胰岛素对糖尿病大鼠下颌下腺内NGF及VEGF表达的影响

目的观察神经生长因子(NGF)及血管内皮生长因子(VEGF)在糖尿病大鼠下颌下腺内表达变化,探讨胰岛素对其表达影响及可能机制。方法 SD大鼠30只,随机分为:对照组、糖尿病组、胰岛素治疗组。大鼠2个月成模后,取血检测血糖;取下颌下腺组织,分别进行HE染色、免疫组织化学染色和计算机图像分析。结果①对照组血糖与糖尿病组比较差异有统计学意义;胰岛素治疗组血糖与糖尿病组比较差异有统计学意义。②糖尿病组腺泡轻度萎缩,排列不规则,颗粒曲管数目减少,直径变小;胰岛素治疗组较对照组变化不明显。③与对照组相比较,糖尿病组NGF表达下调,VEGF表达上调。与糖尿病组相比较,胰岛素治疗组NGF表达上调,VEGF表达下调。结论糖尿病大鼠下颌下腺内NGF表达减少、VEGF表达增高,可能是糖尿病患者神经病变与血管病变的重要原因之一;胰岛素治疗可能与NGF和VEGF的表达改变有内在联系。     

表皮生长因子对大鼠精原干细胞的影响

研究表皮生长因子（EGF）对体外培养的精原干细胞自我更新、增殖过程中所起的调控作用;建立完善的精原干细胞体外培养体系,为精原干细胞的体外大量扩增提供技术和方法．为治疗男性不育等提供相关技术。通过研究证明EGF能够促进精原干细胞的增殖,EGF受体的活化对EGF促进精原干细胞增殖起重要作用。     

诱导回输胰岛素分泌细胞对大鼠糖尿病的疗效

目的:研究胰岛素分泌细胞的体外诱导方法及其对大鼠糖尿病的疗效.方法:分离培养大鼠骨髓干细胞,用尼克酰胺及肠促胰岛素类似物诱导其分化为胰岛素分泌细胞.将24只Wistar大鼠随机分为对照组、糖尿病组和诱导组.后两组建立糖尿病模型,将该胰岛素分泌细胞回输至诱导组体内,监测大鼠体重、血糖(空腹及OGTT 120分血糖)及空腹...     

雌二醇对大鼠颌下腺EGF、NGF生成的影响

为了研究雌二醇（estradiol-17β,E2）对颌下腺表皮生长因子（epidermal growth factor,EGF)和神经生长因子（nerve growth factor,NGF)生成的影响,研究采用免疫组织化学方法,对外源性投予雌二醇后的大鼠颌下腺进行了观察。结果证实E2明显促进颌下腺EGF和NGF的生成。提示雌二醇可能对EGF和NGF的合成起重要的调节作用。     

外源性VEGF促进大鼠Ⅱ度烫伤创面中晚期愈合

目的探究Ⅱ度烫伤时外源性血管内皮生长因子(vascular endothelial growth factor,VEGF)对皮肤愈合及其对表皮干细胞(epidermal stem cells,ESCs)迁移、分化的影响方法健康Wistar大鼠随机分为VEGF组、空白对照组、阿西替尼(Axitinib,VEGF抑制剂)组。采用水浴烫伤法制备Ⅱ度烫伤模型,分别以0.2μg/ml VEGF、PBS溶液和10μg/ml阿西替尼处理各组创面,各组均治疗7d,从烫伤至创面愈合分别在第2d、8d、14d及21d测量创面愈合情况,并取创面组织作组织学检测,运用免疫组化技术检测ECSs的分布及数量。结果①创面愈合率:烫伤后21d VEGF组>对照组>阿西替尼组;②愈合速度:烫伤后1-7d空白对照组>阿西替尼组>VEGF组,其后VEGF组愈合速度逐渐加快,第14d开始愈合速度表现为VEGF组>空白对照组>阿西替尼组;③组织学变化:烫伤后8-21d,VEGF组表皮细胞增殖明显,表皮修复和毛囊再生迅速,均早于空白对照组及阿西替尼组。④ECSs阳性细胞率变化:烫伤后第8-14dVEGF组ECSs阳性细胞率明显高于空白对照组和阿西替尼。结论Ⅱ度烫伤时,外源性VEGF在愈合中晚期加快愈合速度使愈合时间明显缩短,并且促进毛囊汗腺的再生使修复后的创面在外观、功能与正常皮肤相近,有助于提高全层皮肤创面的愈合质量。     

