鸡胚背根神经节培养法测定神经生长因子活性的条件优化

鸡胚背根神经节培养法是测定神经生长因子(NGF)和神经营养因子活性的重要方法。我们通过考察培养基、鸡血浆和鸡胚浸液的比例、背根神经节部位、培养时间等条件对NGF刺激神经节生长的影响,从而建立了一套检测NGF活性的优化条件。在含20％鸡血浆和15％鸡胚浸液的DMEM的培养基中,利用伊莎褐鸡胚腰部的背根神经节,在终浓度为30ng/ml的蛇毒NGF的刺激下,培养36h可以得到理想的实验结果。该条件重复性好、分辨率高、简单实用。     

激活素促进鸡胚神经节神经突起生长作用

为了探讨激活素(activin)促进鸡胚背根神经节(dorsal root ganglia,DRG)突起生长、维持神经节细胞生存作用及其与一氧化氮(NO)释放的关系,实验采用8 d的鸡胚分离背根神经节,原代培养法,观察鸡胚背根神经节的体外生长情况。研究结果表明,添加激活素A培养的背根神经节有明显的神经突起生长,形成密集的网络,背根神经节可存活8~10 d;而阴性对照组几乎无神经突起生长,背根神经节可存活3~4 d。添加激活素A的背根神经节单层培养神经节细胞也可长期存活;而阴性对照组在培养第5 d几乎无神经节细胞生存。NO检测结果显示,添加激活素A培养的背根神经节上清NO分泌水平明显降低,与阴性对照组比较差异显著(P<0.05);激活素A与神经生长因子(nerve growth factor,NGF)具有协同抑制背根神经节NO分泌作用。激活素结合蛋白(follistatin)明显抑制激活素A诱导的背根神经节神经突起生长。研究结果提示,激活素可维持鸡胚神经节细胞存活并刺激神经突起生长,其作用与抑制神经损伤因子NO的释放有关。     

NGF,BDNF和NT-3在培养鸡胚背根节神经元的表达

目的　探讨NGF ,BDNF ,NT - 3在体外培养鸡胚背根节神经元中的表达变化。方法　采用NGF ,BDNF ,NT - 3的兔抗血清分别对培养前后的鸡胚背根节神经元以免疫组化ABC法染色。观察NGF、BDNF和NT - 3在培养前、后鸡胚背根节神经元的表达情况 ,计数并比较培养前、后三种因子免疫阳性神经元百分数。结果　未培养的神经元 ,NGF ,BDNF ,NT - 3的阳性神经元百分数分别是 :10 %± 3%,2 7%± 5 %,2 9%± 7%。培养 48小时后 ,NGF ,BDNF ,NT - 3的阳性神经元百分数分别是 :77%± 6 %,6 4%± 7%,2 4%± 7%。结论　培养后NGF ,BDNF的表达较未培养者增加 (P <0 0 1) ,而NT - 3者则有减少 (P <0 0 5 )。提示在体外培养的鸡胚背根节神经元NT - 3的表达有不同于NGF和BDNF的调节方式。     

神经生长因子细胞测活方法的初步建立

神经生长因子(nerve growth fector,NGF)是神经系统中重要的多肽生长因子,它调控外周交感、感觉神经元和中枢胆碱能神经元的存活和发育,并且对损伤后的部分神经元具有修复作用。神经生长因子经典的生物学活性测定方法是采用8～9天龄鸡胚背根神经节法和PC12细胞法。1996年世界卫生组织(WHO)颁布了新的NGF活性测定的国际标准方法。据此我们初步建立了该方法,该方法具备简便、易行、快速等特点,为NGF活性测定奠定了基础。     

