胎脑中神经生长因子(NGF)的纯化及生物效应研究

神经生长因子(Nervegrowthfactor;NGF)首先由Levi-Montalcin自小鼠颌下腺中纯化[1]。有促进交感、感觉神经细胞、前脑基底核胆碱能神经的存活及突起伸长作用,与机体防御机能相关,不仅对正常神经细胞有营养因子作用,而且具有调节损伤神经修复的机能。近期发 现脑内有合成NGF的机能,但存在量极微,海马中NGF,的含量仅为鼠领下腺的百万分之一[2]。     

大鼠脊神经节感觉神经特异蛋白35kD(SSP-35)的纯化及鉴定(英文)

 以大鼠脊髓背根神经节及背根纤维组织为材料 ,通过离子交换层析 ,FPLC凝胶过滤等技术分离纯化了脊感觉神经特异蛋白 35kD(SSP 35) .经SDS PAGE分析 ,该蛋白特异地存在于脊感觉神经而不存在于脊运动神经 .非还原电泳结果表明 ,该蛋白分子内含有巯基 ,有二聚体存在 .根据HPLC测定 ,蛋白纯度达 90 %以上 .将此蛋白给予培养的PC 1 2细胞 ,观察到它具有神经营养作用 .     

pIRES2-GDNF-NT-3真核表达载体的构建与鉴定(英文)

目的:采用一种简便和高效的方法构建双基因共表达载体pIRES2-GDNF-NT-3。方法:人胶质细胞源性神经营养因子和神经营养素3是采用PCR的方法从人外周血单个核细胞的基因组DNA中获取,将人胶质细胞源性神经营养因子的cDNA片段插入到pIRES2-EGFP多克隆位点构建成为pIRES2-GDNF-EGFP.神经营养素3 cDNA片段通过替换EGFP的方式插入到pIRES2-GDNF-EGFP中构建成为pIRES2-GDNF-NT-3双基因共表达载体。结果:人胶质细胞源性神经营养因子和神经营养素3被克隆,通过测序和酶切鉴定的得知与基因库报道序列一致。结论:人神经生长因子和神经营养素3双基因真核表达载体成功构建,它提供了一个新的表达系统,为进一步研究双基因的功能奠定了基础。     

白眉蝮蛇蛇毒神经生长因子的研究Ⅰ.神经生长因子(NGF)的纯化及其生物学活性鉴定

研究白眉蝮蛇蛇毒神经生长因子纯化方法并行生物学活性鉴定。方法从白眉蝮蛇乌苏里亚种蛇毒中,经硫酸胺分段盐析,提取含有神经生长因子的粗制品,再经二乙氨基乙基纤维素离子交换层析和羧甲基纤维素离子交换层析,分离出蛋白质纯品,经十二烷基磺酸钠——聚丙烯酰胺凝胶电泳进行鉴定,结果纯化的纯品为单一区带,分子量为４３ｋｕ。生物学活性鉴定证明分离出的该蛋白质纯品可促进神经细胞突起密集生长。     

具有生物活性的川牛膝果聚糖的结构(英文)

The structure of a bioactive fructan RCP isolated from the traditional Chinese medicinal herb Cyathula officinalis Kuan was determined by NMR, methylation, reductive-cleavage and GC-MS analyses. It was a highly branched fructan possessing a (2→1)-linked β-D-fructofuranosyl (Fruf) backbone with (2→6)-linked β-D-Fruf side chains, belonging to neokestose family. Of the 93.17% β-D-Fruf residues, 24.15% was terminal, 26.24% was 1-linked, 20.46% was 6-linked, and 22.32% was 1,6-linked. Of the 6.83% α-D-glucopyranosyl (Glcp) residues, 2.14% was terminal and 4.69% was 6-linked. A structural model was postulated for a degree of polymerization (DP) of 15.     

