急性重复低氧预适应小鼠海马组织蛋白磷酸酶1γ表达的变化

目的:检测急性重复低氧预适应小鼠海马脑组织中蛋白磷酸酶(protein phosphatase,pp)1γ的变化,探讨pp1γ的变化在低氧预适应脑保护中的作用。方法:成年昆明小鼠随机分成低氧暴露1次组(H1)或低氧暴露4次(H4)组和常氧组(H0)。低氧结束后取海马组织,应用Real-time PCR技术和蛋白印记技术检测pp1γ表达的变化;应用磷酸酶活性实验对pp1活性的变化进行检测;应用甲基化测序实验对pp1γ启动子区(95~321 bp)甲基化变化进行检测。结果:pp1γ的mRNA在H4组表达较H0组降低,差异有统计学意义(P0.01);pp1γ的蛋白质变化与mRNA变化一致,在H4组的表达较H0组降低,差异有统计学意义(P0.05);pp1γ水解磷酸的活性在H4组和H1组均低于H0组(P0.05);pp1γ启动子区(95~321 bp)DNA甲基化情况在三组间未见差异。结论:pp1γ变化可能参与了低氧预适应小鼠获得的脑保护,但该变化可能与其启动子区(95 bp~321 bp)的甲基化无关。     

低氧预适应过程中小鼠脑内CGRP和AngⅡ含量的变化

近年来有研究表明 ,降钙素基因相关肽 (ｃａｌｃｉｔｏｎｉｎｇｅｎｇ ｒｅｌａｔｅｄｐｅｐｔｉｄｅ,ＣＧＲＰ)在实验性缺血预适应中具有明显的心肌保护作用[1] ,而血管紧张素Ⅱ (ａｎｇｉｏｔｅｎｓｉｎⅡ ,ＡｎｇⅡ )生成的抑制剂则能有效地保护缺血及缺血再灌注引起的脑损伤[2 ] 。本研究观察了反复低氧对小鼠低氧耐受性的影响并监测了小鼠脑内ＣＧＲＰ和ＡｎｇⅡ含量随反复低氧次数增加而发生的动态改变。1　材料与方法1 1　标准耐受时间的测定 :雄性昆明小白鼠体重 18 0～ 2 2 0ｇ(首都医科大学动物部提供 )随机分为对照组、低氧 1、…     

化学预处理对低氧预适应模型小鼠的影响

背景：低氧诱导因子1α调节一些增加抗低氧耐受的基因的表达。氯化钴是一种能够稳定低氧诱导因子1α的化学试剂。 目的：检测氯化钴预处理对急性重复低氧小鼠的影响。 方法：Balb/C小鼠随机分为预处理组和对照组,实验前3 h,两组分别注射氯化钴和生理盐水后, 分别进行0次(正常对照组),1次,4次缺氧暴露,随即分别取海马组织进行指标检测。 结果与结论：预处理组缺氧暴露1次对低氧耐受时间显著高于对照组缺氧暴露1次,预处理组缺氧暴露1次,4次低氧耐受时间差异无显著性意义。对照组中缺氧暴露4次组低氧诱导因子1的DNA结合活力显著高于缺氧暴露1次组和正常对照组,组间比较差异有显著性意义。预处理组中缺氧暴露1次组低氧诱导因子1的DNA结合活力显著高于正常对照组(P < .01),缺氧暴露4次组低氧诱导因子1的DNA结合活力急剧下降,显著低于缺氧暴露1次组和正常对照组。对照组促红细胞生成素和血管内皮生长因子mRNA 在缺氧暴露1次组和缺氧暴露4次组升高,各组间比较差异有显著性意义(P < 0.05)。预处理组促红细胞生成素和血管内皮生长因子mRNA表达组间比较差异无显著性意义。提示氯化钴预处理能够提高低氧耐受时间,降低低氧预适应诱导的耐受时间及低氧诱导因子1 DNA结合能力。     

急性重复低氧预适应小鼠海马组织DNA甲基转移酶表达的变化

目的:检测小鼠脑海马组织中三种DNA甲基转移酶在急性重复缺氧条件下的变化,探讨缺氧预适应过程中DNA甲基转移酶的变化在低氧神经保护中的作用。方法:昆明小鼠随机分成正常对照组(常氧组,H0)、低氧1次组(H1)或低氧4次(H4)组,实验后取海马组织,应用Real-time PCR技术、Western bolt技术和DNA甲基转移酶活性实验对三种DNA甲基转移酶的变化进行研究。结果:DNA甲基转移酶1的mRNA和蛋白质水平在三组之间无明显变化;DNA甲基转移酶3A和3B mRNA和蛋白质水平在三组之间有变化,DNA甲基转移酶3A和3B的mRNA在H4组中表达较H0组降低,差异有统计学意义(P<0.01),DNA甲基转移酶3A和3B的蛋白质变化与mRNA变化相似,H4组与H0组相比有所降低(P<0.05);DNA甲基转移酶活性在H4组较H0组降低。结论:DNA甲基转移酶的变化可能参与缺氧预适应小鼠获得脑保护。     

