bFGF和EGF对体外培养新生大鼠海马神经元促生长作用的研究

ｂＦＧＦ和ＥＧＦ对体外培养新生大鼠海马神经元促生长作用的研究吴承远苏万东张洪华王建刚为了研究碱性成纤维细胞生长因子（ｂＦＧＦ）和表皮生长因子（ＥＧＦ）在中枢神经系统中的生物学作用，本实验采用神经细胞体外原代分离培养的方法，来观察不同浓度ｂＦＧＦ和ＥＧ...     

细胞因子rhIL—1β,rhIL—2及rhIL—6对大鼠海马神经元营养作用的研究

本工作采用显微测量，图像分析和流式细胞分析方法，研究了细胞因子ｒｈＩＬ－１β，ｒｈＩＬ－２及ｒｈＩＬ－６对新生大鼠海马培养神经元突起生长发育，胞体生长状态，神经元存活数及培养过程中蛋白总量影响，结果显示，这３种细胞因子均对海马培养神经元的突起生长，胞体发育以及发育过程中神经元的蛋白质民具有明显的促进作用，并且在提高海马培养神经元存活，延长存活时间方面有较显著的效应，提示白介素类细胞因子ｒｈＩＬ－１     

β—淀粉样蛋白31～35对体外培养的隔区胆碱能神经元的作用

β－淀粉样蛋白是老年斑的主要成分。为探讨其在老年性痴呆患者基底前脑胆碱能神经元选择性溃变中的作用，运用ＭＴＴ自动比色微量分析法、胆碱酯酶组织化学染色及细胞形态学分析方法，研究“老化”的淀粉样蛋白片段３１～３５对体外培养的海马和隔区神经元的作用。结果表明：培养４８ｈ，淀粉样蛋白对海马神经元有毒性作用，减少神经元的存活，呈剂量依赖关系。培养１４ｄ，淀粉样蛋白减少了胆碱能神经元的存活率，抑制突起的延伸，促使神经元胞体和近侧突起的分支增加。表明淀粉样蛋白对隔区胆碱能神经元具有减少存活、抑制突起延伸和促进突起不正常生长的毒性作用。     

白介素-2对海马神经细胞的功能调控

采用形态学、电生理学和生物化学等方法，研究了重组人白介素-2(rhIL-2)对新生大鼠海马培养神经元生长发育、平均蛋白总量、膜电活动以及细胞内游离钙离子浓度的影响。结果显示．rhIL-2对海马培养神经元的突起生长、胞体发育以及发育过程中神经元的蛋白质合成具有明显的促进作用，并且在提高海马培养神经元存活、延长存活时间方面有较为显著的效应。细胞内记录结果显示．rhIL-2可改变海马神经元的膜兴奋性，该作用可被纳洛酮减弱。提示阿片受体可能参与介导rhIL-2的作用过程。另外，采用fura-2荧光探针标记法发现，rhIL-2能够通过胞外钙离子的流入和胞内钙库的释放提高海马神经元胞浆内游离钙离子的浓度。上述研究结果初步表明，细胞因子rhIL-2能够调控海马神经元的功能活动。     

CRMP2在大鼠海马神经元中的表达分布及对神经元突起生长的影响

目的探讨脑衰反应调节蛋白2(CRMP2)在大鼠海马神经元中的表达分布及对神经元突起生长的影响。方法CRMP2及其模拟磷酸化质粒CRMP2-S522D、CRMP2-T555D,转入体外培养的SD大鼠海马神经元,转染72 h后,采用免疫印迹法和免疫荧光染色分析CRMP2、CRMP2-S522D、CRMP2-T555D在神经元中的分布差异以及对神经元突起分枝和突起长度的影响。结果 CRMP2及CRMP2-T555D在神经元胞体和突起均有分布,CRMP2-S522D则主要分布于胞体。CRMP2-T555D对神经元的突起长度和分枝数均没有明显影响;CRMP2-S522D对突起数目没有明显影响,但可以显著降低突起长度。结论 CRMP2的不同磷酸化形式在大鼠海马神经元中存在分布差异,并对神经元的突起长度产生影响。     

