表达VASH1基因的人脑胶质瘤U-87MG细胞对化疗药物敏感性的变化

目的 探讨表达人血管生成抑制因子１（VASH1）的人脑胶质瘤U-87MG细胞对化疗药物的敏感性变化。方法 构建针对VASH1的慢病毒载体pGCL-GFP-VASH1,经测序鉴定后转染293T细胞,筛选出适合浓度的慢病毒转染人脑胶质瘤U-87MG细胞,荧光显微镜下检测转染效率;通过RT-PCR和Western blot分析U-87MG细胞VASH1 mRNA和蛋白表达水平;用CCK-8法检测U-87MG细胞在化疗药物顺铂和替莫唑胺作用下的存活率。流式细胞仪检测U-87MG细胞凋亡。结果 成功构建pGCL-GFP-VASH1慢病毒载体,并成功转染U-87MG细胞,转染率达70％以上;RT-PCR和Western blot结果证实转染VASH1慢病毒载体的U-87MG细胞表达VASH1 mRNA和蛋白。在顺铂或替莫唑胺作用下,表达VASH1的U-87MG细胞存活率均较未表达VASH1的U-87MG细胞明显降低（P<0.01）,而且U-87MG细胞凋亡率明显增加（P<0.01）。结论 VASH1慢病毒载体转染U-87MG细胞可使其稳定表达VASH1,并提高人脑胶质瘤U-87MG细胞对化疗药物敏感性、增加细胞凋亡率。     

利用RNA干扰技术抑制Cyr61表达对人脑胶质瘤细胞药物敏感性和凋亡的影响

目的 探讨RNA干扰技术抑制Cyr61基因表达增强人脑胶质瘤U251细胞对化疗药物敏感性及凋亡能力的研究.方法 针对Cyr61 Mrna序列设计合成小干扰RNA(siRNA)的DNA模板,构建Prnat-Cyr61重组表达载体,转染人脑胶质瘤U251细胞:通过RT-PCR和Western blot分析其对U251细胞内源性Cyr61表达的影响;用四甲基偶氮唑蓝(MTT)法观察U251细胞对化疗药物顺铂和替莫唑胺敏感性的改变;用流式细胞仪检测U251细胞体外凋亡的变化.结果 成功构建Prnat-Cyr61重组质粒,并成功转染U251细胞;RT-PCR和Western blot结果证实重组质粒在Mrna及蛋白水平分别显著抑制Cyr61基因表达(P<0.01);MTT及流式细胞仪结果证明在和顺铂或替莫唑胺联合作用下,Prnat-Cyr61组U251细胞的生长抑制率和凋亡率都明显增高(P<0.01).结论 Prnat-Cyr61可抑制Cyr61在人脑胶质瘤U251细胞中的表达,并增强U251细胞对化疗药物敏感性及其体外凋亡率.     

胶质瘤治疗的靶分子CDC2 RNA干扰的体外实验研究

目的 探讨抑制细胞周期依赖性激酶CDC2基因对人脑胶质瘤细胞生长的影响,并为其作为胶质瘤发生发展相关基因在胶质瘤治疗方面的应用价值提供实验依据.方法 利用本实验室基因重组构建的CDC2 shRNA逆转录病毒表达质粒对体外培养的人脑胶质瘤细胞进行转染.倒置显微镜观察细胞形态和数量变化;RT-PCR及荧光实时定量PCR检测CDC2 mRNA表达;Western blot检测CDC2蛋白表达;流式细胞仪检测细胞周期和凋亡的变化;透射电子显微镜观察细胞超微结构.结果 人脑胶质瘤细胞系U251在转染针对CDC2的shRNA逆转录病毒表达质粒48h后,贴壁生长的细胞逐渐漂浮,细胞生长受到抑制,CDC2 mRNA下调均达98%以上,蛋白表达下调达92%以上,同时出现了明显的细胞G2/M期阻滞,细胞凋亡由0.57%上升到13.01%,并且超微结构受损明显.结论 CDC2 shRNA逆转录病毒表达质粒介导的RNA干扰能有效的抑制U251细胞的增殖,CDC2可作为胶质瘤基因治疗靶分子作进一步研究.     