胰岛素对糖尿病大鼠肝细胞氧化损伤的影响

利用四氧嘧啶建立糖尿病大鼠模型 ,研究了胰岛素对糖尿病大鼠肝细胞及线粒体氧化损伤的保护作用。结果表明 ,胰岛素 1U kg皮下注射 9d ,能明显降低肝组织谷丙转氨酶、谷草转氨酶、乳酸脱氢酶、黄嘌呤氧化酶的活性 ,显著提高肝组织丙二醛的含量及肝线粒体O· -2 (活性氧自由基 )的生成量 ,显著提高抗氧化酶谷胱甘肽过氧化物酶、超氧化物歧化酶的活性 ,提高肝线粒体H+ ATPase的合成活力 ,从而使受损的肝细胞功能得到改善     

人表皮细胞生长因子脂质体制备及促大鼠烫伤修复研究

目的:制备重组人表皮细胞生长因子(rhEGF)脂质体,并考察其促大鼠烫伤创面愈合的作用.方法:采用pH梯度法制备rhEGF脂质体;超滤-离心法分离rhEGF脂质体混悬液中的游离rhEGF,ELISA法测定rhEGF含量,计算脂质体包封率;采用透射电镜观察脂质体的外观形态;采用纳米粒度及Zeta电位分析仪分别测定脂质体的粒径和Zeta电位;以大鼠烫伤模型观察给药后各试验组创面愈合过程中的形态、愈合时间和愈合率的变化.结果:制备的rhEGF脂质体包封率为57.7±1.1％;脂质体形状较为规则,呈完整圆球形或椭圆形的单室囊泡;脂质体粒度分布均匀,呈正态分布,平均粒径为63.7 nm;脂质体的Zeta电位为+9.2mV,带正电荷;rhEGF脂质体高、中剂量组能显著性促进大鼠烫伤创面愈合,使创面愈合时间明显提前,低剂量组促烫伤修复效应不明显.结论:pH梯度法制备的rhEGF脂质体包封率较高,rhEGF脂质体对大鼠烫伤创面的愈合有明显促进作用.     

表皮生长因子对精原干细胞的作用研究

应用不连续Percoll梯度液和选择性贴壁法分离纯化精原干细胞:c-kit细胞免疫组化鉴定细胞类型;MTT法研究EGF对精原干细胞增殖的效应;加入MAPK-ERK信号通路特异性抑制剂PD98059,探讨EGF对精原干细胞增殖作用的可能机制.证明:1)c-kit细胞免疫组化结果显示分离得到细胞为精原千细胞;2)MTT结果显示各实验组比对照组细胞数量均有显著增多(p＜0.01),且20 ng/mL剂量组的增殖作用最明显;3)与对照组相比,加入PD98095组的活细胞数有显著下降(p＜0.01).结论:EGF能够促进精原干细胞的增殖,并且可以通过MAPK-ERK信号通路起作用.     

糖尿病大鼠膀胱和骶髓背根神经节中NGF的表达与尿流动力学改变的研究

目的：探讨糖尿病大鼠膀胱与骶髓背根神经节（DRG）中神经生长因子（NGF）的表达与尿流动力学改变。方法：建立糖尿病大鼠模型10只,对照组10只,应用酶联免疫吸附试验（ELISA）法,分别检测大鼠膀胱组织及骶髓DRG中NGF的变化情况,结合代谢笼及尿流动力学改变,探讨糖尿病膀胱病变的可能发病机制。结果：造模12周后,糖尿病大鼠膀胱容量较正常对照组明显增大（1.47±0.28vs0.71±0.12,p〈0.05）,残余尿量明显增多（0.52±0.18vs0.07±0.08,p〈0.01）,排尿效率明显下降,膀胱及骶髓DRG中NGF表达水平明显降低。结论：NGF在糖尿病大鼠膀胱和骶髓背根神经节中低表达,在糖尿病膀胱病变中发挥着重要作用。     