有氧训练对阿尔茨海默病模型大鼠海马齿状回神经发生的影响

目的：观察有氧训练对β淀粉样蛋白25-35（Aβ25-35）诱导的阿尔茨海默病（AD）大鼠齿状回（DG）神经发生的影响。方法：SD大鼠48只,随机分为假手术组（侧脑室注射生理盐水）,有氧训练组（侧脑室注射Aβ25-35＋4周有氧训练）和实验组（侧脑室注射Aβ25-35）。3组大鼠行Morris水迷宫行为学检测,免疫组织化学方法检测海马DGBrdU阳性细胞数,以确定有氧训练对细胞存活的影响;微管相关蛋白免疫组化染色观察新生神经元的突起生长。结果：①与假手术组比,实验组逃避潜伏期显著延长（P〈0.05）;有氧训练组与实验组比,逃避潜伏期明显缩短（P〈0.05）;有氧训练组逃避潜伏期与假手术组比,差异无统计学意义。②与假手术组比,实验组DGBrdU阳性细胞数显著减少（P〈0.05）。③与假手术组比,实验组DG新生神经元突起的数量明显减少（P〈0.05）。④与实验组比,有氧训练可改善DGBrdU阳性细胞的显著减少（P〈0.05）、保护神经元突起的生长（P〈0.05）。结论：Aβ25-35能诱导AD大鼠模型的建立,损害新生神经元突起生长和新生神经元的存活;有氧训练可改善Aβ25-35损害的大鼠新生神经元突起生长和新生神经元的存活。     

丹参在缺血再灌注时的神经保护作用──成纤维细胞生长因子上调

丹参作为传统中药治疗缺血性脑血管病具有良好的疗效，本实验研究丹参是否通过影响成纤维细胞生长因子（ｂＦＧＦ）起修复作用。用大鼠线栓法缺血再灌注模型及免疫组化方法，发现大鼠缺血９０ｍｉｎ、再灌流４８ｈ后，缺血侧皮层、尾壳核及海马区ｂＦＧＦ样免疫反应增加及神经细胞变性；缺血后给予丹参（１５ｇ／　ｋｇ）大鼠，ｂＦＧＦ样免疫反应加强，并且缺血对应侧脑区也可见轻度的ｂＦＧＦ样免疫反应改变，且神经细胞变性程度较轻。提示丹参对脑缺血再灌流的保护作用可能与它加强ｂＦＧＦ的修复作用有关。     

碱性成纤维细胞生长因子对脑缺血后神经元特异性烯醇化酶的影响

目的研究碱性成纤维细胞生长因子（ｂＦＧＦ）对局灶脑缺血的作用。方法采用线栓法制备右侧大脑中动脉闭塞局灶性脑缺血模型。术后即刻分别腹腔注射ｂＦＧＦ和生理盐水。测定缺血前后血压、心率，缺血前、缺血后３０分钟～２４小时的局部脑血流量（ｒＣＢＦ），缺血２４小时血清神经元特异性烯醇化酶（ＮＳＥ），观察缺血２４小时病理学改变和行神经功能评分。结果发现ｂＦＧＦ可降低血清ＮＳＥ，改善组织病理和ｒＣＢＦ，促进神经功能恢复，但不影响血压、心率。结论ｂＦＧＦ能减轻缺血性脑损害，保护脑组织。     

基因工程重组TGF-α-PE40融合蛋自治疗人脑胶质瘤的实验研究

目的评价基因工程重组ＴＧＦ－α－ＰＥ４０融合蛋白在体外、体内对人脑胶质瘤的治疗作用。方法体外使用改良ＭＴＴ法测定ＴＧＦ－α－ＰＥ４０融合蛋白对体外培养的恶性胶质瘤细胞生长抑制作用，同时检测各细胞株表皮生长因子受体（ＥＧＦＲ）的表达情况；体内利用Ｕ２５１裸鼠移植瘤模型观察不同剂量的融合蛋白对移植瘤体积增长的影响。结果　ＴＧＦ－α－ＰＥ４０融合蛋白对体外培养恶性胶质瘤细胞有明显的生长抑制作用，其作用依赖于肿瘤细胞ＥＧＦＲ的表达；对裸鼠移植瘤，２５０ｕｇ／ｋｇ组用药后２１日内瘤体积无明显增长，抑瘤率为４６．４％。结论ＴＧＦ－α－ＰＥ４０融合蛋白可以选择性地抑制表达ＥＧＦＲ的胶质瘤的生长。     