低氧预适应增加小鼠脑组织蛋白激酶C膜转位

初步探讨蛋白激酶C(PKC)在脑低氧预适应发生机制中的作用，以我室建立的小鼠低氧预适应模型，结合SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白印迹(Western bolt)等生化技术，观察了低氧预适应对小鼠大脑皮层及海马组织内PKC膜转位的影响。结果表明，在小鼠大脑皮层和海马组织内的PKC膜转位均随着低氧次数(小鼠低氧耐受时间)的增加而增高。特别是低氧3次的海马组织和低氧4次的大脑皮层和海马组织内，PKC膜转位的增加有统计学显著意义。结果提示，PKC的激活可能在脑低氧预适应发生机制中具有重要的作用。     

脑低氧／缺血性预适应的机制

氧是维持生命活动必不可少的物质。由于各种原因导致机体氧供不足及／或机体氧利用障碍，皆可导致缺氧或低氧性病理过程，缺氧或低氧性心脑肺疾患尤为常见。研究和克服缺氧既是临床医学、也是航空航天医学、高原医学以及运动医学等面临的重大课题。克服与战胜缺氧的一个主要策略是对之进行服习或适应。人或动物通过间断或连续地处于高原或低氧环境，可以产生或形成对缺氧的适应。事先短暂的缺血或轻度低氧的重复预处理或预适应（preconditioning）亦可触发或动员机体内在的防护能力，从而对随后的严重缺氧或缺血损伤产生强大的防御和保护作…     

高原习服与低氧预适应

高原低氧易导致急性高原反应,高原习服是机体为适应高原低氧环境发生的一系列代偿适应性变化。寻找加快高原习服的有效措施,提高机体高原习服能力,是预防和减轻急性高原反应的最好策略。低氧预适应是指机体经一次或多次短暂、非致死性低氧刺激后,机体获得的对后续更长时间或更严重低氧性损伤的耐受性。低氧预适应作为一种通过内源性保护机制来提高机体缺氧耐力的有效方法,是促进高原习服的重要措施。     

低氧预适应提高脑缺氧耐受性的研究进展

低氧预适应可提高脑耐缺氧能力,产生低氧预适应的保护性因素包括能量代谢降低;腺苷水平升高;氨基酸类递质含量变化;ATP敏感性钾通道开放;超氧化物歧化物活性增强;一氧化氮的双重调节作用;Bcl-2高表达等诸多因素.     

The adaptive effects of hypoxic preconditioning of brain neurons

A preventive short-term hypoxia (preconditioning) increases neuronal resistance against subsequent strong hypoxic effects. Literature review and authors' own data on molecular-cellular mechanisms of the hypoxic preconditioning, are presented. Participation of intracellular signal transduction, genome, stress-proteins, and neuromodulating peptides in this process, is discussed. The role of glutamatergic as well as calcium and phosphoinositide regulatory systems and neuromodulating factors as the components of a volume signal transmission are analyzed in hypoxic precondition-associated induction of functional tolerance mechanisms against acute harmful effects in neurones of olfactory slices.     

Missense mutations to the TSC1 gene cause tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34 or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, interact to form a protein complex that inhibits signal transduction to the downstream effectors of the mammalian target of rapamycin (mTOR). Here we investigate the effects of putative TSC1 missense mutations identified in individuals with signs and/or symptoms of TSC on TSC1-TSC2 complex formation and mTOR signalling. We show that specific amino-acid substitutions close to the N-terminal of TSC1 reduce steady-state levels of TSC1, resulting in the activation of mTOR signalling and leading to the symptoms of TSC.     

Functional assessment of variants in the TSC1 and TSC2 genes identified in individuals with Tuberous Sclerosis Complex

The effects of missense changes and small in-frame deletions and insertions on protein function are not easy to predict, and the identification of such variants in individuals at risk of a genetic disease can complicate genetic counselling. One option is to perform functional tests to assess whether the variants affect protein function. We have used this strategy to characterize variants identified in the TSC1 and TSC2 genes in individuals with, or suspected of having, Tuberous Sclerosis Complex (TSC). Here we present an overview of our functional studies on 45 TSC1 and 107 TSC2 variants. Using a standardized protocol we classified 16 TSC1 variants and 70 TSC2 variants as pathogenic. In addition we identified eight putative splice site mutations (five TSC1 and three TSC2). The remaining 24 TSC1 and 34 TSC2 variants were classified as probably neutral.     