糖尿病大鼠膀胱和骶髓背根神经节中NGF 的表达与尿流动力学 改变的研究

目的:探讨糖尿病大鼠膀胱与骶髓背根神经节(DRG)中神经生长因子(NGF)的表达与尿流动力学改变。方法:建立糖尿病大鼠模型10只,对照组10只,应用酶联免疫吸附试验(ELISA)法,分别检测大鼠膀胱组织及骶髓DRG中NGF的变化情况,结合代谢笼及尿流动力学改变,探讨糖尿病膀胱病变的可能发病机制。结果:造模12周后,糖尿病大鼠膀胱容量较正常对照组明显增大(1.47±0.28vs0.71±0.12,p<0.05),残余尿量明显增多(0.52±0.18vs0.07±0.08,p<0.01),排尿效率明显下降,膀胱及骶髓DRG中NGF表达水平明显降低。结论:NGF在糖尿病大鼠膀胱和骶髓背根神经节中低表达,在糖尿病膀胱病变中发挥着重要作用。     

Nerve Growth Factor Effect on Adenosine Transport in Cultured Chromaffin Cells

Chromaffin cells both recently isolated or in culture present a high-affinity adenosine transporter with a Km value of 1 microM. When cells were exposed to nerve growth factor (NGF; 10 ng/ml), the adenosine transporter affinity decreased to 3 microM. This value was maintained from 3 days after plating to the end of the culture period. A change in the transport capacity was observed, with a significant increase (approximately 200-260%) in NGF-cultured cells throughout the period studied.     

碱性成纤维细胞生长因子对大鼠晚期周围神经再生作用的实验研究

目的探讨外源性碱性成纤维细胞生长因子(bFGF)对晚期周围神经再生的作用.方法50只SD大鼠随机分治疗组、对照组各25只,切断右侧坐骨神经,12周后予以修复,修复术后每日分别给予bFGF和生理盐水,行神经电生理和组织学检查.结果治疗组和对照组修复处远段神经均有不同程度再生,4周时已可见到再生轴突,且治疗组多见.计量分析治疗组运动神经传导速度、神经肌肉动作电位幅值、髓鞘厚度、再生轴突直径和截面积明显优于对照组.治疗组与对照组相比,差异有显著性.结论bFGF能促进晚期周围神经再生.     

Nerve Growth Factor Promotes Neurite Regeneration in PC12 Cells by Translational Control

Abstract: When PC12 cells are primed with nerve growth factor (NGF) for periods of ≥1 week, they acquire the ability to regenerate neurites rapidly in response to NGF. It is not known how NGF promotes this regeneration, but it does not require ongoing RNA synthesis. Previous studies have suggested that NGF directs the accumulation of precursor molecules that are rapidly assembled to form the regenerated neurites. To address the nature of these precursor molecules, we have treated PC12 cells with macromolecular synthesis inhibitors during the priming and regeneration phases of neurite growth. Here we show that NGF promotes neurite regeneration by inducing the synthesis of new proteins. These proteins are encoded by short-lived mRNAs that are generated during the NGF priming period. The isolation and identification of these mRNAs will allow a further understanding of how NGF promotes neurite regeneration.     

Nerve Growth Factor, Epidermal Growth Factor, and Insulin Differentially Potentiate ATP-Induced [Ca2+]i Rise and Dopamine Secretion in PC12 Cells

Abstract: To study how growth factors affect stimulus-secretion coupling pathways, we examined the effects of nerve growth factor (NGF), epidermal growth factor (EGF), and insulin on ATP-induced [Ca²⁺]_i rise and dopamine secretion in PC12 cells. After a 4-day incubation of cells, all three factors increased ATP-induced dopamine secretion significantly. We then examined which step of ATP-induced secretion was affected by the growth factors. Cellular levels of dopamine-β-hydroxylase and catecholamines were increased by NGF treatment but were not affected by EGF or insulin. The ATP-induced [Ca²⁺]_i rise was also enhanced after growth factor treatment. The EC₅₀ of ATP for inducing [Ca²⁺]_i rise and dopamine secretion was increased by NGF treatment but not by treatment with EGF or insulin. Accordingly, the dependence on [Ca²⁺]_i of dopamine secretion was increased significantly only in NGF-treated cells. Our results suggest that for EGF- and insulin-treated PC12 cells, the increase in secretion is mainly due to increased potency of ATP in inducing [Ca²⁺]_i rise. NGF treatment not only increased the potency of ATP but also decreased the Ca²⁺ sensitivity of the secretory pathway, which as a result becomes more tightly regulated by changes in [Ca²⁺]_i.     