重组人神经生长因子β亚基的原核融合表达及其活性研究

目的：通过原核融合表达,获得具有生物活性的重组人神经生长因子（hNGF）的B亚基。方法：分别以大肠杆菌二硫键形成蛋白家族（Dsb）中的DsbA、DsbC蛋白及硫氧还蛋白（Trx）为融合分子,与hNGFB亚基在原核表达系统进行融合表达,优化融合蛋白的复性条件,获得可溶性rhNGFp亚基融合蛋白;通过鸡胚背根神经节培养实验鉴定各融合蛋白的生物活性。结果：在获得的3种融合蛋白中,只有DsbA-L-NGF表现较高的、类似小鼠NGF的生物活性,可观察到其促进鸡胚被根神经节突起生长。结论：人神经生长因子B亚基与DsbA融合蛋白具有良好的生物活性。     

Neurturin嵌合神经营养因子重组腺病毒的构建及体外活性测定

Neurturin是神经营养因子家族成员,研究发现体外表达的重组Neurturin能够促进多巴胺能神经元的存活,对帕金森氏病的基因治疗有重要意义。但有研究指出野生型Neurturin在细胞中不能完成加工进而不能被分泌到胞外。因此实验对野生型Neurturin进行改造,将NGF的信号肽与Neurturin的成熟肽进行拼接,形成一种嵌合的NGF/NTN基因,并将该基因插入重组腺病毒基因组中,在AD-293细胞中包装获得重组腺病毒。通过免疫荧光和Western blotting证实Neurturin能够在胞内表达,并且能够对其进行加工最后分泌至胞外。最后通过鸡胚背根神经节培养实验证明该嵌合基因能够表达具有明显活性的Neurturin。     

低浓度神经生长因子存在下GPI-1046刺激鸡胚神经节突起生长

对GPI-1046是否具有神经营养作用目前有两种不同的认识。Steiner等认为GPI-1046能促进体外培养的感觉神经节神经元突起生长。但Harper等却没能证明GPI-1046有这样的作用。由于GPI-1046在临床上具有重要应用价值和前景,我们重新评价了GPI-1046对体外培养鸡胚神经节的神经营养作用,发现在低浓度神经生长因子（nerve growth factor,NGF）存在下,GPI-1046能明显促进鸡背根神经节神经突起的生长。     

定量测定重组人睫状神经营养因子活性的新方法

目的：建立一种能定量测定重组人睫状神经营养因子（ciliary neurotrophic factor,CNTF）生物学活性的新方法。方法：从鸡胚中分离出背根神经节并制成神经细胞,将重组人睫状神经营养因子加入到细胞中继续培养64h后,用酸性磷酸酶法检测活细胞内酸性磷酸酶的活性,从而定量测定重组人睫状神经营养因子的生物活性。结果：重组人睫状神经营养因子有促原代鸡胚背根神经细胞存活作用,细胞存活率与加入重组人睫状神经营养因子的量成正相关。结论：通过检测存活的原代鸡胚背根神经细胞内酸性磷酸酶的含量来定量测定重组人睫状神经营养因子生物活性的实验方法具有干扰因素少、定量准确、重复性好等优点。     

睫状神经营养因子突变体蛋白的活性研究

为了进一步研究我室应用计算机分子模拟设计并表达纯化的睫状神经营养因子突变体蛋白的生物学活性,分别采用鸡胚背根神经节无血清培养法、TF-1细胞增殖法、正常小鼠减重法对其活性进行研究。结果是突变体蛋白能促进鸡胚背根神经节的生长;促进TF-1细胞增殖,MTT测定法表明突变体蛋白与国际参考品相比,比活不低于2.0×106U/mg;使正常小鼠的体重减轻,摄食量减少,脂肪指数下降,并且体重的减轻与突变体蛋白的给药剂量呈现良好的剂量依赖关系,其ED50为：150.986?g/kg/d。以上实验表明CNTF突变体蛋白具有促神经生长、促TF-1细胞增殖和减重的生物学活性。从而为其进一步的应用和开发提供了线索。     

具有NGF活性通过二硫键连接的 2个小分子多肽的制备及鉴定(英文)