丹参在缺血再灌注时的神经保护作用——成纤维细…

丹参作为传统中药治疗缺血性脑血管病具有良好的疗效，本实验研究丹参是否通过影响成纤维细胞生长因子起修复作用，用大鼠线栓法缺血再灌注模型及免疫组化方法，发现大鼠缺血９０ｍｉｎ，再灌流４８ｈ后，缺血侧皮层，尾壳核及海马区ｂＦＧＦ样免疫反应增加及神经细胞变性；缺血后给予丹参大鼠，ｂＦＧＦ样免疫反应加强，并且缺血对应侧脑区也可见轻度的ｂＦＧＦ样免疫反应改变，且神经细胞变性程度较轻，提示丹参对脑缺血再灌流的保     

Basic fibroblast growth factor and epidermal growth factor exert differential trophic effects on CNS neurons

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) are potent mitogenic proteins capable of inducing cell division in a wide variety of cell types. In addition to their mitogenic properties, both proteins have recently been shown to enhance survival and process outgrowth from neurons of central nervous system origin. The full spectrum of neuronal subtypes responding to these factors has not been elucidated. In the present study, EGF was found to enhance survival and process outgrowth of primary cultures of cerebellar neurons of neonatal rat brain. This effect was dose-dependent and was observed with EGF concentrations as low as 100 pg/ml. In marked contrast, bFGF was ineffective in enhancing survival or neurite elongation from cerebellar neurons when tested in the range of 0.1 to 10.0 ng/ml. However, within this concentration range, bFGF did prove effective in stimulating an increase in [3H]thymidine incorporation into primary cultures of cerebellar astrocytes, demonstrating that bFGF was active and that cells in the cerebellum do respond to bFGF. These results suggest that EGF or an EGF-like peptide may act as a neurite elongation and maintenance factor for cerebellar neurons. EGF has now been shown to support striatal, cortical, and cerebellar neurons, suggesting that this factor may have trophic activity throughout the central nervous system. bFGF, in contrast, appears to exert its effects on limited populations of neurons.     

Epidermal growth factor and basic fibroblast growth factor: effects on an overlapping population of neocortical neurons in vitro

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) have trophic effects on rat neocortical neurons in vitro. Concentration-response studies reveal that EGF maximally stimulates neuronal survival and process outgrowth at approximately 10 ng/ml, while the maximal effect of bFGF is seen at 10-30 ng/ml. Treatment with maximal concentrations of bFGF results in cultures containing a greater number of neurons with long processes, as well as greater branching of processes, than does treatment with EGF. When EGF and bFGF are added together to cultures the effects are not additive. In addition, bFGF is capable of supporting the survival of neurons previously treated with EGF. These findings indicate that EGF and bFGF affect a largely overlapping population of neocortical neurons, but that bFGF may be a more effective trophic agent for these cells.     

Protection from 1-methyl-4-phenylpyridinium (MPP) toxicity and stimulation of regrowth of MPP-damaged dopaminergic fibers by treatment of mesencephalic cultures with EGF and basic FGF