Production of 1,2-Diacylglycerol in PC12 Cells by Nerve Growth Factor and Basic Fibroblast Growth Factor

The addition of nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) to PC12 cells prelabeled with [3H]inositol and preincubated for 15 min in the presence of 10 mM LiCl stimulated the production of inositol phosphates with maximal increases of 120-180% in inositol monophosphate (IP), 130-200% in inositol bisphosphate (IP2), and 45-50% in inositol trisphosphate (IP3) within 30 min. The majority of the overall increase (approximately 85%) was in IP; the remainder was recovered as IP2 and IP3 (approximately 10% as IP2 and 5% as IP3). Under similar conditions, carbachol (0.5 mM) stimulated about a 10-fold increase in IP, a sixfold increase in IP2, and a fourfold increase in IP3. The mass level of 1,2-diacylglycerol (DG) in PC12 cells was found to be dependent on the incubation conditions; in growth medium [Dulbecco's modified Eagle's medium (DME) plus serum], it was around 6.2 mol %, in DME without serum, 2.5 mol %, and after a 15-min incubation in Dulbecco's phosphate-buffered saline, 0.62 mol %. The addition of NGF and bFGF induced an increase in the mass level of DG of about twofold within 1-2 min, often rising to two- to threefold by 15 min, and then decreasing slightly by 30 min. This increase was dependent on the presence of extracellular Ca2+, and was inhibited by both phenylarsine oxide (25 microM) and 5'-deoxy-5'-methylthioadenosine (3 mM). Under similar conditions, 0.5 mM carbachol stimulated the production of DG to the same extent as 200 ng/ml NGF and 50 ng/ml bFGF. Because carbachol is much more effective in stimulating the production of inositol phosphates, the results suggest that both NGF and bFGF stimulate the production of DG primarily from phospholipids other than the phosphoinositides.     

肌注神经生长因子及血小板衍生生长因子对胫骨干闭合骨折大鼠早期骨愈合的影响及机制探究

摘要 目的：探讨肌肉注射神经生长因子（NGF）和血小板衍生生长因子（PDGF）对胫骨干闭合骨折大鼠早期骨愈合的效果及潜在机制。方法：采用随机数字表法将80只健康成年雄性SD大鼠分为模型组、NGF组、PDGF组和NGF+PDGF组,各20只。建立胫骨干闭合骨折模型后,给予NGF组大鼠肌注0.8 μg NGF;给予PDGF组大鼠肌注0.8 μg PDGF,给予NGF+PDGF组大鼠肌注0.8 μg NGF和0.8 μg PDGF;给予模型组大鼠肌注等体积生理盐水。分别在治疗第2周（T0）、第4周（T1）、第6周（T2）通过X线检查计算骨痂体积,采用酶联免疫吸附法检测血清中碱性磷酸酶（AKP）水平。颈椎脱臼法处死大鼠后采用苏木精-伊红（HE）染色观察胫骨骨折端病理学改变,采用实时荧光定量PCR法检测骨痂组织骨形态发生蛋白2（BMP2）、血管内皮生长因子（VEGF）和胰岛素样生长因子-1（IGF-1）mRNA相对表达水平。结果：NGF+PDGF组大鼠在T1时骨折断端愈合,骨痂体积大于其他三组;NGF组、PDGF组大鼠在T2时骨折断端愈合,骨痂体积大小均大于模型组（P<0.05）。模型组大鼠T2时骨折断端尚未完全愈合,骨痂体积显著大于其他三组（P<0.05）。NGF+PDGF组大鼠T0~T1时血清AKP水平均显著高于其他三组,NGF组和PDGF组大鼠血清AKP水平显著高于模型组（P<0.05）。T2时4组大鼠血清AKP水平比较无显著差异（P>0.05）。T1时,NGF组、PDGF组和NGF+PDGF组大鼠均可见骨小梁形态更加粗大、致密,呈栅栏状排列,骨小梁间的间隙变小,NGF+PDGF组大鼠骨断裂处被新生骨填满,NGF组、PDGF组骨断裂处仍有少量间隙。T1时NGF+PDGF组大鼠BMP2、VEGF和IGF-1相对表达水平均显著高于其他三组（P<0.05）,NGF组和PDGF组大鼠各指标mRNA相对表达水平比较无显著差异（P>0.05）,但均显著高于模型组（P<0.05）。T2时各组大鼠骨痂组织中BMP2、VEGF和IGF-1 mRNA相对表达水平比较无显著差异（P>0.05）。结论：NGF和PDGF对胫骨干闭合骨折大鼠早期骨愈合有协同促进作用,可能与促进BMP2、VEGF和IGF-1表达上调有关。     