过表达钙整合素结合蛋白CIB诱导SHG44胶质瘤细胞凋亡的实验研究

目的 观察CIB基因对人胶质瘤SHG44细胞体外生长和细胞凋亡的影响.方法 构建重组质粒pcDNA3-CIB,转染人胶质瘤细胞株SHG44,MTT观察各组细胞的体外生长情况,流式细胞术(FCM)测定各组细胞的细胞周期和凋亡细胞数量,Western-blot检测CIB转染后凋亡相关蛋白的表达变化.结果 RT-PCR、Western印迹显示pcDNA3-CIB转染组CIB的mRNA及CIB蛋白表达水平明显高于对照组;与对照组细胞相比,转染CIB的SHG44-CIB组细胞牛长明显减缓(P＜0.01),SHG44-CIB组细胞G_1、S期细胞减少,G_2期细胞增多,凋亡细胞增加至19.2%;凋亡相关蛋白Bcl-2表达下调,Bax表达上调.结论 CIB在体外明显抑制SHG44细胞的生长并诱导其发生凋亡,CIB诱导的凋亡可能与Bcl-2及Bax表达的变化相关.     

RNAi沉默Gli1基因对人胶质瘤细胞U251增殖与凋亡的影响

目的 研究RNA干扰技术(RNAi)沉默Hedgehog(Hh)信号转导途径中核心转录因子Gli1基因表达后对U251细胞增殖与凋亡以及下游因子Bcl-2、Bax、cycin D1表达的影响。 方法 合成、转染4对针对Gli1 mRNA不同位点序列的siRNA (58、59、60、61)至人胶质瘤U251细胞,RT-PCR检测细胞Gli1 mRNA的表达,筛选最有效抑制Gli1 mRNA表达的siRNA干扰片段(siRNA-Gli1);RT-PCR、Western blotting分别检测转染SiRNA-Gli后不同时间U251细胞Gli1mRNA和蛋白的表达,确定转染干扰的时间规律;实验分为3组：siRNA-Gli1组（转染筛选后siRNA-Gli1片断）、siRNA-NC组（转染阴性对照siRNA片断）、siRNA-N组（空白对照）。RT-PCR、Western blotting分别检测各组转染48 h后U251细胞cyclin D1、Bcl-2及Bax mRN和蛋白的表达,MTT、流式细胞术分别检测细胞增殖、凋亡及细胞周期的变化。 结果 干扰片断58、59、60、61、NC的转染效率均达69.2％;RT-PCR检测显示转染后48 h U251-60细胞无明显Gli1 mRNA表达,选择60作为最佳干扰片段siRNA-Gli1转染U251细胞,48h时无明显Gli1 mRNA和蛋白表达,确定48 h为转染干扰的最佳时间;转染48 h后与siRNA-N和siRNA-NC组相比,siRNA-Gli1组U251细胞Bcl-2、cyclin D1 mRNA和蛋白表达下调、Bax mRNA和蛋白上调,差异均有统计学意义(P＜0.05)。MTT检测显示24、48和72 h时siRNA-Gli1组细胞增殖抑制率均明显高于siRNA-NC和siRNA-N组,差异均有统计学意义(P＜0.05)。流式细胞术结果显示siRNA-Gli1组细胞凋亡率高于siRNA-NC和siRNA-N组,差异有统计学意义(P＜0.05),且siRNA-Gli1组G1/G0期细胞比例增加,S期细胞明显减少。 结论 针对Gli1基因设计合成的siRNA-Gli1能明显抑制人胶质瘤U251细胞生长并能诱导其凋亡,其机制可能与下调cyclin D1、Bcl-2的表达,上调Bax的表达有关。     

LRIG2基因全长及胞外段对胶质瘤细胞系U251细胞增殖和凋亡的影响

目的探讨富含亮氨酸重复序列免疫球蛋白样蛋白2（LRIG2）基因全长及胞外段对脑胶质瘤细胞系U251细胞增殖和凋亡的影响。方法将LRIG2全长,LRIG2胞外段（LRIG2_ecto）及空白质粒（对照）经慢病毒法分别转染胶质瘤细胞系U251细胞,筛选稳定细胞株,实时PCR及免疫印迹法检测mRNA及蛋白表达,细胞增殖检测试剂盒检测细胞增殖,流式细胞分析检测细胞周期及细胞凋亡。结果两转染组Flag标签蛋白表达成功;两转染组LRIG2全长及LRIG2_ectomRNA表达较对照组明显升高（氏0．01）;两转染组细胞增殖率均明显高于对照组（P〈0．01）;两转染组s期与G2/M期细胞数之和（即细胞增殖指数）均明显高于对照组（P〈0．01）,而细胞凋亡率均明显低于对照组（P〈0．05）。结论LRIG2基因全长及LRIG2_ecto促进胶质瘤细胞增殖,并将细胞周期阻滞于G2/M期,抑制细胞凋亡。     

