缺氧诱导因子-1α在老年性痴呆模型鼠海马中的表达

目的 观察老年性痴呆(AD)大鼠行为学、海马神经细胞凋亡率及缺氧诱导因子(HIF)-1α表达水平的变化.方法 侧脑室注射Aβ25～35构建AD大鼠模型,Morris水迷宫实验测试大鼠空间学习记忆能力,流式细胞术检测海马神经细胞凋亡率, Western印迹技术检测大鼠海马HIF-1α蛋白的表达.结果 Morris水迷宫实验结果显示AD组大鼠平均逃避潜伏期显著延长(P<0.05),流式细胞分析结果显示,AD组海马神经细胞凋亡百分率较正常对照组显著升高(P<0.05),Western印迹检测结果显示,AD组海马神经细胞HIF-1α蛋白表达水平较正常对照组显著升高(P<0.05).结论 侧脑室注射Aβ25～35成功构建AD大鼠模型并上调海马神经细胞HIF-1α水平表达.     

远志皂苷对APP/PS1双转基因小鼠海马神经细胞线粒体的保护作用

目的观察远志皂苷对APP/PS1双转基因小鼠海马神经细胞线粒体功能的调控,探讨远志皂苷的神经保护作用机制。方法将APP/PS1双转基因小鼠分为模型组,远志皂苷低、中和高剂量组(18.5、37.0和74.0 mg·kg~(-1)·d~(-1)),选同月龄C57BL/6小鼠为空白对照组,采用免疫荧光检测海马神经细胞线粒体膜电位,流式细胞术检测细胞凋亡率,比色法等检测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)活力和丙二醛(MDA)含量。结果与空白对照组相比,模型组线粒体膜电位降低,神经细胞凋亡率增高,SOD,过氧化氢酶(CAT)和(GSH-PX)活性明显降低,MDA含量明显增高;与模型组相比,远志皂苷各剂量组的线粒体膜电位升高,神经细胞凋亡率下降,SOD,CAT和GSH-PX活性明显升高,MDA含量明显降低(P<0.05)。结论远志皂苷可能通过调控线粒体功能,发挥保护海马神经元的作用。     

改良型TAT-VP3融合蛋白对膀胱癌的抑制效应研究

目的探讨改良型TAT-VP3融合蛋白对膀胱癌细胞的抑制效应,为其临床应用于肿瘤的生物大分子药物治疗提供实验基础。方法运用免疫细胞化学染色法确定改良型TAT-VP3融合蛋白进入到膀胱癌细胞EJ的细胞核内,CCK-8检测EJ细胞的生长抑制率,通过透射电镜观察凋亡细胞的超微结构变化,流式细胞术检测细胞周期和细胞凋亡率。结果改良型TAT-VP3融合蛋白进入膀胱癌EJ细胞核内;经该融合蛋白作用后,EJ细胞凋亡率呈时间和剂量依赖关系,细胞周期随着药物作用浓度和时间的增加而改变,EJ细胞的生长抑制率随其浓度的增加和作用时间的延长而呈明显上升趋势。结论改良型TAT-VP3融合蛋白对膀胱癌EJ细胞的增殖具有一定的抑制作用,并对该细胞具有凋亡诱导作用。     

血管性痴呆大鼠学习记忆障碍及发病机制

目的 研究血管性痴呆(VD)大鼠学习记忆障碍及发病机制.方法 4血管阻断法制作模型,Morris水迷宫法检测学习记忆能力,免疫组化法观察Bcl-2和Bax表达,流式细胞术检测海马神经细胞凋亡率,亚甲蓝比色法检测血清中H2S含量.结果 模型组大鼠学习记忆能力明显减退.Bcl-2/Bax在缺血再灌注(I/R)开始时升高,随后开始下降.模型组大鼠海马神经细胞凋亡率明显增加.模型组大鼠血清中H2S含量明显减少.血清H2S含量与海马神经细胞凋亡率间呈明显负相关. 结论 VD可导致学习记忆障碍,海马神经细胞凋亡和血清中H2S含量减少;海马神经细胞凋亡程度可能与内源性H2S生成减少有关.     