茼蒿素类似物对菜粉蝶幼虫的生物活性及作用机理研究(英文)

以菜粉蝶幼虫为对象对茼蒿素类似物进行了拒食活性筛选,结果表明,12号和20号化合物的活性最高。生测结果表明,在非选择性条件下,12号和20号化合物对菜粉蝶3龄幼虫处理后24h后的AFC50分别为370.00μg/mL和226.93 μg/ml;在选择性条件下,对菜粉蝶4龄幼虫处理后24h后的AFC50分别为398.88?g/ml和280.54?g/ml。20号化合物能明显延迟菜粉蝶卵的孵化,降低菜粉蝶幼虫血淋巴及其蛋白质的含量。盆栽试验结果表明,20号化合物具有较好的保叶效果并对菜粉蝶幼虫具有较好的防效。     

人β_2m转基因小鼠的制备及鉴定(英文)

制备人 β2m转基因小鼠 ,研究HLA B2 70 4基因的表达 .应用显微注射将人 β2m基因注入C5 7BL 6×昆明鼠和昆明鼠×昆明鼠F1代受精卵 .出生动物及其后代经PCR筛选 ,采用斑点杂交和Southern杂交对阳性鼠基因组DNA标本进行进一步鉴定和测定整合拷贝数 ,利用RT PCR检测阳性鼠中人 β2m转基因的表达 .6只原代仔鼠及 7只它们的下一代鼠 (F1)带有人 β2m基因 .由微注射基因后移卵出生的 86只小鼠中 ,C5 7BL 6×昆明鼠杂交仔鼠 35只 ,其中 4只阳性 (11 4 % ) ,昆明鼠×昆明鼠杂交仔鼠 5 1只 ,其中 2只阳性 (3 9% ) ,含有人 β2m基因的原代鼠×昆明鼠杂交仔鼠 2 0只 ,其中 7只阳性 .整合的转基因均为单拷贝 .Southern杂交证实上述阳性鼠确有转基因整合 .阳性鼠的皮肤、结肠、睾丸和脾脏组织中均有人β2m转基因mRNA的表达 .在转基因动物制备中 ,C5 7BL 6×昆明鼠F1代明显优于昆明鼠×昆明鼠F1代 .与人HLA B2 70 4基因相比 ,人 β2m基因不易整合 ,其整合率与整合拷贝数均较低 .得到的人 β2m转基因小鼠能够将人 β2m基困传给下一代 ,并可与人HLA B2 70 4转基因鼠交配 ,研究它的致病性     

人β_2m转基因小鼠的制备及鉴定(英文)

 制备人 β2m转基因小鼠 ,研究HLA B2 70 4基因的表达 .应用显微注射将人 β2m基因注入C5 7BL 6×昆明鼠和昆明鼠×昆明鼠F1代受精卵 .出生动物及其后代经PCR筛选 ,采用斑点杂交和Southern杂交对阳性鼠基因组DNA标本进行进一步鉴定和测定整合拷贝数 ,利用RT PCR检测阳性鼠中人 β2m转基因的表达 .6只原代仔鼠及 7只它们的下一代鼠 (F1)带有人 β2m基因 .由微注射基因后移卵出生的 86只小鼠中 ,C5 7BL 6×昆明鼠杂交仔鼠 35只 ,其中 4只阳性 (11 4 % ) ,昆明鼠×昆明鼠杂交仔鼠 5 1只 ,其中 2只阳性 (3 9% ) ,含有人 β2m基因的原代鼠×昆明鼠杂交仔鼠 2 0只 ,其中 7只阳性 .整合的转基因均为单拷贝 .Southern杂交证实上述阳性鼠确有转基因整合 .阳性鼠的皮肤、结肠、睾丸和脾脏组织中均有人β2m转基因mRNA的表达 .在转基因动物制备中 ,C5 7BL 6×昆明鼠F1代明显优于昆明鼠×昆明鼠F1代 .与人HLA B2 70 4基因相比 ,人 β2m基因不易整合 ,其整合率与整合拷贝数均较低 .得到的人 β2m转基因小鼠能够将人 β2m基困传给下一代 ,并可与人HLA B2 70 4转基因鼠交配 ,研究它的致病性     