外源性Bax基因过表达对神经母细胞瘤SH-SY5Y细胞株Smac蛋白表达的影响

目的观察Bcl-2相关X蛋白（Bax）基因过表达对人神经母细胞瘤SH-SY5Y细胞第二个线粒体来源的半胱氨酸酶的激活剂（Smac）蛋白表达的影响。方法采用脂质体Lipofectamine 2000将携带人Bax基因的过表达质粒转入SH-SY5Y细胞（转染组）,同时设空载体组和未转染组。通过G-418筛选后获得转入目的基因的细胞克隆。Hoechst33258染色观察凋亡细胞,免疫印迹法法检测细胞中Bax、Smac蛋白表达,免疫荧光染色检测Bax、Smac蛋白在细胞中的分布。结果转染后用G-418持续筛选2周可获得G418抗性的细胞克隆。Smac主要分布于胞质,Bax主要位于细胞核。转染组SH-SY5Y细胞Bax蛋白表达水平（1.067±0.036）较空载体组（0.436±0.035）和未转染组（0.444±0.046）显著增高（P〈0.05）;转染组Smac蛋白表达水平（1.408±0.044）相比空载体组（0.708±0.021）和未转染组（0.748±0.041）显著增高（P〈0.05）;空载体和未转染组细胞Bax和Snac蛋白表达均无显著差异（P〉0.05）。转染组较未转染组细胞凋亡率显著增加（P〈0.05）。结论外源性Bax基因转入人神经母细胞瘤细胞中并稳定过表达,能够上调Smac蛋白表达,促进细胞凋亡。     

华蟾素对人脑胶质瘤细胞SHG44增殖及凋亡的影响

目的初步探讨华蟾素对人脑胶质瘤细胞SHG44增殖、细胞周期分布及凋亡的影响机制。方法利用MTT法、流式细胞仪检测华蟾素对人脑胶质瘤细胞SHG44增殖及细胞周期变化的影响。AO／EB染色、透射电镜观察人脑胶质瘤细胞SHG44凋亡细胞形态学的变化。Westernblot检测Bcl-2与Caspase8蛋白的表达。结果华蟾素（1μg／ml、10μg／ml）对细胞株增殖具有显著抑制作用（P〈0．05）,并呈时间和浓度依赖性。沆式细胞仪检测可见凋亡峰,Go／G。期细胞明显增多（P〈0．05）;华蟾素作用后可使Bcl-2蛋白表达降低．Caspase8蛋白表达升高。结论华蟾素可调控人脑胶质瘤细胞SHG44的周期进程,诱导Bcl-2及Caspase8差异性表达,且具有抑制肿瘤细胞增殖及促凋亡的作用。     

替尼泊苷诱导脑胶质瘤细胞凋亡的检测指标评估

目的：观察化疗药物替尼泊苷在脑胶质瘤化疗过程中诱导的细胞凋亡,探讨进行临床疗效有效监控的评价指标。方法：从40例脑胶质瘤组织中分离胶质瘤细胞,用替尼泊苷对培养的胶质瘤细胞分别干预24、48和72h。用流式细胞技术测定胶质瘤细胞线粒体膜电位（MMP）、细胞周期、Bcl-2和Bax水平。结果：随着替尼泊苷干预时间的延长,MMP降低的百分率和细胞周期凋亡峰比例均增加,Bcl-2／Bax的比值下降。结论：替尼泊苷在脑胶质瘤化疗过程中诱导细胞凋亡的效果显著。在凋亡检测中MMP为最敏感和快捷的检测指标。     

