联合应用ATP和NGF对体外培养乳鼠脊髓神经元影响的研究

目的　探讨三磷酸腺苷 (ATP)和神经生长因子 (nervegrowthfactor ,NGF)联合使用时对体外培养的乳鼠脊髓神经元的作用。　方法　利用神经细胞培养技术 ,根据不同干预分为NGF组、ATP组、ATP加NGF组以及单纯对照组。相差倒置显微镜观察细胞生长情况 ,并测量细胞突起的长度 ;用MTT法测定培养细胞的存活率。　结果　实验各组神经元的存活率和轴突的长度均明显优于对照组 ,差异有非常显著性 (P <0 .0 1)。比较MTT值 :ATP组与ATP加NGF组之间差异有非常显著性 (t =4.2 5 ,P <0 .0 1) ;NGF组与ATP加NGF组之间差异有显著性 (t =3 .5 0 3 ,P <0 0 5 )。比较细胞突起长度 :ATP加NGF组分别与ATP组、NGF组之间差异有非常显著性 (P <0 .0 1)。　结论　ATP、NGF对于体外培养的脊髓神经元的存活均有较强的维持作用 ,并能促进轴突生长 ;而两者联合使用作用明显增强。     

不同营养因子组合对大鼠脊髓神经元的作用研究

[目的]探讨NGF、bFGF和BDNF的不同组合对大鼠脊髓神经元的作用。[方法]利用神经元培养技术，将新生1d的SD大鼠脊髓神经元接种于培养板，根据不同因子两两组合分为3个实验组、空白对照组和单因子对照组，因子终浓度均为50ng／ml。倒置相差显微镜动态观察，接种后第3、7d细胞爬片行β-tubulin3免疫荧光染色和Hoechst染色。测量阳性细胞最长突起长度，计算阳性细胞率，第1、3、5、7、9d行MTT检查，以OD值绘制生长曲线。[结果]各实验组神经元突起长度均优于对照组（P〈0．05），联合应用组优于单因子组，其中以A组（BDNF＋bFGF）突起最长，各实验组β-tubulin3阳性细胞率均高于对照组，MTT结果与染色结果一致。[结论]特定浓度下NGF、bFGF及BDNF可以有效促进大鼠脊髓神经元轴突的生长，并可有效维持神经元细胞存活，联合应用作用增强，BDNF＋bFGF作用最强。     

神经生长液含药血清对神经元生长和雪旺细胞增殖的影响

目的 观察神经生长液含药血清对体外培养的背根神经节神经元生长和雪旺细胞增殖的影响,探讨其促损伤神经修复的机制.方法 制备神经生长含药血清;采用新生大鼠背根神经节及雪旺细胞体外培养,通过形态学分析,观察神经生长液含药血清对背根神经节神经元突起生长的影响;采用MTr、BrdU标记和细胞计数法,观察神经生长液含药血清对雪旺细胞活力及其增殖的影响.结果 高剂量的神经生长液含药血清,可有效地促进大鼠背根神经节神经元生长;高、低剂量的神经生长液含药血清均能显著增加大鼠雪旺细胞的活力,促进雪旺细胞增殖.结论 神经生长液含药血清具有促进神经元生长、增强雪旺细胞活力、促进雪旺细胞增殖作用.     

神经生长因子促神经再生的研究进展

神经生长因子(Nerve Growth Factor,NGF) 是最早被发现、兼有神经元营养和促突起生长双重生物学功能的一种细胞调节因子。它对中枢和周围神经元的发育、分化、生长、再生和功能特性的表达均具有重要的调控作用,可促进受损神经再生、免受继发性损害、减少细胞死亡、支持神经元存活。本文就NGF的作用机理,对神经系统的生物效应,对神经再生的影响及外源性神经生长因子的应用途径等相关研究进展作一综述。     

细胞外ATP对体外培养乳鼠脊髓神经元生长的影响

目的 探讨三磷酸腺苷(adenosine triphosphate，ATP)对培养乳鼠脊髓神经元活性及生长的影响。方法 将不同浓度的ATP加入培养的大鼠乳鼠脊髓神经元培养液中，使终浓度分别为0．1、1．0和10．0mmol／L，倒置相差显微镜观察细胞生长及形态。用四唑盐比色法(MTT)和乳酸脱氢酶(LDH)法分别检测不同浓度组神经元的活性和细胞损伤程度。用图像分析仪分析不同浓度ATP对神经元突起生长的影响。结果 0．1mmol／LATP可提高细胞活性，但对细胞突起生长无影响；1．0mmol／LATP提高神经元活性，促进突起生长；10．0mmol／LATP降低神经元活性，抑制突起生长。结论 细胞外ATP能剂量依赖性地影响培养神经元活性，适宜浓度的ATP能提高神经元活性并促进神经元突起的生长。     