羊胎盘免疫调节活性因子(SPIF-Ⅰ)的分离纯化和鉴定研究

为探讨研究羊胎盘的活性组分,将羊胎盘绞碎通过匀浆、冻融、超虑等步骤制备羊胎盘提取液,经Sephadex G-50和Sephadex G-25凝胶过滤、Sephadex G-10凝胶柱脱盐,反相高效液相色谱纯化出单一组分并进行理化性质、生物活性初步研究.结果得到紫外特征吸收峰为273nm、分子质量为303.8的羊胎盘免疫调节活性因子-Ⅰ(Sheep placental immunoreglating activity factor-Ⅰ,SPIF-Ⅰ).体外生物学活性试验证明,SPIF-Ⅰ能显著影响小鼠脾淋巴细胞增殖分裂,具有明显的剂量-效应依赖关系.SPIF-Ⅰ是羊胎盘提取液中一种具有免疫调节的小分子活性多肽.     

小牛胸腺新胸腺活性因子(TAF-Ⅰ)的纯化和鉴定

以小牛胸腺为原料 ,通过匀浆、冻融、超滤、SephadexG 15凝胶过滤、DEAE SephadexA 2 5阴离子交换层析和反相高效液相色谱等步骤 ,分离得到了胸腺活性因子Ⅰ (thymusactivityfactorⅠ ,TAF Ⅰ ) .5 0 0g小牛胸腺组织中纯化得到了 0 92mg胸腺活性因子Ⅰ .经ESI MS质谱鉴定 ,结果显示其分子量为 6 18 8.氨基酸组成分析显示它由Ala、Gly、Glu、Gln、Lys、Ser 6种氨基酸残基组成 .体外生物学活性实验证明 ,TAF Ⅰ能显著促进人外周血淋巴细胞的E 玫瑰花结的形成率和促进小鼠脾淋巴细胞的分裂增殖     

Single-Step Purification and Biological Activity of Human Nerve Growth Factor Produced from Insect Cells

Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus-infected Spodoptera frugiperda (SF-9) insect cell system. Culture supernatants contained 2-3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single-step affinity chromatography procedure using a high-affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1-2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transfected into the SF-9 cells coded for pro-NGF, the NGF recovered after purification was greater than 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 +/- 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 +/- 20 pM and 9.6 +/- 1.5 pM for a 3-day primary response and a 1-day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 +/- 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from submaxillary glands.     

神经生长因子启动哮喘神经-内分泌-免疫网络功能失衡

神经生长因子是一种对神经生长、分化起到营养作用的肽类,其在哮喘发病过程中被认为是连接神经-内分泌-免疫网络的桥梁,作用机制可能如下:a.神经生长因子引起气道神经解剖结构和功能变化,促进气道神经末梢合成和释放递质,有助于气道重构的形成;b.神经生长因子能够增强变应原特异性IgE抗体的表达,促进肥大细胞、嗜酸性粒细胞、淋巴细胞等在气道聚集,诱导其释放炎症介质,改变免疫应答平衡状态;c.神经生长因子可能启动肾上腺髓质细胞冗余性,使其向神经细胞转变,导致髓质细胞内分泌功能削弱,使肾上腺素合成、释放和再摄取功能障碍,最终导致循环中肾上腺素达不到维持气道舒张状态所需水平.     