Several peptide growth factors can maintain survival or promote recovery of injured central neurons. In the present study, the effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the toxicity produced by the dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), were investigated in rat mesencephalic dopaminergic neurons in culture. High affinity [3H]DA uptake and morphometric analyses of tyrosine hydroxylase immunostained neurons were used to assess the extent of MPP+ toxicity, dopaminergic neuronal survival and growth of neurites. Consistent with previous reports, EGF and bFGF treatments stimulated neuritic outgrowth in dopaminergic neurons, increased DA uptake and enhanced their long-term survival in vitro. These growth factors also stimulated proliferation of astrocytes. The time course of EGF and bFGF effects on dopaminergic neurons coincided with the increase in glial cell density, suggesting that proliferation of glia mediates their trophic effects. Several findings from our study support this possibility. When MPP+ was applied to cultures at 4 days in vitro, before glial cells had proliferated, the damage to dopaminergic neurons was not affected by EGF or bFGF pretreatments. However, when cultures maintained in the presence of the growth factors for 10 days were exposed to MPP+, after they had become confluent with dividing glial cells, the MPP(+)-induced decreases in DA uptake and cell survival were significantly attenuated. Furthermore, when glial cell proliferation was inhibited by 5-fluoro-2'-deoxyuridine, the protective effects of EGF and bFGF against MPP+ toxicity were abolished. Continuous treatment of MPP(+)-exposed cultures with EGF or bFGF resulted in the stimulation of process regrowth of damaged dopaminergic neurons with concomitant recovery of DA uptake, suggesting that the injured neurons are able to respond to the trophic effects of EGF and bFGF. In summary, our study shows that the trophic effects of EGF and bFGF on mesencephalic dopaminergic neurons include protection from the toxicity produced by MPP+ and promotion of recovery of MPP(+)-damaged neurons. Stimulation of glial cell proliferation is necessary for these effects.     

Epidermal growth factor exerts neuronotrophic effects on dopaminergic and GABAergic CNS neurons: comparison with basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) have been described to exert neuronotrophic effects on central nervous system neurons in culture. To study the selectivity of trophic actions of these growth factors, neurotransmitter-identified populations of embryonic rat mesencephalon were used. At 20 days in vitro, EGF (3 ng/ml) promoted survival and neurite outgrowth from these neurons. The neuritogenic effect of bFGF (3 ng/ml) was, however, more robust. Quantitative analysis with the neurofilament monoclonal antibody RR97 and ELISA confirmed the differential response, bFGF being 2-2.5 times more effective at all concentrations tested (ED100: 3-10 ng/ml for both EGF and bFGF). At 10 days in vitro, EGF displayed no trophic activity--even at 30 ng/ml. Treatment of mesencephalic cultures with EGF (3 ng/ml) for 20 days stimulated [3H]dopamine and [14C]GABA uptakes about 4-fold. While bFGF (3 ng/ml) also stimulated GABA uptake some 4-fold, dopamine uptake was increased almost 20-fold. Thus, EGF is also capable of enhancing the transmitter traits of selected central neuronal populations; however, the actions of bFGF appear to preferentially address dopaminergic cells.     

Developmental changes in progenitor cell responsiveness to cytokines.

Multipotent progenitor cells have been identified within periventricular generative zones of the developing and adult brain. To determine whether the environmental responsiveness of these cells changes during development, progenitor cells were cultured from embryonic, postnatal, and adult rat brain in the presence of either basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). Embryonic cells cultured as intact progenitor neurospheres proliferated more robustly in response to bFGF than to EGF, whereas proliferation of postnatal and adult progenitor cells was enhanced more by EGF than bFGF. Progenitor cells generated in the presence of either bFGF or EGF had the capacity to generate neurons, astrocytes, and oligodendrocytes at all developmental stages. Most embryonic and neonatal bFGF-generated cells differentiated predominantly into neurons, whereas late stage embryonic and neonatal EGF-generated progenitors largely remained in an undifferentiated state. However, later postnatal and adult progenitor species, irrespective of whether they were generated in the presence of bFGF or EGF, gave rise preferentially to astrocytes. Treatment with bone morphogenetic protein (BMP)2 or BMP7 enhanced astroglial differentiation and suppressed oligodendroglial differentiation of both EGF- and bFGF-generated progenitor species, suggesting that the effects of the BMPs are not dependent on EGF receptor activation. Thus, while central nervous system (CNS) progenitor cells retain multipotent capacity and responsiveness to the BMPs throughout development, they exhibit significant changes in other cellular response properties, perhaps reflecting differences in the requirements for specific generative versus regenerative events.     