培养中的雪旺氏细胞对脊髓前角神经元的营养作用

通过隔玻片ＳＤ大鼠瓦勒氏变性远段坐骨神经获得的雪旺氏细胞（ＳＣ）与胎龄１４天的ＳＤ大鼠胚胎脊髓前角神经元（ＳＡＨＮ）联合培养，用倒置显微镜、Ｎｉｓｓｌ染色、ＳＡＨＮ免疫学观察，证实培养中的ＳＣ具有维持神经元成活和促进突起生长的作用。在这二种神经营养因子作用下，脊髓前角神经元胞体增大，突起生长可达数倍胞体长度。为认识和研究运用神经营养学说促进神经再生提供了新资料。     

嗅鞘细胞对脊髓后角神经元突起生长的影响

目的:观察嗅鞘细胞对胚胎脊髓后角神经元突起生长的影响。方法:分离孕15d胚胎大鼠脊髓,取纵向分开的后半制备成细胞悬液,培养于:(1)原代培养的嗅鞘细胞单细胞层上;(2)嗅鞘细胞培养上清中;(3)原代培养的成纤维细胞单细胞层上;(4)单纯培养。24h后终止培养,行抗神经丝-200免疫细胞化学染色。每组随机取15个神经元,在LeicaQ-570图像分析仪中测量各组神经元的平均总突起长度及平均单个最长突起长度,进行组间比较。结果:神经元平均总突起长度(1)～(4)组分别为408.62±126.91滋m、336.24±106.34滋m、199.37±76.14滋m和64.61±24.49滋m;平均单个最长突起长度(1)～(4)组分别为180.13±83.54滋m、141.22±53.68滋m、107.29±43.35滋m和23.04±5.30滋m。第(1)、(2)组神经元平均总突起长度和平均单个最长突起长度明显高于第(3)、(4)组(P<0.01),而(1)、(2)组之间没有明显差异(P>0.05)。结论:与嗅鞘细胞共培养能明显促进胚胎脊髓后角神经元突起的生长速度。     

重组腺相关病毒转导NIH3T3建立神经生长因子真核表达细胞株的研究

目的探讨重组腺相关病毒(recombinantadeno-associatedvirus,rAAV)转导美国国家健康研究中心建系的小鼠胚胎成纤维细胞(mouseembryofibroblast,NIH3T3)建立神经生长因子β(nervegrowthfactorsubunitβ,NGFβ)真核表达细胞株可行性,并检测其生物活性。方法使用表达NGFβ的重组腺相关病毒感染NIH3T3细胞,取感染重组病毒的3T3细胞培养基上清,经表达蛋白提取和浓缩,肝素-SepharoseCL-6B亲和层析柱的纯化;酶免疫斑点法检测表达蛋白的NGFβ抗原活性;Coomassic亮蓝G250法测定表达蛋白的浓度;十二烷基硫酸-聚丙烯酰胺凝胶电泳(sodiumdodecylsulfat-polyarylanidegelelectrophoresis,SDS-PAGE)分析检测表达蛋白的分子量,计算表达蛋白占总蛋白的比率;鸡胚背根神经节突起生长实验和小鼠嗜铬瘤细胞(mousea鄄drenalpheochromocytomacells,PC12)无血清培养生长刺激5溴-2-脱氧尿嘧啶(5-bromo-2-deoxyurididne,Brdu)掺入实验观察真核表达蛋白的生物学活性。结果转导的3T3细胞具有13.6kd的表达蛋白,每升细胞培养基中可以获得400~500μgNGFβ蛋白,表达蛋白纯度可达95.4%以上。表达蛋白具有很强的NGF抗原活性。鸡胚背根神经节突起生长实验显示真核表达的NGFβ在10ng/ml时有明显的促进突起生长作用,PC12细胞的Brdu掺入实验显     

