific for NaCh6. Stellate cells with processes and flat, pancake-like astrocytes are the two morphological types predominantly present in these cultures. The antibody to NaCh6 labeled clusters at the cell body and along the length of the processes in stellate, process-bearing cells. Weak staining was observed in the flat, pancake-like astrocytes. Together with previous studies (Black et al., Mol Brain Res 23:235-245, 1994, Glia 14:133-144, 1995) that show that stellate cells express NaChs II and III (but not NaCh I) and flat cells express NaCh II, these results support the conclusions that there are different patterns of sodium channel expression between flat and stellate astrocytes and that multiple channel isoforms are expressed within the same cell. This study also suggests that NaCh6 may contribute to the electrical properties found in stellate astrocytes.     

Neuronal and non-neuronal catechol-O-methyltransferase in primary cultures of rat brain cells

Previous biochemical and histochemical studies have suggested that catechol-O-methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers. COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18-day-old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double-staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, flat cells displaying type-1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type-2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane-bound 28-kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.     

A monoclonal antibody against vimentin: characterization

A monoclonal antibody was developed using rat cerebral cortex astrocytes purified in vitro as the antigenic material. Screening was done by labeling antibodies bound to cerebellum slices with fluorescent tagged secondary antibodies. The monoclonal antibody (RBA1) bound to intracellular filaments in all cells examined in culture. The coloration of tissue sections by RBA1 was identical to that described for polyclonal antibodies against the intermediate filament protein vimentin. In the brain this included binding to meninges, blood vessels, ependymal cells, choroid plexus lining cells and a subpopulation of astrocytes. The latter included Bergman glial fibers, white matter astrocytes and tanycytes. Müller cells in the retina and fibroblast-like cells in the skin, tongue and intestine were RBA1-positive. In immunoblots in which purified vimentin and desmin were run on SDS and transferred to nitrocellulose paper, RBA1 bound to vimentin but not desmin. When cultured astrocyte proteins were blotted, the antibody bound to both GFAP and vimentin, but no GFAP staining was observed in any of the tissue section staining. Purified vimentin blocked tissue and cultured cell binding of the antibody. Therefore RBA1 is considered to be an antibody specific for the intermediate filament protein vimentin.     

Purification of astrocytes from adult human optic nerve heads by immunopanning

Most in vitro studies in the CNS require pure cultures of astrocytes. Astrocytes from the human optic nerve head (ONH, type 1B) represent a specialized population of astrocytes. Primary cells grown from human optic nerve head explants were cultured for 3-4 weeks. To select astrocytes by immunopanning, cell suspensions were placed on a P100 panning dish coated with C5 anti-neuroepithelial antibody and allowed to attach for 30 min. Nonadherent cells were plated on a second dish coated with anti-Thy1.1 antibody to deplete microglia and meningeal cells. Finally, remnant nonadherent cells were plated on a noncoated dish. Purified cells were immunostained with astrocyte markers: GFAP, vimentin, Pax2, A2B5, nestin and NCAM. Other cell types were characterized by HLA-DR for microglia and smooth muscle actin for vascular smooth muscle. The proportion of GFAP+ astrocytes in the cultures was determined by flow cytometry. About 95% of the cells that adhered to the C5 dish were GFAP+ astrocytes. GFAP+ astrocytes expressed vimentin, Pax2, nestin and NCAM, but not A2B5. From the Thy1.1 dish, 60-75% cells were GFAP+ astrocytes and the remainder cells were GFAP- cells. Using cloning rings, we eliminated fibroblast-like cells, smooth muscle and meningeal cells from astrocyte cultures. Smooth muscle cells and fibroblasts grew on the noncoated dish. In conclusion, immunopanning is an efficient method to get high yields of viable type 1B astrocytes from adult human ONH. The current described culture system may provide a valuable tool in studying human optic nerve head biology and disease.     

Monoclonal antibody to intermediate filament proteins in astrocytes

A monoclonal antibody was developed using rat astrocytes purified in vitro as the starting antigenic material. Selection of the monoclonal was on the basis of astrocyte binding specificity in brain sections using indirect immunofluorescence techniques. The antibody (RBA2) that was chosen was specific for astrocytes in that it did not stain neurons or oligodendrocytes in frozen brain sections. It did, however, show binding to vascular smooth muscle and meningeal cells. The antigenic determinant(s) was determined to be on filaments of the intermediate-size class in cultured astrocytes and fibroblasts. From analysis of binding patterns in various tissues and in immunoblots, it was found that RBA2 cross-reacted strongly with glial fibrillary acidic protein (GFAP) and desmin. There was a weaker cross-reactivity to a vimentin-associated component. It is proposed that this antibody can be used as an astrocyte and blood vessel marker in brain sections, a vimentin marker in cultures and as a probe of intermediate filament composition and distribution.