神经生长因子与冻干异体神经桥接大鼠神经缺损的研究

实验采用冻干处理的异体神经与外源性神经生长因子（ＮＧＦ）结合来桥接大鼠的坐骨神经１．０ｃｍ的缺损。用雄性Ｗｉｓｔａｒ大鼠进行的四组实验结果表明：冻干处理的异体神经可降低其抗原性,但处理后并不损害雪旺氏细胞（ＳＣ）基底膜的完整性,在移植后可能成为轴突再生的通道和支架;外源性ＮＧＦ与冻干神经结合形成的复合体,可为神经的再生提供一个较好的微环境,具有成为理想桥接材料的可能性     

TF-1细胞增殖法测定神经生长因子生物学活性的建立与应用

目的：建立并优化用于检测神经生长因子（NGF）生物学活性的TF-1细胞增殖法,并应用于重组人NGF和鼠NGF制品的活性检测。方法：将梯度稀释的人NGF国际参考品与TF-1细胞相作用,通过MTT法测定细胞增殖情况,建立测定NGF活性的TF-1细胞增殖法;从粒细胞巨噬细胞集落刺激因子（CM-CSF）残留、NGF加样稀释率、TF-1细胞接种浓度、培养时间等多个环节对实验条件进行优化;将建立的TF-1细胞增殖法应用于重组人NGF和鼠NGF的活性检测。结果：建立了用于NGF活性检测的TF-1细胞增殖法;实验条件优化结果表明,在无GM-CSF残留的情况下,将稀释至1／4的NGF样品与4×10^5／mL的细胞相互作用,培养48h,可以得到更为典型的的四参数曲线;利用条件优化后建立的检测方法对重组入NGF和鼠NGF各2批样品分别进行4次测定,结果平均值分别为10．01×10^5、10．81×10^5和3．55×10^5、3．30×10^5U／mg,变异系数分别为7．5％、7．2％和7．2％、9．1％,检测结果稳定均一,表明检测方法具有很好的重复性。结论：建立并优化了用于NGF活性检测的TF-1细胞增殖法,该方法操作简便、定量准确、重复性好、稳定可靠,可有效应用于人NGF和鼠NGF制品的活性检测。     

Disulfide Bond Formation Between Nerve Growth Factor and the Nerve Growth Factor Receptor from Embryonic Sensory Neurons

Recent studies with sympathetic neurons using radiolabeled nerve growth factor have indicated that a high-molecular-weight covalent complex is formed. This complex is between the nerve growth factor and the high-affinity (type I) receptor and occurs through the formation of a disulfide bond. Studies presented in the present article demonstrate a similar complex is formed on chicken embryonic sensory neurons. The formation of this complex is inhibited by the addition of unlabeled nerve growth factor, metabolic energy inhibitors (dinitrophenol and NaF), and of sulfhydryl reagents. On the other hand, formation of this complex is not inhibited by temperature, or by the addition of insulin or epidermal growth factor. The receptor involved in the covalent complex formation is the high-affinity (type I) receptor. The molecular weight of this complex is approximately 232,000 daltons. Evidence indicates that this covalent complex may be required for the biological activity of the nerve growth factor.     

神经生长因子药效学研究

目的考察神经生长因子对视神经损害的疗效。方法腹腔注射二硫化碳致视神经损害的大鼠模型,给药组球后注射或肌内注射神经生长因子,每天一次,每周6d,共计3周,空白对照组球后注射等量生理盐水,于治疗前后测定大鼠模式翻转（FREP）和闪光视觉诱发电位。结果球后注射高剂量组大鼠在治疗10d和20d时各波潜伏时比对照组有显著缩短,而球后注射低剂量组和肌内注射高剂量组大鼠于治疗10d时FVEP各波潜伏时也显著缩短,肌内注射低剂量组大鼠于治疗20d时FEP的P1和P2波潜伏时也显著或非常显著缩短。结论神经生长因子对大鼠视神经损害有明显的治疗作用。     