神经生长因子 (NGF)作用广泛 ,β NGF是神经生长因子的活性部分 .由于 3对二硫键的影响 ,体外表达NGF很难形成正确折叠的肽链 .由于神经系统血脑屏障的存在 ,大分子肽链的给药途径是一个不可忽视的难题 .根据NGF的氨基酸序列及其晶体构象资料 ,选择CNBr在 9位Met处 ,胰蛋白酶在Arg或Lys处裂解 β NGF .经过SephadexG 5 0层析、DE 5 2纤维素离子交换层析和反相高压液相层析分离纯化后获得NGF活性片段 .氨基酸组成分析及氨基酸序列分析结果表明 ,此片段由 16肽GEFSVCDSVSVWVGDK与 14肽HWNSYCTTTHTFVK通过 1对二硫键连接而成 .8日龄鸡胚背根神经节生物活性分析表明 ,其最佳作用浓度为 0 0 0 1～ 0 1μg L .它的成功分离和鉴定为合成或表达高活性小分子神经营养物质奠定了关键而重要的基础 .     

The neurite growth stimulating effect of terrilytin on sensory neurons and the spinal cord in tissue culture

Terrilytin (a protease) was found to promote neurite extension in chick embryo dorsal root ganglia and spinal cord in vitro. This protease was active at the concentration 10-50 ng/ml, which provided extensive neurite outgrowth in the bioassay, compared to the control. Terrilytin may be used for stimulating regenerating processes in nervous tissue.     

The stimulating effect of prolactin on the neurite growth of sensory neurons in an organotypic culture

Prolactine was found to promote neurite extension in chick embryo dorsal root ganglia in vitro. This hormone was active at the concentration 10-50 ng/ml provided extensive neurite outgrowth in the bioassay, compared to the control. The liquor from patients with prolactinoma was also found to promote neurite extension. This fact may be used for prolactinoma diagnostics.     

Chick embryo nerve growth factor : Fractionation and biological activity

A nerve growth factor (NGF)-like factor initiating nerve fibre outgrowth from sympathetic ganglia in culture was partially purified from chick embryo extract by cation-exchange chromatography followed by hydrophobic interaction chromatography on octylsulfide agarose. The NGF-like factor was markedly activated upon gel filtration in the presence of 6 M urea. Further analysis of the activated chick NGF by immunoblotting following SDS-PAGE, and by inhibition of bioassay response using antibodies to mouse beta NGF demonstrated a distinct antigenic cross-reactivity. The size of the chick embryo NGF was also indistinguishable from that of the mouse beta NGF with a molecular weight (MW) of about 14,000. The findings demonstrate directly the presence of biologically active NGF protein in the developing 18-day chick embryo.     

The effect of nerve growth factor (NGF) on the synthesis of gangliosides

—Dorsal root ganglia from 8-day- and 14-day-old chick embryos contained gangliosides with a pattern qualitatively similar to that of embryonic chick brain. The pattern of gangliosides from dorsal root ganglia changed with age, there being a decrease in polysialogangliosides with increasing age. When isolated, dorsal root ganglia were incubated in the presence of a concentration of nerve growth factor (NGF) sufficient to promote the outgrowth of nerve fibres, there was increased incorporation of d -[1-¹⁴C]glucosamine into gangliosides. There was, however, no difference in the pattern of incorporation into gangliosides by control ganglia and those exposed to NGF.     

Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor

Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+     

Rapid retraction of neurites by sensory neurons in response to increased concentrations of nerve growth factor

The phenomenon of growth cone (GC) and neurite retraction resulting from a rapid incrase in concentration of the trophic molecule NGF was studied. Neurite outgrowth from explants of 8-d chick embryo dorsal root ganglia was achieved at very low NGF concentrations with heart conditioned medium during overnight culture. Quickly incrasing the NGF concentration in the growth medium dramatically affected GC and neurite morphology: the majority of GCs and neurites collapsed and retracted towards the cell body over a course of approximately 2-5 min. Retraction was elicited by increasing NGF levels from 0 to 0.05 ng/ml to as little as 0.5 ng/ml but did not occur if the NGF concentration during the initial overnight culture period exceeded 0.8 ng/ml, regardless of how much the concentration was elevated. Similar concentration changes of cytochrome c or insulin did nt result in retraction. Neurites that had been separated from their cell bodies by cutting close to their exit from the explant still retracted when NGF levels were raised. Cytochalasin B reversible inhibits retraction, whereas colchicine allows retraction to occur. Observation of cell- substratum adhesion during retraction revealed that some adhesion points remain during retraction and that they correspond to the ends of NGF leels and that it may involve microfilaments in the neurite cytoskeleton. The NGF concentration changes that elicit neurite retraction suggest that a primary event in retraction may be increased occupancy of a high-affinity NGF receptor on neurites.     

