脑红蛋白在成年男性生殖系统的表达及意义

目的:探讨脑红蛋白(neuroglobin,Ngb)在成年男性生殖系统的表达及其意义。方法:用免疫组织化学SP法观测Ngb在成人男性生殖系统的表达、分布与定位。结果:Ngb在生殖系统内神经节细胞、睾丸间质细胞有较强表达,在睾丸支持细胞、生精细胞、附睾管腔上皮、前列腺腺上皮及精囊黏膜上皮呈弱阳性表达。结论:Ngb在男性生殖系统中分布广泛,其功能值得研究。     

脑红蛋白在绵羊视网膜中的分布

目的:探讨脑红蛋白在绵羊视网膜的分布特征。方法:利用免疫组织化学显色SP法,观察脑红蛋白在健康成年绵羊视网膜中的分布情况。结果:脑红蛋白在绵羊视网膜的视神经纤维层、内网状层、外网状层和光感受器内节段中有强阳性表达,在视网膜的内核层和节细胞层有弱阳性表达,在视网膜外核层、光感受器外节段和色素上皮层中未见有阳性表达,内界膜、外界膜和视神经中亦有脑红蛋白阳性表达。绵羊视网膜脑红蛋白阳性表达的细胞类型主要有节细胞、双极细胞和光感受器细胞,其中节细胞的阳性表达定位于细胞质,胞核中未见表达。结论;除外核层、光感受器外节段和色素上皮层外,脑红蛋白在绵羊视网膜其他各层中均有表达,提示脑红蛋白在维持视网膜中氧平衡状态时发挥重要作用。     

米诺环素对脊髓损伤大鼠凋亡机制探索和神经红蛋白表达的影响

目的 探讨米诺环素对脊髓横断损伤大鼠凋亡蛋白 Bcl-2、Bax和 Caspase-3的表达及神经红蛋白 Ngb表 达的影响。方法 36只健康成年雄性 Wistar大鼠随机分为假手术组、脊髓损伤组、米诺环素干预组,每组 12只。假 手术组只暴露脊髓,脊髓损伤组和米诺环素干预组制备胸3~4脊髓节段全横断损伤模型。米诺环素干预组于术前腹 腔注射米诺环素（90 mg/kg）,1次/d,连续5 d,脊髓损伤组和假手术组腹腔注射等体积生理盐水。术后22 d处死所有 大鼠,采用免疫组化染色和 Western blot法检测脊髓组织 Bcl-2、Bax和 Caspase-3蛋白的表达;Hoechst 染色检测凋亡 细胞;免疫荧光染色检测神经红蛋白（Ngb）的表达。结果 与假手术组相比,脊髓损伤组脊髓组织中 Bcl-2表达增 多,Bax和 Caspase-3蛋白的表达增多,凋亡细胞数明显增多,Ngb荧光强度明显增强（均P＜0.05）。与脊髓损伤组相 比,米诺环素干预组Bcl-2表达进一步增高（P＜0.05）,Bax和Caspase-3表达显著降低（P＜0.05）,而凋亡细胞数有所 减少,Ngb的平均荧光强度较其明显增强。结论 米诺环素干预可通过降低 Bax和 Caspase-3的表达,升高 Bcl-2的 表达来抑制细胞凋亡,提高神经红蛋白表达。     

When the brain goes diving: glial oxidative metabolism may confer hypoxia tolerance to the seal brain

Deep diving mammals have developed strategies to cope with limited oxygen availability when submerged. These adaptations are associated with an increased neuronal hypoxia tolerance. Brain neurons of the hooded seal Cystophora cristata remain much longer active in hypoxic conditions than those of mice. To understand the cellular basis of neuronal hypoxia tolerance, we studied neuroglobin and cytochrome c in C. cristata brain. Neuroglobin, a respiratory protein typically found in vertebrate neurons, displays three unique amino acid substitutions in hooded seal. However, these substitutions unlikely contribute to a modulation of O₂ affinity. Moreover, there is no significant difference in total neuroglobin protein levels in mouse, rat and seal brains. However, in terrestrial mammals neuroglobin resided exclusively in neurons, whereas in seals neuroglobin is mainly located in astrocytes. This unusual localization of neuroglobin is accompanied by a shift in the distribution of cytochrome c. In seals, this marker for oxidative metabolism is mainly localized in astrocytes, whereas in terrestrial mammals it is essentially found in neurons. Our results indicate that in seals aerobic ATP production depends significantly on astrocytes, while neurons rely less on aerobic energy metabolism. This adaptation may imbue seal neurons with an increased tolerance to hypoxia and potentially also to reactive oxygen species, and may explain in part the ability of deep diving mammals to sustain neuronal activity during prolonged dives.     