神经生长因子及其受体在大鼠雪旺细胞中的表达及对先天性巨结肠的影响

先天性巨结肠(HD)是一种起源于神经嵴的组织发育障碍所致的疾病,其病因学研究很多,有基因缺陷和胚胎发育异常等[1-2].雪旺氏细胞(SCs)起源于胚胎时期的神经峭,神经嵴细胞在明显表达各自的表型之前就广泛地迁移并精确地在胚胎各处定位,这一事实曾引起人们极大的兴趣并提出许多假想.SCs可作为神经营养因子基因的遗传工程细胞用于神经系统损伤性和退行性疾病的基因治疗,但利用SCs临床治疗HD尚无报道.有学者在实验中发现SCs能分泌数种神经营养因子,如神经生长因子(NGF)、脑源性神经营养因子(BDNF)、成纤维细胞生长因子(FGF)等具有维持神经元存活并促进神经突起生长的作用[3].     

NGF与雌激素对离体培养的人头皮毛囊影响的实验研究

目的　观察神经生长因子 (nervegrowthfactor,NGF)、雌激素 (17β E2 )与米诺地尔对离体培养人头皮毛囊生长的影响。方法　建立离体人头皮毛囊培养模型 ,加入NGF、17β E2 和米诺地尔 ,通过目镜测微器测量毛囊长度 ,利用3H TdR掺入检测毛囊 2 4hDNA合成率。结果　NGF(10 0ng ml)与米诺地尔 (12 5 μg ml)对离体培养的人头皮毛囊的生长有促进作用 (P <0 0 5 ) ;而 17β E2 (0 5μg ml)则显示有抑制作用 (P <0 0 5 ) ;NGF与雌激素组毛囊的生长长度与阴性对照比则无明显变化(P >0 0 5 ) ;3H TdR掺入检测与长度检测结果完全一致。结论　NGF(10 0ng ml)与米诺地尔 (12 5 μg ml)对离体培养的人头皮毛囊的生长有促进作用 ,而 17β E2 (0 5ng ml)则显示有抑制作用 ,NGF(10 0μg ml)可干预 17β E2 (0 5 μg ml)抑制毛囊生长的影响。     

雷公藤服用者精液中乳酸脱氢酶及其同功酶C_4活性的研究

本文对19例中药雷公藤服用者精子、精浆中乳酸脱氢酶(LDH)及同功酶C_4(LDHC_4)的活性进行了测定分析。发现服药后以单位数量精子表示的LDHC_4活性在精子、精浆中均有明显升高(双侧非参数秩和检验,分别为P<0.01和P<0.02);通过酶活性与精子计数的相关分析,显示服药后精浆LDHC_4活性(mU/ml)与精子计数的正相关性(r=0.2279)较服药前(r=0.5505)弱,可能与精液中精子以外的其他LDHC_4来源贡献增大有关,诸如生精细胞的分解增多等。本文认为服药后精子LDHC_4活性上升,精液中非成熟精子比例增高是其原因之一。     

Evaluation of a commercially available kit for the colorimetric determination of zinc in human seminal plasma

A kit from Wako Pure Chemical Industries for colorimetric determination of zinc has been evaluated for its possible use in the determination of zinc in human seminal plasma. The within-assay variation for 15 replicates of each of two seminal plasma samples having zinc concentrations (mM) of 0.43 +/- 0.025 and 6.06 +/- 0.125 (mean +/- SD) was 5.7% and 2.1%, respectively. The between-assay variation after analysis of 15 replicates of a seminal plasma sample (zinc conc. 5.6 mM) on different days was 2.3%. No interference from other metal ions present in seminal plasma was observed. The average % recovery of zinc added to seminal plasma was 102.7 +/- 1.77 (mean +/- SD). A close correlation (r = 0.996, n = 105) was found between the levels of zinc determined by the colorimetric method and that determined by atomic absorption spectrophotometry as reference method. It is concluded that the present colorimetric method, which is fast, sensitive and linear over the entire concentration range of zinc present in human seminal plasma, can be recommended for use in semen analysis laboratories.     