肺癌平药物血清对A549人肺癌细胞凋亡及基因表达的影响

目的探讨肺癌平治疗肺癌的作用机制。方法采用血清药理学方法孵育A549人肺癌细胞，通过电镜、流式细胞术、免疫细胞化学方法检测肺癌平药物血清作用后对细胞凋亡及相关基因bcl-2、p53表达的影响。结果肺癌平药物血清作用后电镜下可见凋亡小体形成，流式细胞仪检测可见亚二倍体核型峰的特征，免疫细胞化学法显示抑癌基因p53主要定位在细胞核，部分细胞浆也有淡染。癌基因bcl-2主要表现为胞浆着色，流式细胞仪分析药物血清作用后，p53表达增强，bcl-2表达降低，随着作用时间延长，两种基因表达明显增强。结论肺癌平治疗肺癌的作用机制是通过诱导肺癌细胞凋亡及影响相关基因bcl-2、p53表达实现的。     

三氧化二砷对人恶性黑色素瘤A375细胞增殖及新癌基因PPO表达的影响

目的 探讨三氧化二砷对人恶性黑素瘤A375细胞株细胞周期、增殖、凋亡及新癌基因PPO(proliferation and phosphorylation oncogene)表达的影响.方法 培养人恶性黑色素瘤A375细胞株,采用 MTT法检测三氧化二砷在不同浓度和不同时间下对A375细胞增殖的影响,流式细胞术分析三氧化二砷对细胞周期及凋亡的影响,倒置相差显微镜观察药物处理后对人A375细胞的细胞分化及形态的影响,免疫细胞化学(SP)法检测三氧化二砷对PPO蛋白表达的影响.结果 MTT法证实三氧化二砷在1～10 μmol/L浓度范围内对A375细胞有明显的增殖抑制作用,呈现明显的剂量和时间依赖效应关系.流式细胞术检测发现,随着药物浓度的增加,G0/G1的细胞逐渐减少(P＜0.01),S期的比例显著增加,出现S期阻滞.倒置显微镜观察发现细胞出现典型凋亡细胞形态学改变.免疫细胞化学法检测PPO蛋白主要表达于A375细胞胞浆内,染色呈棕黄色.三氧化二砷作用48 h后,随着药物浓度的增加,PPO的染色程度逐渐减弱,差异具有显著性(P＜0.01).结论 三氧化二砷能够显著抑制A375细胞株增殖,并诱导凋亡发生,且与三氧化二砷的浓度和作用时间成正相关,其作用机制可能是通过下调新癌基因PPO的表达发挥作用.     

丹参对衰老鼠脑海马神经细胞凋亡作用的研究

目的：研究丹参对衰老鼠脑海马神经细胞凋亡的作用。方法：用TUNEL法及流式细胞仪观察衰老鼠的神经细胞凋亡,并用免疫组织化学法检测相关基因表达变化。以丹参注射液干预衰老的大鼠模型,观察了丹参对衰老鼠神经细胞凋亡的作用。结果：衰老鼠海马细胞有典型的凋亡特征,与丹参组比较海马细胞的凋亡率有明显差异,衰老鼠bcl-2表达下调,bax的表达量明显增加,基因表达变化与流式细胞仪测定的细胞凋亡率一致。结论：丹参对衰老鼠的脑神经细胞凋有明显的抑制作用。     

Aβ和氧自由基诱导的培养神经元凋亡及维生素E的保护作用

目的研究β淀粉样肽(Aβ)、反应活性氧(ROS)对原代培养鼠脑皮层神经细胞凋亡的诱导及维生素E(VE)可能的保护作用。方法通过形态学观察、MTT法检测、流式细胞技术分析、DNA琼脂糖凝胶电泳、免疫细胞化学技术研究Aβ、ROS对培养皮层神经细胞的损伤。结果经Aβ、活性氧的作用,神经细胞出现明显的凋亡特征,OD值降低,DNA电泳显示出现断裂DNA片段。凋亡细胞数增多。免疫细胞化学显示Bcl2表达下降,Bax表达增加(均P<0.05),而VE组可明显改变上述凋亡特征。结论Aβ、活性氧诱导培养皮层神经元凋亡机制可能与氧化损伤有关,维生素E能减缓这一作用。     

甘肃党参水提物对衰老模型小鼠脑细胞凋亡及相关基因表达的影响

目的 研究甘肃党参水提物对衰老模型小鼠脑细胞凋亡及相关基因表达的影响,初步明确其延缓衰老的作用机制.方法 50只昆明种小鼠随即分为5组,分别为青年对照组,D-半乳糖衰老模型组及党参低、中、高剂量组; 采用D-半乳糖致亚急性衰老小鼠模型,测定各组小鼠各剂量胸腺指数和脾脏指数;流式细胞仪观察海马细胞凋亡的变化;免疫组化法检测Bcl-2基因在海马及皮质神经元中的表达.结果 甘肃党参水提取物与衰老模型组比较,小鼠胸腺、脾指数明显升高(P＜0.05);抑制脑细胞凋亡;使细胞凋亡抑制基因Bcl-2的表达上调,党参水提物各组海马结构及皮质神经元内Bcl-2表达阳性比衰老模型组强.结论 党参水提物可提高机体免疫功能,上调Bcl-2的表达,抑制神经细胞凋亡.     