利用SEC—HPLC测定鼠神经生长因子的纯度

目的：建立检测鼠神经生长因子（mNGF）纯度的分子排阻高效液相色谱法（SEC-HPLC）。方法：利用TSK G3000 SWXL分析柱和Waters 2695/2487高效液相色谱系统检测mNGF标准品纯度,并对方法的专属性、线性、重复性、中间精密度、检测限及耐用性等指标进行评估。结果：空白溶剂在mNGF积分范围内无特异峰出现;样品浓度与吸收峰之间线性回归系数R2=0.9998;6次进样峰面积相对标准偏差（RSD）为1.18%;中间精密度分析RSD为0.45%;检测限为0.2μg;耐用性实验表明磷酸盐浓度在0.2~0.3 mol/L之间变动对检测结果无影响。结论：各项指标符合规定,SEC-HPLC法可用于检测mNGF的纯度。     

Nerve Growth Factor of Red Nucleus Involvement in Pain Induced by Spared Nerve Injury of the Rat Sciatic Nerve

Nerve growth factor (NGF), a member of the neurotrophin family, is essential for the development and maintenance of sensory neurons and for the formation of central pain circuitry. The current study was designed to evaluate the expression of NGF in the brain of rats with spared nerve injury (SNI), using immunohistochemical technique. The results showed that the level of NGF in the Red nucleus (RN) of SNI rats was apparently higher than that of sham-operated rats. To further study the effect of NGF in the development of neuropathic pain, different doses of anti-NGF antibody (20, 2.0 and 0.2 μg/ml) were microinjected into the RN contralateral to the nerve injury side of SNI rats. The data suggested that the higher doses of anti-NGF antibody (20 and 2.0 μg/ml) significantly attenuated the mechanical allodynia of neuropathic rats, while the 0.2 μg/ml antibody showed no analgesic effect. These results suggest that the NGF of RN is involved in the development of neuropathic allodynia in SNI rats.     

Biochemical Characterization of Recombinant Human Nerve Growth Factor

Recombinant human nerve growth factor (rhNGF) was expressed and secreted by Chinese hamster ovary cells and purified to homogeneity using ion-exchange and reversed-phase (RP) chromatography. The isolated product was shown to be consistent with a 120-amino-acid residue polypeptide chain by amino acid composition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), RP-HPLC, and mass spectrometry and with an N-terminal sequence consistent with that expected from the cDNA for human nerve growth factor. By size-exclusion chromatography, rhNGF behaves like a noncovalent dimer. Limited enzymatic digests of the 120-residue monomer produced additional species of 118 (trypsin, removal of the C-terminal Arg119-Ala120 sequence) and 117 (trypsin plus carboxypeptidase B, removal of the C-terminal Arg118-Arg119-Ala120 sequence) residues. Each of these species was isolated by high-performance ion-exchange chromatography and characterized by amino acid and N-terminal sequence analyses, SDS-PAGE, RP-HPLC, and mass spectrometry. All three species were present in the digests as both homodimeric and heterodimeric combinations and found to be equipotent in both the chick dorsal root ganglion cell survival and rat pheochromocytoma neurite extension assays.     

Growth inhibition, morphological differentiation and stimulation of survival in neuronal cell type (Neuro-2a) treated with trophic molecules

Trophic molecules are key regulators of survival, growth and differentiation of neural cells. Neuronal cell type Neuro-2a is a good model to study development and molecules modulating this process, and retinoic acid (RA) and neurotrophins (NGF, BDNF, NT-3 and NT-4) have been shown to be active in this modulation. The purpose of the present study was the functional analysis of these trophic molecules in our short-term bioassay of Neuro-2a cells, an immortalised murine neuroblastoma cell line. Through cell counting, image process and arithmetic combination of digital parameters of treated and untreated cultures, we show that RA inhibits growth and induces morphological neuronal phenotype of treated cells. Through DNA labelling with BrdU we also show that NGF, BDNF, and NT-3 increase survival and proliferation of cells, grown in serum-deprived media. From these results we conclude that neurotrophins have manifest trophic effects on cells improving survival, growth and proliferation and we also confirm the growth arrest and differentiation properties of RA on Neuro-2a cells.