Structural Features of the Nerve Growth Factor Inducible Large External Glycoprotein of PC12 Pheochromocytoma Cells and Brain

Abstract: We have examined the oligosaccharide structure of a major M_r= 230,000 cell surface glycoprotein from rat PC12 pheochromocytoma cells, and of the immunochemically cross-reactive species present in brain. In response to nerve growth factor (NGF) the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. These morphological changes are accompanied by a 3- to 5-fold increase in the concentration and labeling of this cell surface glycoprotein, which has previously been named the NGF-inducible large external, or NILE, glycoprotein. Tri- and tetraantennary complex oligosaccharides are the predominant carbohydrate units present in the NILE glycoprotein from both brain and PC12 cells, where they represent 77–90% of the biosynthetically labeled oligosaccharides. Most of these are not substituted by fucose on the core N -acetylglucosamine which is linked to asparagine, and are accompanied by smaller proportions of biantennary and high-mannose oligosaccharides. Sequential lectin-agarose affinity chromatography employing concanavalin A, lentil lectin, and the leukoagglutinating lectin of Phaseolus vulgaris , together with neuraminidase treatment of the fractionated glycopeptides, demonstrated a moderate degree of microheterogeneity among the predominant tri- and tetraantennary oligosaccharide units with respect to the presence of core fucose, outer galactose and sialic acid residues, and the substitution positions on the α-linked mannose residues. NGF treatment of the PC12 cells had no significant effect on the oligosaccharide structure of the NILE glycoprotein. The greater molecular size of the PC12 cell NILE glycoprotein as compared to the immunochemically cross-reactive species present in brain (M_r= 205,000) is apparently due to the greater size of the PC12 cell tri- and tetraantennary complex oligosaccharides.     

Nerve Growth Factor Receptors in Human Neuroblastoma Cells

Receptors for the nerve growth factor protein (NGFR) present in the human neuroblastoma cell line LAN-1 were characterized. LAN-1 cells display high-affinity (type I, with KD value of 5.9 X 10(-11) M) and low-affinity (type II, with KD value of 9.2 X 10(-9) M) binding to NGF. NGFR were fractionated by preparative isoelectric focusing in a granulated gel (PEGG). High-affinity binding was found in the 5.9-6.2 pH region of the PEGG, and low-affinity binding in the 4.6-4.8 and 8.8-9.3 pH ranges. After further analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we observed both 92.5- and 200-kDa molecular species associated with NGF binding activity. The 200-kDa protein was found in fractions displaying high-affinity NGF binding and the 92.5-kDa protein in fractions displaying low-affinity NGF binding. Equilibrium binding analysis of NGF in PEGG fractions confirmed the presence of two specific saturable binding sites with KD values similar to those observed for whole dissociated cells. When NGFR II activity from the acidic region of the PEGG chromatogram was incubated with NGFR II from the basic region of the PEGG chromatogram, there was no change in NGF binding or in the number of apparent NGF receptors. However, incubation of these same fractions with a fraction having only NGFR I showed an apparent increase in high-affinity NGF binding and a decrease in low-affinity NGF binding. Immunoprecipitation of this "mixed" fraction and analysis on SDS-PAGE under reduced and nonreduced conditions showed 200-kDa and 92.5-kDa proteins under nonreduced conditions and a 92.5-kDa protein under reduced conditions. Our findings are consistent with the hypothesis that there are two distinct NGF receptors in NGF-responsive cells. The interconvertibility of low- and high-affinity receptors and the possible existence of a modulator type protein or of "silent" type receptors are also in agreement with our findings.     

Insulin-Like Growth Factor I Receptor Expression and Function in Nerve Growth Factor-Differentiated PC12 Cells

Abstract: Receptors for insulin-like growth factor I (IGF-I) were studied on PC12EY cells, a subclone of PC12. Differentiation of PC12EY cells with nerve growth factor (NGF) did not alter either the number of IGF-I receptors nor their affinity for IGF-I. IGF-I receptors remained fully functional during differentiation, promoting increases in thymidine incorporation, glucose uptake, amino acid uptake, and the phosphorylation of the S6 protein of the ribosomes. IGF-I also increased the proportion of differentiated cells found in S-phase. But although the addition of IGF-I to naive cells caused an increase in cell number, there was no comparable increase when IGF-I was added to differentiated cells. Thus, although the receptor for IGF-I continues to be present and functional, IGF-I fails to induce cell proliferation in differentiated PC12 cells.