Nerve growth factor in skeletal tissues of the embryonic chick

Summary This study demonstrates, via immunohistochemistry and bioassay, the presence of NGF in embryonic bone and cartilage of the chick. Embryos were killed on days 6–9 of incubation at 12 h intervals, and on days 10–18 at 24 h intervals. Paraffin-embedded sections of hind limbs or buds were immunostained with a polyclonal antibody against NGF and the biotin-avidin-horseradish peroxidase technique. Immunostaining was positive in both bone and cartilage, with cartilage staining more intensely. For bioassay, bones from the hind limbs of 9- and 12-day embryos were fast-frozen, lyophilized, and homogenized with Medium 199 (M199). Dorsal root ganglia from 8-day embryos were cultured for 24–36 h with rooster plasma, M199, and varying concentrations of bone homogenate. Significant neurite outgrowth was seen, with the greatest response elicited by 12-day bone homogenate. Addition of anti-NGF to the cultures abolished neurite outgrowth. The results indicate that NGF is present in cartilage and bone of the chick embryo; it may determine the density of sympathetic innervation to the developing skeletal tissues.     

Use of ELISA to G4 Antigen to Quantitate Neurite Outgrowth in the Chick Both In Vivo and In Vitro

Abstract: The G4 glycoprotein is found on the earliest developing neurite tracts of the chick embryo. An ELISA is introduced here to quantify the amount of G4-expressing neurites in the picogram range. In this double-sandwich assay, an anti-G4 monoclonal antibody fixes the G4 antigen to the plastic surface, which then is detected by a polyclonal antiserum; nonspecific background is decreased by competitive displacement. The sensitivity of the assay allows us to follow quantitatively the very first neurite growth in embryonic heads, trunks, retinae, and brains. G4-based neurite growth is shown to occur earlier in heads than in trunks; in brain it is nearly 10-fold higher than in the retina by embryonic day 8. By determination of acetylcholinesterase (AChE) activities from the same homogenates, our earlier histochemical findings are verified now on a quantitative basis, again showing that AChE consistently precedes G4 antigen. Moreover, as an in vitro example, the G4 ELISA is applied to the nerve growth factor (NGF) standard bioassay on dorsal root ganglia; the half-maximal response is reached at ∼10 ng/ml of NGF for G4-based neurite growth and at ∼1 ng/ml of NGF for AChE expression, respectively.     

Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. II. Sensory neurons

Studies were carried out in dissociated cell cultures on the nerve growth factor (NGF) requirement of chick embryo dorsal root ganglionic (DRG) neurons. Findings were: (i) The minimum level of 2.5 S NGF required to sustain the survival of maximal numbers of process-bearing cells derived from 8-day (E8) embryonic DRGs is 0.5 ng/ml (～2 × 10^?11M). (ii) Cultures derived from chick embryos of increasing ages (E8 to E18) showed a progressive increase in the proportion of process-bearing cells which survived in the absence of NGF. While few process-bearing cells survived in cultures of E8 ganglia in the absence of NGF, survival of neurons in cultures derived from E17 and E18 ganglia was not affected by the absence of the factor. Comparable results were obtained with cultures in which the number of non-neuronal cells was greatly reduced. (iii) Neurons derived from E8 ganglia lost their NGF requirement in culture at a conceptual age similar to that which they appear to do so in vivo. These results are discussed with respect to the role of NGF in development of sensory neurons.