Electron microscopic study of the secretion process in bovine reproductive organs

The origin and mechanism of the secretion of membrane-bound particles in bovine seminal plasma were studied with transmission (TEM) and scanning (SEM) electron microscopy of the epididymis, vas deferens, ampulla, and seminal vesicle of adult bulls. In the SEM study, all these organs were found to contain apical protrusions in the lining of the epithelial cells. Eventually the protrusions became detached and formed secretory bodies within the lumina of these organs. In the epididymis, the TEM study disclosed a granular and rather homogeneous content in the protrusions and bodies, whereas in the vas deferens they contained dilated cisternae of smooth endoplasmic reticulum. In the ampulla and seminal vesicle, the formation of the apical protrusions was associated with an accumulation of membrane-bound vesicles. These vesicles were found to be released from the storage bodies into the secretory fluid of the lumen. Both could be harvested from isolated seminal vesicle secretions by Percoll gradient centrifugation. It was concluded that various parts of the bovine reproductive organs discharge their secretory products at least partly by an apocrine mechanism. The membrane-bound particles in the seminal plasma, however, appear to be mainly derived from the ampulla and seminal vesicle.     

Free L-carnitine in human seminal plasma

It has often been suggested that determination of free L(—)-carnitine in seminal plasma may provide a good indication of epididymal function. However, there has been disagreement regarding the origin of L(—)-carnitine (epididymis and seminal vesicles) and its concentration in human seminal plasma. In this study, free L(—)-carnitine was determined after deproteinization with an enzymatic spectrophotometric method. In 29 semen samples from fathers and with normal spermiograms (semen volume between 2 and 6 ml, sperm count over 20.10⁶/ml, more than 50% motile spermatozoa), the total free L(—)-carnitine in the seminal plasma was 1010 nmoles (SD:±480), in 16 samples from vasectomized men it was 131 nmoles (SD:±77), and in 5 from men with agenesis of the vas deferens and seminal vesicles it was 21 nmoles (SD:±25). These results suggest that free L(—)-carnitine in the seminal fluid is predominantly of epididymal origin. The results of free L(—)-carnitine determinations in split ejaculates and the absence of a correlation between L(—)-carnitine and fructose concentrations in semen from normal subjects indicate that the seminal vesicles make only on minor contribution to L(—)-carnitine in the seminal plasma.     

全自动精浆γ-L-谷氨酰转肽酶检测方法的建立及评价

目的:建立精浆γ-L-谷氨酰转肽酶(GGT)全自动检测方法并对其准确性、重复性和线性范围等进行评价。方法:利用速率法检测精浆GGT活性,并在全自动生化分析仪上建立检测参数,并评估该方法的试剂空白吸光度、准确性、重复性和线性范围,同时与目前临床上常用的精浆GGT检测化学比色法试剂盒(南京欣迪)进行比较。结果:试剂空白吸光度平均为0.047 6,空白吸光度变化率(△A/min)平均为0.000 168。3份高、中、低GGT活性的精浆样本重复测定10次的变异系数值分别为0.26%、4.83%和1.60%。采用比对试验的方法来评价准确性,40份精浆标本每个浓度点的相对偏差范围为-13.38%¹1.05%,均低于15%的要求。精浆GGT活性在29911.05%,均低于15%的要求。精浆GGT活性在299¹ 833 U/L范围时,具有良好的线性关系(r>0.99)。以精浆GGT检测化学比色法试剂盒作为对照试剂,全自动检测法作为试验试剂,两者对115例精浆样本的检测结果呈显著正相关(r=0.981,P<0.01),Kappa值为0.776(P<0.05),符合率为90.43%。结论:本研究建立的精浆GGT全自动检测法有较低的试剂空白,重复性和准确性较好,且与目前临床上使用的化学比色法有很好的一致性。该法操作简单、快速,精浆样本无需稀释,适合临床上大批量样本的检测筛查,大大节省了人力和试剂成本。     

Bovine seminal plasma osteopontin: Structural modelling,recombinant expression and its relationship with semen quality

Osteopontin (OPN) is a multifunctional phosphoprotein that has been linked to fertility in bulls. However, the exact mechanism by which OPN contributes to fertilisation is yet unknown. The biotechnological use of OPN in bovine reproduction is promising but some gaps remain unfilled. The present work aimed: (a) to verify whether the seminal plasma OPN is associated with seminal traits and a standard breeding soundness exam; (b) to predict OPN interactions with integrins, CD44 and glycosaminoglycans through molecular docking; and (c) to develop a protocol for recombinant expression of OPN from vesicular gland cDNA. Ejaculates from top ranked bulls had higher amounts of seminal plasma OPN in comparison with bulls classified as questionable (p < .01). The structural modelling and molecular docking predictions indicated that bovine OPN binds to heparin disaccharide, hyaluronic acid and hyaluronan. In addition, docking studies described the binding complexes of OPN with CD44 and the integrin heterodimers α5β1 and αVβ3. Finally, expression of rOPN-6His was successfully obtained after 3 hr of induction with 0.5 mM IPTG at 37°C and a denaturing purification protocol resulted in efficiently purified recombinant OPN. The present results contribute to the development of biotechnological uses of OPN as a biomarker in bovine reproduction.     