β-淀粉样肽(25-35)对PC12细胞前列腺凋亡反应蛋白-4和bcl-2基因表达的影响

目的观察β-淀粉样肽（25-35）（β-amyloid peptide 25-35，Aβ-25-35）诱导PC12细胞凋亡及对前列腺凋亡反应蛋白-4（prostate apoptosis response-4，par-4）和bcl-2基因表达的影响。方法采用MTT比色法分析细胞存活率，Hoechst33258-PI荧光染色观察细胞核凋亡的形态学改变，RT-PCR检测par-4和bcl-2基因mRNA表达变化，Western blot检测Par-4和Bcl-2蛋白表达变化。结果 随Aβ23-35浓度的增加，PC12细胞存活率降低，荧光染色可见细胞核固缩、核碎裂，par-4 mRNA及蛋白表达增加，bcl-2 mRNA及蛋白表达降低。结论 在Aβ25-35诱导PC12细胞凋亡过程中Par-4和Bcl-2蛋白分子可能起重要作用。     

L-ascorbic acid-induced DNA strand breaks and cross links in human neuroblastoma cells

The effect of high concentrations of L-ascorbic acid on the in vivo and in vitro growth of human neuroblastoma has been investigated. Directly implemented into cell culture it decreased the DNA, RNA and protein synthesis, and mitosis of neuroblastoma cells, without affecting normal neuronal cells. In vivo treatment of young nude mice bearing human neuroblastoma with 500 mg/kg L-ascorbic acid for the first 10 days markedly inhibited the growth of tumor mass. As determined by alkaline elution, both DNA strand breaks and DNA cross links were observed in tumor cells treated with 1 X 10(-4) M L-ascorbic acid for 2 h. DNA-DNA and DNA-protein cross links in cells treated with L-ascorbic acid were revealed by the proteinase potassium assay. The results indicated that L-ascorbic acid can be a very effective and selective agent against human neuroblastoma.     

l-Ascorbic acid-induced DNA strand breaks and cross links in human neuroblastoma cells

The effect of high concentrations ofl-ascorbic acid on the in vivo and in vitro growth of human neuroblastoma has been investigated. Directly implemented into cell culture it decreased the DNA, RNA and protein synthesis, and mitosis of neuroblastoma cells, without affecting normal neuronal cells. In vivo treatment of young nude mice bearing human neuroblastoma with 500 mg/kgl-ascorbic acid for the first 10 days markedly inhibited the growth of tumor mass. As determined by alkaline elution, both DNA strand breaks and DNA cross links were observed in tumor cells treated with 1 × 10⁻⁴ Ml-ascorbic acid for 2 h. DNA-DNA and DNA-protein cross links in cells treated withl-ascorbic acid were revealed by the proteinase potassium assay. The results indicated thatl-ascorbic acid can be a very effective and selective agent against human neuroblastoma.     

Genistein induces receptor and mitochondrial pathways and increases apoptosis during BCL‐2 knockdown in human malignant neuroblastoma SK‐N‐DZ cells

The potent antiapoptotic molecule Bcl‐2 is markedly up‐regulated in a majority of cancers, including neuroblastoma. Genistein is an isoflavone with antitumor properties. The present study sought to elucidate the molecular mechanism of genistein‐induced apoptosis and also to examine the effect of genistein in increasing apoptosis during Bcl‐2 knockdown in human malignant neuroblastoma SK‐N‐DZ cells. The cells were transfected with Bcl‐2 siRNA plasmid vector, treated with 10 μM genistein, or the combination, and subjected to TUNEL staining and FACS analysis. Semiquantitative and real‐time RT‐PCR experiments were performed for examining expression of Fas ligand (FasL), tumor necrosis factor‐α (TNF‐α), Fas‐associated death domain (FADD), and TNFR‐1‐associated death domain (TRADD). The cell lysates were analyzed by Western blotting for levels of molecules involved in both receptor‐ and mitochondria‐mediated apoptotic pathways. Treatment with the combination of Bcl‐2 siRNA and genistein resulted in more than 80% inhibition of cell proliferation. TUNEL staining and FACS analysis demonstrated apoptosis in 70% of cells after treatment with the combination of both agents. Apoptosis was associated with increases in Bax:Bcl‐2 ratio, mitochondrial release of cytochrome c, and activation of caspases through the mitochondria‐mediated apoptotic pathway. Genistein triggered the receptor‐mediated apoptotic pathway through upregulation of TNF‐α, FasL, TRADD, and FADD and activation of caspase‐8. Combination of Bcl‐2 siRNA and genistein triggered a marked increase in cleavage of DFF45 and PARP that resulted in enhanced apoptosis. Our study demonstrates that Bcl‐2 knockdown during genistein treatment effectively induced apoptosis in neuroblastoma cells. Therefore, this strategy could serve as a potential therapeutic regimen to inhibit the growth of human malignant neuroblastoma. © 2009 Wiley‐Liss, Inc.     