银杏内酯A对海马神经细胞凋亡保护作用的研究

目的探讨银杏内酯A(GKA)对凋亡海马神经细胞的影响及机制。方法用MTT法分析GKA对原代培养海马神经细胞的毒性作用,用浓度分别为0.1、1、10μmol/L的GKA预处理神经细胞6h,再用50μmol/LSNP孵育神经细胞24h;用流式细胞仪分析各组凋亡情况,用RT-PCR和Westernblot分析bcl-2、bax和cpp32 mRNA和蛋白表达水平,用试剂盒测定各组Cpp32活性。结果流式细胞术结果显示GKA实验组凋亡率均显著低于SNP组(P<0.01);RT-PCR结果和WesternBlot结果表明,GKA可以对抗SNP,上调bcl-2表达,下调bax表达,不影响Cpp32表达,但能显著降低Cpp32的活性(P<0.01)。结论GKA可以对抗SNP,对海马神经细胞有保护作用。     

氟对原代培养大鼠海马细胞氧化应激及凋亡的影响

目的观察氟诱导的大鼠原代培养海马细胞氧化应激损伤和凋亡及活性氧在细胞凋亡中的作用。方法采用原代细胞培养的方法,将大鼠海马细胞暴露于20、40、80 mg／L氟化钠中,24 h后检测海马细胞存活率、活性氧(ROS)、丙二醛(MDA)及细胞凋亡。结果与对照组比较,各染氟组细胞存活率降低(9．1％、 12．2％、25．0％),但组间比较差异无统计学意义(F=106．8．P>0．05)。各染氟组MDA(5．06、7．66、 9_82 nmol／mg Pr)明显高于对照组(2．34 nmol／mg Pr),组间比较差异有统计学意义(F=7．151,P<0．05)。氟对海马细胞的损伤呈明显的剂量-效应关系,染氟40、80 mg／L组细胞内ROS和细胞凋亡率高于20 mg／L组和对照组,细胞凋亡率与ROS水平存在明显正相关关系(r=0．913,P<0．05)。结论氟可引起大鼠海马细胞发生氧化应激反应及凋亡,活性氧可能在氟诱导细胞凋亡中起重要作用。     

过量碘化物诱导原代培养的大鼠仔鼠皮层神经元细胞凋亡的实验研究

目的观察过量碘化物对原代培养的大鼠仔鼠皮层神经元细胞凋亡的诱导作用，并对其机制作初步探讨。方法取新生0～1d的大鼠仔鼠皮层神经元培养24h后，加入不同浓度的碘化钾，采用光镜及荧光显微镜观察细胞的形态改变；流式细胞术分析DNA的降解；分光光度法检测细胞的一氧化氮（NO）含量及一氧化氮合酶（NOS）活性。结果实验组加入过量碘化物12h可见细胞形态发生改变，经荧光染色后可见凋亡细胞，染色质浓集，呈致密的斑块状或新月状，并可见凋亡小体及核碎片，48h左右达到高峰。流式细胞术分析细胞凋亡率随碘化物浓度的升高而升高。实验组细胞的NO含量及NOS活性较正常对照组增高。结论过量碘化物可以诱导原代培养的大鼠仔鼠皮层神经元细胞凋亡，并呈剂量依赖关系，其机制可能与NOS活性及NO含量增高有关。     