多细胞因子诱导成年鼠骨髓基质细胞向神经细胞分化的实验研究

目的观察大鼠骨髓基质细胞(rBMSCs)的生长特点及诱导条件下分化成神经细胞的能力,并对其机制进行初步探讨。方法以密度梯度离心分离骨髓基质细胞,在神经干细胞培养液中培养,采用四唑盐(MTT)法观察在培养液中添加碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)对BMSCs增殖的影响;观察添加脑源性神经生长因子(BDNF)、神经生长因子(NGF)和维甲酸(RA)对rBMSCs的诱导分化情况;采用免疫组织化学法(ABC)检测诱导后的细胞表达神经元特异性烯醇化酶(NSE)、神经元核蛋白(NeuN)和胶质原性纤维酸性蛋白抗体(GFAP)等特异性标志物的情况;以流式细胞分选确定神经元的比例。结果bFGF和EGF能在体外促进rBMSCs增殖,BDNF、NGF和RA能诱导rBMSCs来源的神经干细胞(NSCs)表达NSE、GFAP等特异性标志物。结论EGF、bFGF、BDNF、NGF、RA及适宜的培养液可使rBMSCs定向转化为NSCs,获得足够的目的细胞,进而分化为神经元样和神经胶质样细胞。     

Effects of epidermal growth factor and basic fibroblast growth factor on generation of long-term potentiation in the dentate gyrus of fimbria-fornix-lesioned rats

The effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on long-term potentiation (LTP) in the dentate gyrus in vivo were investigated in fimbria-fornix (FF)-lesioned rats. Transection of FF resulted in decreased frequency of LTP generation. Intracerebroventricular injection of EGF (50 ng) and bFGF (50 ng) significantly facilitated LTP generation in the FF-lesioned rats. These results suggest that EGF and bFGF can promote the hippocampal LTP impaired by loss of subcortical afferents.     

胚胎大鼠嗅神经干细胞的培养及分化特性

目的建立胚胎大鼠嗅神经干细胞(NSCs)体外培养方法,研究其增殖和分化特性.方法采用添加丝裂原的无血清培养基分离、培养胚胎14 d(E14)大鼠嗅球NSCs,应用免疫细胞化学方法鉴定培养的NSCs及自然分化为特异性神经细胞的类型,测定NSCs的生长曲线.结果从E14大鼠嗅球分离、培养出表达nestin,并能分化为神经元、星形胶质细胞和少突胶质细胞的NSCs.嗅NSCs的增殖依赖表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF),其中EGF的促分裂增殖作用明显优于bFGF.结论从E14大鼠嗅球培养出具有自我增殖和多向分化潜能的NSCs.     

Basic FGF in astroglial, microglial, and neuronal cultures: characterization of binding sites and modulation of release by lymphokines and trophic factors.

The present study characterizes whether basic fibroblast growth factor (bFGF) is present and released from astroglia, microglia, and hippocampal neurons in vitro. For cell content, bFGF-like immunoreactivity (IR) of cell extracts was measured, whereas release was determined by assessing the levels of bFGF-like IR in media. In addition, the effects of lymphokines and trophic factors that are known to be released from these cells on bFGF release were examined. For all three cell types, bFGF-like IR in extracts of cell lysates was detectable. In addition, media content was highest in astroglial cultures and lowest in neuronal cultures. Although bFGF-like IR of neuronal and microglial media appeared to increase with time in culture, this was likely due to significant astroglial proliferation. Thus, notable levels of bFGF are released by astroglia in vitro. In astroglia, bFGF release was enhanced by interleukin-1 (IL-1), IL-6, and epidermal growth factor (EGF), but not by other lymphokines or NGF. In contrast, bFGF in microglial media was reduced by IL-3, EGF, and NGF, but slightly augmented by gamma-interferon (IFN); other lymphokines were ineffective. In addition, no effects were seen in the neuronal cultures. It is likely that the bFGF found in glial media interacts with bFGF receptors since in both glial and neuronal cell types, a single class of low-capacity (Bmax), high-affinity (Kd) bFGF binding sites was evident. The possibility that endogenous bFGF acts as an autocrine factor for astroglia was further supported by experiments that tested the mitogenic effects of exogenous bFGF on glial cells. bFGF significantly enhanced 3H-thymidine uptake into astroglial, but not microglial, cells in vitro. Thus, the present study demonstrates that a complex regulation of glial bFGF release by astroglia and microglia occurs in vitro. Moreover, the results are consistent with an autocrine role for bFGF in astroglial cultures.