Cultured neurons release an inhibitor of astroglia proliferation (astrostatine)

Using in vitro techniques, we looked for a possible downregulation of rat astroglia proliferation by neuronal cells. We demonstrate that medium conditioned by 7-day-old rat cerebellar granule neurons or by 16-day-old rat embryo hippocampal neurons strongly inhibits the proliferation of cultured astroglial cells. Two neuronal cell lines, the PC12 rat pheocromocytoma and the neuro 2A (N2A) murine neuroblastoma also release such an activity. This release in N2A-conditioned medium (CM) occurs when the cells are at high density and show a low proliferation rate. This activity is present in media conditioned by neuronal cells, but not in media conditioned by normal astrocytes, by two glioma cell lines, or by one fibroblastic cell line. This proliferation inhibitor addresses normal astrocytes: the proliferation of two glioma cell lines, of a fibroblastic cell line, and of the two neuronal cell lines (PC12, N2A) is not inhibited by N2A CM. Moreover, this activity is directed against type 1 astrocytes, but not against type 2. Using three different assays, we demonstrate that DNA synthesis by astroglial cells is inhibited. N2A CM has no cytotoxic effect on astrocytes and does not modify their overall protein synthesis. Using affinity and gel filtration chromatography, we show that this activity is associated with a protein whose molecular weight ranges between 15 and 20 kDa. The possible relationship between this N2A cell-derived astroglia proliferation inhibitor and other types of potential glial proliferation inhibitors has been investigated. A brain glycoprotein immunologically related to epidermal growth factor receptor (EGFR) was reported to inhibit astroglial cell proliferation in vitro. Using polyclonal and monoclonal antibodies against EGFR, we were unable to immunoprecipitate the astrocyte proliferation inhibitor in N2A CM or to demonstrate by immunoblotting the presence of an EGFR-like immunoreactivity in the N2A CM or in the active chromatographic fractions of N2A CM. Transforming growth factor beta (TGF beta) is a well-known modulator of the proliferation of various cell types and was shown to be present in N2A CM. Using a polyclonal anti-TGF beta antibody that recognizes TGF beta on Western blots of N2A CM, we were unable to immunoprecipitate the astrocyte proliferation inhibitor of N2A CM. It seems thus far that the neuronal astroglia proliferation inhibitor is a new protein for which we propose the name astrostatine.     

Endogenous inhibitors of InsP3-induced Ca2+ release in neuroblastoma cells

Cerebellar Purkinje neurons and neuroblastoma N1E-115 cells require 10-50 times more InsP3 to induce Ca2+ release than do a variety of non-neuronal cells (including astrocytes, hepatocytes, endothelial cells, or smooth muscle cells). Given the importance of InsP3-induced Ca2+ release for the development of synaptic plasticity in Purkinje neurons, a low InsP3 sensitivity may facilitate the integration of numerous synaptic inputs before initiating a change in synaptic strength. In the present study, attention is directed at the mechanism underlying this low InsP3 sensitivity of Ca2+ release. We show that permeabilization of neuroblastoma cells with saponin increased InsP3 sensitivity of Ca2+ release, indicating the presence of a diffusible, cytosolic inhibitor(s) of Ca2+ release. Consistent with this hypothesis, gel filtration of the neuroblastoma cytosol yielded three peaks that inhibited InsP3-induced Ca2+ release from permeabilized cells. The prominent inhibitory peak decreased the InsP3 sensitivity of Ca2+ release from permeabilized cells, did not bind 3H-InsP3, and was present in sufficient levels to account for the low InsP3 sensitivity of Ca2+ release in intact neuroblastoma cells. Purification of this prominent inhibitory fraction yielded a protein band that was identified by mass spectrometry as stress-induced phosphoprotein 1 (mSTI1). Furthermore, immunoprecipitation of mSTI1 decreased the inhibitory activity of N1E-115 cytosol, indicating that mSTI1 contributes to the inhibition of InsP3-induced Ca2+ release. Thus, the low InsP3 sensitivity of Ca2+ release in neuroblastoma cells can be explained by the presence of cytosolic inhibitors of Ca2+ release and include stress-induced phosphoprotein 1.     