Effect of arsenic trioxide on rat hepatocarcinoma and its renal cytotoxicity

AIM: To study the effect of arsenic trioxide (As2O3) on rat experimental hepatocarcinoma and its renal cytotoxicity.METHODS: The hepatocarcinoma model was established by diethaylnitrosamine perfusion in stomach of 120 Wistar rats, and the treatment began at the end of 20 weeks.Before the treatment, the rat models were randomly divided into 5 groups. In the treatment groups, three doses of As2O3 were injected into rat abdominal cavity, the total time of drug administration was 4 weeks. Cisplatin control or the blank group was injected into abdominal cavity with equal amount of cisplatin or saline at the same time,respectively. On the 7th, 14th and 28th day after the treatment, the hepatocarcinoma nodules were obtained and the morphologic changes of hepatocarcinoma cells were observed under light and electron microscopes;Immunohistochemistry (S-P methods) was employed to detect the expression of bcl-2, bax and PCNA in hepatocarcinoma tissues; flow cytometry (TUNEL assay)was used to detect the apoptosis of liver cancer cells and the change of cytokinetics. On the 28th day, the kidneys were obtained and their histologic changes were observed under light microscope, and immunohistochemistry (SP stain) was also employed to detect the expression of bcl-2and PCNA. Cisplatin and saline solution were used as the control.RESULTS: As2O3 could induce the apoptosis of rat liver cancer cells and exhibited typical morphologic changes.The incidence of apoptosis of hapatocarcinoma cells was elevated (P=0.001). The elevation was the most higher in the group of middle-dose of As2O3 (1 mg.kg-1), significantly higher than that of the other arsenic groups and the controls (P=0.001). Large dose of As2O3 (5 mg.kg-1) was able to arise the incidence of apoptosis, but also produced a large amount of necrosis and inflammatory reaction. Middle dose of As2O3 dramatically increased the cell number in G2/M phase (P=0.0001), and apoptosis happened apparently.The expression of bcl-2 and bax was related to the dose of As2O3. With the up-regulation of apoptotic incidence, the ratio of bcl-2/bak decreased. But the incidence of apoptosis was not the highest status and the ratio of bcl-2/bax was at the lowest when the highest-dose of As2O3 was used.There was significant difference among the PCNA indexes (PCNA L1) of the five groups. Of them, three arsenic groups all showed decrease of different degrees, and this downregulation was most obvious in group A. There was significant difference among the three groups (P=0.016).Under the light microscope, the rat kidney in the cisplatin group exhibited tubular epithelium swelling and degeneration, protein casts in collecting tubules; While all arsenic groups didn't show the significant changes (P=0.013).In the arsenic groups, the expression of bcl-2 in the renal tubular epithelium was increased (P=0.005), no obvious changes happened to PCNA L1. But in the group of cisplatin,the PCNA L1 increased significantly (P=0.001).CONCLUSION: AS2O3 can induce apoptosis of rat hepatocellular carcinoma cells. And there is optimum dose;too high dose will induce the cytotoxic effect, while certain dose of As2O3 is able to block the cell cycle at G2/M phase.As2O3 had the most remarkable influence on G2/M cells,and it can also induce apoptosis to cells at other phases.As2O3 can restrain the proliferation of rat hepatocellular carcinoma cells, in a dose-time dependent manner.Compared with cisplatin, As2O3 didn't show obvious renal toxicity, which was related to the increasing expression of bcl-2 in renal tubular epithelium, the inhibition of apoptosis and the anti-oxidation effects.     

Effect of arsenic trioxide on rat hepatocellular carcinoma and its renal cytotoxity