大鼠脑组织缺血后Ngb mRNA表达与缺血时间推断的研究

目的 探讨大鼠脑组织缺血后不同时间脑红蛋白（Ngb）mRNA表达及法医学意义。方法 建立Wistar大鼠脑组织缺血模型，应用一步法逆转录-聚合酶链式反应（one step RT-PCR）技术与计算机图像分析技术检测脑组织缺血后不同时间Ngb mRNA表达的差别。结果 脑缺血后Ngb mRNA表达迅速升高，缺血1min达高峰，缺血5min表达迅速降低，但仍高于假手术组水平，10min后随缺血时间延长表达再度逐渐增强。结论大鼠脑组织缺血后Ngb mRNA的表达呈现时相性的变化，可在法医实践中用于早期脑缺血时间的推断。     

氯化血红素对大鼠局灶性脑缺血组织的作用探讨

目的探讨氯化血红素(Hemin)在局灶性脑缺血脑组织中作用及其诱导脑红蛋白(Ngb)表达的可能机制.方法将SD大鼠随机分为3组(n=20),分别为假手术对照组、手术组、Hemin处理组,3组大鼠于术后24 h检测脑含水量、脑梗死体积、观察组织病理学改变,免疫组织化学技术了解脑红蛋白表达情况.结果 Hemin处理组脑含水量、脑梗死体积较手术组有显著性差异(P<.05).病理切片观察,Hemin处理组水肿程度较手术组减轻,变性神经元数量较手术组减少.免疫组化分析,Hemin处理组脑红蛋白阳性神经元数量较手术组增多.结论 Hemin能减轻局灶性脑缺血时脑组织的损伤,其可能通过诱导脑红蛋白表达达到脑保护作用.     

大鼠神经球蛋白的原核表达及多克隆抗体制备

目的 克隆Wistar大鼠神经球蛋白(NGB)基因,通过原核表达制备其多克隆抗体,为进一步研究其功能奠定基础.方法 从雄性Wistar大鼠脑组织中提取总RNA,经逆转录PCR得到cDNA,测序证明该基因序列正确后,亚克隆人pET-28a( )载体中,通过双酶切和测序确定序列和阅读框正确.转化大肠杆菌BL21菌株,获得可溶性表达产物,经鉴定、纯化后免疫新西兰兔,分离血清,ELISA法测定抗体效价.结果 神经球蛋白多克隆抗体效价达1:100 000,此多抗可以与293细胞内表达的重组蛋白发生很好的抗原抗体反应.结论 成功制备了兔抗NGB的多抗,为后续研究提供了重要的实验材料.     

阻塞性睡眠呼吸暂停低通气综合征患者血清脑红蛋白水平的变化与早期脑损伤的相关性

目的探讨阻塞性睡眠呼吸暂停低通气综合征（OSAHS）患者血清中脑红蛋白水平的变化与早期脑损伤的相关性。方法采用酶联免疫吸附法检测经多导睡眠监测仪监测确诊的40例OSAHS患者和16例健康体检者血清脑红蛋白水平。结果轻、中、重度OSAHS组脑红蛋白水平较对照组明显增高（P〈0.05或P〈0.01）,OSAHS患者3组间脑红蛋白水平比较,有显著性差异（P〈0.01）,与呼吸暂停低通气指数呈正相关（r=0.948,P〈0.01）,与最低血氧饱和度呈负相关（r=-0.800,P〈0.01）,与血氧饱和度低于90%的时间占总睡眠时间的百分比无明显相关性（r=-0.005,P〉0.05）。结论 OSAHS患者的血清脑红蛋白水平升高,可能与早期脑损伤有一定的相关性。     

Disulfide bond influence on protein structural dynamics probed with 2D-IR vibrational echo spectroscopy

Intramolecular disulfide bonds are understood to play a role in regulating protein stability and activity. Because disulfide bonds covalently link different components of a protein, they influence protein structure. However, the effects of disulfide bonds on fast (subpicosecond to approximately 100 ps) protein equilibrium structural fluctuations have not been characterized experimentally. Here, ultrafast 2D-IR vibrational echo spectroscopy is used to examine the constraints an intramolecular disulfide bond places on the structural fluctuations of the protein neuroglobin (Ngb). Ngb is a globin family protein found in vertebrate brains that binds oxygen reversibly. Like myoglobin (Mb), Ngb has the classical globin fold and key residues around the heme are conserved. Furthermore, the heme-ligated CO vibrational spectra of Mb (Mb-CO) and Ngb (Ngb-CO) are virtually identical. However, in contrast to Mb, human Ngb has an intramolecular disulfide bond that affects its oxygen affinity and protein stability. By using 2D-IR vibrational echo spectroscopy, we investigated the equilibrium protein dynamics of Ngb-CO by observing the CO spectral diffusion (time dependence of the 2D-IR line shapes) with and without the disulfide bond. Despite the similarity of the linear FTIR spectra of Ngb-CO with and without the disulfide bond, 2D-IR measurements reveal that the equilibrium sampling of different protein configurations is accelerated by disruption of the disulfide bond. The observations indicate that the intramolecular disulfide bond in Ngb acts as an inhibitor of fast protein dynamics even though eliminating it does not produce significant conformational change in the protein's structure.     

阿司匹林对化学性缺氧状态下新生大鼠脑神经细胞[Ca~(2+)]i和NGB的影响

目的 探讨阿司匹林(ASA)对化学性缺氧大鼠脑神经细胞的保护作用及细胞外钙离子对ASA神经保护作用的影响。方法 分别在含钙和去钙培养液中加入连二亚硫酸钠（Na2S2O4）诱导化学性缺氧,用ASA预处理体外培养的大鼠脑细胞,激光共聚焦显微镜观察[Ca2+]i和脑红蛋白（NGB）的变化。结果 化学性缺氧使大鼠脑细胞[Ca2+]i和NGB表达增高（P＜0.05, n=5）。ASA预处理可明显缓解缺氧组和无钙缺氧组大鼠脑细胞[Ca2+]i和NGB的增高（P＜0.05, n=5）。结论 ASA可能通过减少大鼠脑细胞的钙超载,抑制缺氧诱导的NGB表达增高,发挥神经细胞保护作用。