Brain-derived neurotrophic factor mediates macrophage migration inhibitory factor to protect neurons against oxygen-glucose deprivation

Macrophage migration inhibitory factor(MIF)is a chemokine that plays an essential role in immune system function.Previous studies suggested that MIF protects neurons in ischemic conditions.However,few studies are reported on the role of MIF in neurological recovery after ischemic stroke.The purpose of this study is to identify the molecular mechanism of neuroprotection mediated by MIF.Human neuroblastoma cells were incubated in Dulbecco’s modified Eagle’s medium under oxygen-glucose deprivation(OGD)for 4 hours and then returned to normal aerobic environment for reperfusion(OGD/R).30 ng/mL MIF recombinant(30 ng/mL)or ISO-1(MIF antagonist;50μM)was administered to human neuroblastoma cells.Then cell cultures were assigned to one of four groups:control,OGD/R,OGD/R with MIF,OGD/R with ISO-1.Cell viability was analyzed using WST-1 assay.Expression levels of brain-derived neurotrophic factor(BDNF),microtubule-associated protein 2(MAP2),Caspase-3,Bcl2,and Bax were detected by western blot assay and immunocytochemistry in each group to measure apoptotic activity.WST-1 assay results revealed that compared to the OGD/R group,cell survival rate was significantly higher in the OGD/R with MIF group and lower in the OGD/R with ISO-1 group.Western blot assay and immunocytochemistry results revealed that expression levels of BDNF,Bcl2,and MAP2 were significantly higher,and expression levels of Caspase-3 and Bax were significantly lower in the MIF group than in the OGD/R group.Expression levels of BDNF,Bcl2,and MAP2 were significantly lower,and expression levels of Caspase-3 and Bax were significantly higher in the ISO-1 group than in the OGD/R group.MIF administration promoted neuronal cell survival and induced high expression levels of BDNF,MAP2,and Bcl2(anti-apoptosis)and low expression levels of Caspase-3 and Bax(pro-apoptosis)in an OGD/R model.These results suggest that MIF administration is effective for inducing expression of BDNF and leads to neuroprotection of neuronal cells against hypoxic injury.     

Annals of Neurology: Volume 86, Number 3, September 2019

A fluorescence photomicrograph showing mitochondria in a human neuroblastoma cell line, SH‐SY5Y. The nuclei of the cells are stained blue with DAPI and the mitochondria are stained gold with a combination of mitotracker red and antibody against ssbp1, a single‐stranded DNA binding protein that is necessary for replication of mitochondrial DNA. Green punctate structures may indicate the presence of mitochondrial nucleoids. Mutations in SSBP1 may result in autosomal dominant optic atrophy with variable degree retinal degeneration. See article by Jurkute N and colleagues in this issue (pp. 368–383 ).     

Differential sensitivity of neural cells to bilirubin toxicity

Two established neural cell lines were used to examine the cytotoxicity of bilirubin in vitro. N2AB-1 is a subclone of Neuro 2a from the original C1300 mouse neuroblastoma and C6 glioma is a rat astrocytoma cell line. These cells were grown in monolayer cultures in medium with 10% fetal calf serum containing doses of 5 to 42 microM bilirubin. Morphologic and biochemical characteristics of cell viability were monitored for 72 h. Additional cultures of N2AB-1 were differentiated by treatment with prostaglandin E1/cyclic adenosine monophosphate before exposure to various bilirubin concentrations. The cells were examined every 24 h by phase contrast microscopy and protein synthesis was measured by incorporation of tritiated leucine for appropriate times. N2AB-1 cells were extremely sensitive to bilirubin within 24 h at doses greater than 11 microM. Cells showed swelling, vacuole formation, pigment accumulation, and lift off from the growing surface. Growth of N2AB-1 over 3 days was inhibited in a dose-dependent manner. Moreover, protein synthesis 6 h after bilirubin exposure and 24 h after bilirubin exposure was inhibited in the same dose dependency as cell growth. In contrast, N2AB-1 cells morphologically differentiated by drug treatment showed no effect of bilirubin exposure. Also, mitotically active rat glial cells were resistant to bilirubin toxicity under similar conditions. This study demonstrates that marked differences exist among neural cells in susceptibility in vitro to bilirubin toxicity and that mitotically active neuronal cells are more sensitive to bilirubin treatment than "mature" neurons.