AIM: To study the effect of arsenic trioxide (As2O3) on rat experimental hepatocarcinoma and its renal cytotoxicity.METHODS: The hepatocarcinoma model was established by diethaylnitrosamine perfusion in stomach of 120 Wistar rats, and the treatment began at the end of 20 weeks.Before the treatment, the rat models were randomly divided into 5 groups. In the treatment groups, three doses of As2O3 were injected into rat abdominal cavity, the total time of drug administration was 4 weeks. Cisplatin control or the blank group was injected into abdominal cavity with equal amount of cisplatin or saline at the same time,respectively. On the 7th, 14th and 28th day after the treatment, the hepatocarcinoma nodules were obtained and the morphologic changes of hepatocarcinoma cells were observed under light and electron microscopes;Immunohistochemistry (S-P methods) was employed to detect the expression of bcl-2, bax and PCNA in hepatocarcinoma tissues; flow cytometry (TUNEL assay)was used to detect the apoptosis of liver cancer cells and the change of cytokinetics. On the 28th day, the kidneys were obtained and their histologic changes were observed under light microscope, and immunohistochemistry (SP stain) was also employed to detect the expression of bcl-2 and PCNA. Cisplatin and saline solution were used as the control.RESULTS: As2O3 could induce the apoptosis of rat liver cancer cells and exhibited typical morphologic changes.The incidence of apoptosis of hapatocarcinoma cells was elevated (P=0.001). The elevation was the most higher in the group of middle-dose of As2O3 (1mg&#183;kg^-1), significantly higher than that of the other arsenic groups and the controls(P=0.001). Large dose of As2O3 (5mg&#183;kg^-1) was able to arise the incidence of apoptosis, but also produced a large amount of necrosis and inflammatory reaction. Middle dose of As2O3 dramatically increased the cell number in G2/M phase (P=0.0001), and apoptosis happened apparently.The expression of bcl-2 and bax was related to the dose of As2O3. With the up-regulation of apoptotic incidence, the ratio of bcl-2/bak decreased. But the incidence of apoptosis was not the highest status and the ratio of bcl-2/bax was at the lowest when the highest-dose of As2O3 was used.There was significant difference among the PCNA indexes(PCNA L1) of the five groups. Of them, three arsenic groups all showed decrease of different degrees, and this downregulation was most obvious in group A. There was significant difference among the three groups (P=0.016).Under the light microscope, the rat kidney in the cisplatin group exhibited tubular epithelium swelling and degeneration, protein casts in collecting tubules; While all arsenic groups didn‘t show the significant changes (P=0.013).In the arsenic groups, the expression of bcl-2 in the renal tubular epithelium was increased (P=0.005), no obvious changes happened to PCNA L1. But in the group of cisplatin,the PCNA L1 increased significantly (P=-0.001).CONCLUSION: As2O3 can induce apoptosis of rat hepatocellular carcinoma cells. And there is optimum dose;too high dose will induce the cytotoxic effect, while certain dose of As2O3 is able to block the cell cycle at G2/M phase.As2O3 had the most remarkable influence on G2/M cells,and it can also induce apoptosis to cells at other phases.As2O3 can restrain the proliferation of rat hepatocellular carcinoma cells, in a dose-time dependent manner.Compared with cisplatin, As2O3 didn‘t show obvious renal toxicity, which was related to the increasing expression of bcl-2 in renal tubular epithelium, the inhibition of apoptosis and the anti-oxidation effects.     

冷诱导RNA结合蛋白在亚低温下的神经保护作用研究

目的研究亚低温下冷诱导RNA结合蛋白(CIRP)对受损海马神经元的保护作用机制。方法将原代培养的海马神经元分为3组:对照组、损伤组和治疗组。氧糖剥夺55min制作海马神经元损伤模型,损伤组和治疗组在氧糖剥夺55min后分别置于37℃和32℃培养24h。收集各组细胞,MTT检测各组细胞的存活率,荧光定量PCR和Western blot检测CIRP的mRNA和蛋白的表达水平,流式细胞术检测各组细胞凋亡率,Western blot检测Caspase-3蛋白表达水平。结果与对照组比较,损伤组细胞生存率明显下降[(43.3±8.1)%vs(100.0±12.7)%,P<0.05],治疗组细胞生存率明显升高[(71.8±13.6)%vs(43.3±8.1)%,P<0.05],与损伤组比较,治疗组细胞凋亡率下降[(19.5±0.6)%vs(30.7±2.5)%],CIRP的mRNA蛋白表达明显升高,Caspase-3蛋白表达水平降低,差异有统计学意义(P<0.05)。结论亚低温下CIRP表达水平升高,通过抑制细胞凋亡发挥神经元保护作用,是亚低温脑保护作用途径之一。     

溶血磷脂酸损伤小脑颗粒神经元并诱导细胞凋亡

目的　研究溶血磷脂酸 (LPA)对原代培养大鼠小脑颗粒细胞的细胞毒性作用及其损伤机制。方法　将原代培养的大鼠小脑颗粒细胞暴露于不同剂量LPA中 ,测定噻唑蓝 (MTT) ;应用流式细胞仪、透射电镜、激光共聚焦显微镜等技术观察细胞的凋亡率、凋亡细胞的核形态及细胞胞质和线粒体内活性氧自由基 (ROS)的形成。结果　LPA对小脑颗粒细胞的损伤呈剂量依赖效应。经 5 0 μmol/LLPA作用后 ,细胞生存率为正常细胞的 (5 4.8± 11.5 ) % ,细胞凋亡率达 (4 0 .5± 2 .3) % ,细胞染色质发生浓缩和形成凋亡小体 ,细胞胞质和线粒体内ROS形成增加 ,而经四甲基吡嗪 (TMP)预处理 ,细胞生存率升至 (84.7± 8.8) % ,细胞凋亡率减至 (16 .7± 5 .8) % ,细胞核形态无明显改变 ,细胞胞质和线粒体内ROS形成被抑制。结论　LPA具有神经毒性作用。通过增加线粒体内ROS形成 ,继而诱导神经元凋亡可能是其损伤机制之一。能减少内源性ROS形成的抗氧化剂可对抗LPA诱导的神经元凋亡     