Tissue inhibitors of metalloproteinases 1 and 2 in human seminal plasma and their association with spermatozoa

A 24-kDa heparin binding protein recently identified as tissue inhibitor of matrix metalloproteinases 2 (TIMP-2) in bovine seminal fluid was suggested to play an important role in bull fertility. As no data are present for men, the concentrations of tissue inhibitors of metalloproteinases 1 (TIMP-1) and TIMP-2 were quantified in human seminal plasma of normozoospermic and azoospermic men using enzyme-linked immunosorbent assay methods. TIMP-1 and 2 were not significantly different in both groups and there were no relationships between the concentrations of both TIMPs and other sperm characteristics. It is assumed that TIMPs are released from accessory sex glands.     

Inhibition of human and bovine sperm acrosin by divalent metal ions. Possible role of zinc as a regulator of acrosin activity

Human and bovine spermatozoa have been collected and washed repeatedly with isotonic saline to remove seminal plasma inhibitors and activate the acrosin. Then the acrosin activity of the cells was assayed with α-N-Benzoyl-DL-Arg-β-naphthylamide (BANA). It was found that the surface-bound enzyme was not inhibited by high molecular weight inhibitors of trypsin but was markedly inhibited by low molecular weight trypsin inhibitors. Divalent metals (Zn⁺⁺, Cu⁺⁺, Hg⁺⁺, Co⁺⁺, Cd⁺⁺) were all efficient inhibitors of acrosin on the washed cells. It was shown that the removal of zinc or copper from acrosin completely restored activity. It is proposed that the different levels of zinc in the male and female genital tract regulate acrosin activity. Aged cells released a soluble acrosin which was inhibited by serum and seminal plasma inhibitors of trypsin-like enzymes as well as by zinc ions in an identical manner to the surface-bound enzyme.     

Dipeptidyl peptidases in bovine reproductive organs and secretions

Dipeptidyl peptidases (DPP) I-IV were analysed in homogenates of bovine reproductive organs as well as in seminal vesicle secretions and seminal plasma. The presence of various molecular forms of these enzymes was studied by fractionation using gel filtration, anion exchange chromatography and chromatofocusing. The eluting enzymes were pooled, and their biochemical properties were briefly characterized. The histochemical localization of DPP II and IV was carried out with the most active tissues. DPP I and III were absent from seminal plasma, but their highest activity was found in the epididymis and increased during sexual maturation. DPP II was found mainly in a single molecular form and displayed a wide distribution in the reproductive organs. Its activity in seminal plasma may be derived from various organs, although the major sources are probably the apical activity in the epididymis, ampulla and seminal vesicle. DPP IV activity was high in the cauda epididymis, and ampulla, and in the seminal vesicles and their secretions. The high activity of DPP IV in seminal plasma appeared to derive from these organs, which showed a strong apical reaction of the epithelial lining. In seminal vesicles the enzyme was mainly secreted attached to membrane particles called vesiculosomes.     

精浆抗精子抗体阳性不育患者顶体酶、一氧化氮合酶及超氧化物歧化酶活力变化的研究

目的:研究精浆抗精子抗体(AsAb)阳性对精子顶体酶、精浆一氧化氮合酶(NOS)及超氧化物歧化酶(SOD)活力的影响。方法:精浆AsAb阳性不育者40例,对照组为40例正常生育男性。通过吸光度变化分别计算顶体酶活力(BAEE/ADH联合法)、NOS活力(氧化还原反应)、SOD活力(黄嘌呤氧化酶法)。结果:精浆As-Ab阳性组与正常生育组比较,精子顶体酶活力明显降低(P<0.01),NOS活力明显升高(P<0.01),精浆中SOD活力明显降低(P<0.01)。结论:精浆AsAb阳性引起不育可能与精子顶体酶、精浆中SOD及NOS活力改变有关。