Evaluation of adipocyte apoptosis by laser scanning cytometry

OBJECTIVE: Adipocyte apoptosis plays an important role in adipose tissue homeostasis and can be altered under a variety of physiological and pathological conditions. This study was carried out to determine whether laser scanning cytometry (LSC) can be used to measure changes in apoptosis of adipocytes over time. DESIGN: LSC was used to investigate adipocyte apoptosis induced by tumor necrosis factor-alpha (TNF-alpha), a cytokine that is associated with obesity and insulin resistance. LSC, a slide-based solid phase cytofluorometer, provides quantitative flow fluorescence data together with morphological information for apoptotic detection. Both 3T3-L1 cells and rat adipocytes from primary cell culture were incubated with 0 or 25 nM TNF-alpha for up to 24 h. Both the FITC-conjugated annexin V/propidium iodide assay and the TUNEL assay were used to distinguish cells with apoptotic characteristics from nonapoptotic cells. RESULTS: Apoptosis did not increase over time in the absence of TNF-alpha for both 3T3-L1 cells and rat primary adipocytes. For both 3T3-L1 cells and rat primary adipocytes, a significant increase in the percentage of apoptotic cells was observed by 3-4 h incubation with TNF-alpha (P<0.05). By 24 h, more than 50% of cells incubated with TNF-alpha were apoptotic (P<0.001). This process was also associated with morphological changes typical of adipocytes undergoing apoptosis. By estimating the percentage of cell subpopulations after different times of incubation with TNF-alpha, we were able to develop grading parameters, based on the adipose apoptotic measurements. CONCLUSION: With morphological information, LSC can be a useful tool to evaluate adipocyte apoptosis.     

腺相关病毒介导的脑源性神经营养因子基因对大鼠海马神经元树突生长的影响

目的研究重组腺相关病毒介导的脑源性神经营养因子基因（rAAV-BDNF）的表达对体外培养的大鼠海马神经元树突生长的影响。方法取孕18d大鼠建立体外培养的海马神经元模型,感染rAAV-BDNF病毒。在检测了BDNF稳定的表达水平后,用MAP2染色法来标示神经元树突形态。用荧光显微镜采样,统计胞体约15～20μm的MAP2阳性细胞初级树突的数目和长度。对照组神经元为感染表达绿色荧光蛋白的腺相关病毒。结果感染rAAV-BDNF的海马神经元初级树突数目为（14±3）个,对照组的初级树突数目为（6±1）个。rAAV-BDNF感染的海马神经元树突总长度为（1500±150）μm,对照组的树突总长度为（700±50）μm,两组无论是树突数目还是长度,差异均有统计学意义（t=3.088和6.238,均P〈0.05）。结论rAAV-BDNF转染大鼠体外培养的海马神经元后,能起到促进树突生长的作用。     

Proliferation, differentiation, and long-term culture of primary hippocampal neurons.

Primary embryonic hippocampal neurons can develop morphologically and functionally in culture but do not survive more than a few weeks. It has been reported that basic fibroblast growth factor (bFGF) promotes the survival of and neurite elongation from fetal hippocampal neurons. We report that bFGF, in a dose-dependent manner, can induce the survival (50 pg to 1 ng/ml) and proliferation (10-20 ng/ml) of embryonic hippocampal progenitor neurons in vitro. In serum-free medium containing high concentrations of bFGF, neurons not only proliferated (4-day doubling time) and differentiated morphologically but also could be passaged and grown as continuous cell lines. The neuronal nature of the proliferating cells was positively established by immunostaining with several different neuron-specific markers and by detailed ultrastructural analyses. The proliferative effect of bFGF was used to generate nearly pure neuronal cell cultures that can be passaged, frozen, thawed, and cultured again. Neurons have been maintained > 5 months in culture. The ability to establish long-term primary neuronal cultures offers the possibility that clonal lines of distinct neuronal cell types may be isolated from specific areas of the central nervous system. Such long-term neuronal cultures should prove valuable in studying neurons at the individual cell level and also in exploring interactions between neurons in vitro. The observed dose dependence raises the possibility that cell survival and proliferation in vivo may be influenced by different levels of bFGF.