Thioredoxin-1 expression levels in rat hippocampal neurons in moderate hypobaric hypoxia

Previous studies have demonstrated that preconditioning (PC) with three sessions of moderate hypoxia significantly increases the expression of the antioxidant protein thioredoxin-1 (Trx-1) in the rat hippocampus by 3 h after subsequent acute severe hypoxia as compared with non-preconditioned animals. However, it remained unclear whether this increase in Trx-1 accumulation during PC is induced before severe hypoxia or is a modification of the response to severe hypoxia. This question was addressed in the present investigation using experiments on 12 adult male Wistar rats with studies of Trx-1 expression after PC without subsequent severe hypoxia. Immunocytochemical studies were performed 3 and 24 h after three episodes of moderate hypobaric hypoxia (three sessions of 2 h at 360 mmHg with 24-h intervals). Immunoreactivity to Trx-1 24 h after the last session was significantly decreased in neurons in all the areas of the hippocampus studied (CA1, CA2, CA3, and the dentate gyrus). Immunoreactivity in CA3 was also decreased 3 h after hypoxia. These results provide evidence that moderate preconditioning hypoxia itself not only does not increase, but even significantly decreases Trx-1 expression. Thus, increases in Trx-1 contents in the hippocampus of preconditioned animals after severe hypoxia are not associated with the accumulation of this protein during PC, but with a PC-induced modification of the reaction to severe hypoxia. Translated from Morfologiya, Vol. 133, No. 1, pp. 20–24, January–February, 2008.     

The possible use of hypoxic preconditioning for the prophylaxis of post-stress depressive episodes

The protective effects of hypoxic preconditioning on the development of depressive states in rat models were studied. Three episodes of intermittent preconditioning using hypobaric hypoxia (360 mmHg, 2 h) prevented the onset of depressive behavioral reactions, hyperfunction of the hypophyseal-adrenal system, and impairments in its suppression in the dexamethasone test in rats following unavoidable aversive stress in a model of endogenous depression. The anxiolytic and antidepressant actions of hypoxic preconditioning in experiments on rats were no less marked than those of the tetracyclic antidepressant ludiomil. The results obtained here provide evidence that preconditioning with intermittent hypobaric hypoxia increases resistance to psychoemotional stresses, has marked anxiolytic and antidepressant effects, and can be used for the prophylaxis of depressive episodes.     

Functional assessment of TSC1 missense variants identified in individuals with tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 genes. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a complex that inhibits the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Previously, we demonstrated that pathogenic amino acid substitutions in the N-terminal domain of TSC1 (amino acids 50-224) are destabilizing. Here we investigate an additional 21 unclassified TSC1 variants. Our functional assessment identified four substitutions (p.L61R, p.G132D, p.F158S, and p.R204P) between amino acids 50 and 224 that reduced TSC1 stability and prevented the TSC1-TSC2-dependent inhibition of TORC1. In four cases (20%), our functional assessment did not agree with the predictions of the SIFT amino acid substitution analysis software. Our new data confirm our previous finding that the N-terminal region of TSC1 is essential for TSC1 function.     

低氧预适应对HT22细胞自噬的影响

目的:研究低氧预适应(HPC)对小鼠海马HT22细胞自噬的影响。方法:将HT22细胞并分为对照组(control)、低氧组(hypoxia)、低氧预适应组(HPC)和低氧预适应与3-甲基腺嘌呤联合处理组(HPC+3-MA)。MTS法检测HT22细胞活性,采用real time RT-PCR检测HT22细胞中自噬及微管相关蛋白1轻链3(LC3)mRNA表达,利用Western Blot检测LC3蛋白的表达及LC3Ⅱ/LC3Ⅰ的比值变化,转染GFP-LC3进一步检测LC3Ⅱ表达。结果:HPC可增加低氧状态下HT22细胞的活性,与HPC组相比,HPC+3-MA组HT22细胞活性降低;HPC可以诱导低氧状态下HT22细胞中LC3的mRNA的表达,并增加LC3Ⅱ/LC3Ⅰ的比值。结论:HPC通过促进低氧状态下HT22细胞自噬提高细胞活性。     

Mutational spectrum of the TSC1 gene in a cohort of 225 tuberous sclerosis complex patients: no evidence for genotype-phenotype correlation

Tuberous sclerosis complex is an inherited tumour suppressor syndrome, caused by a mutation in either the TSC1 or TSC2 gene. The disease is characterised by a broad phenotypic spectrum that can include seizures, mental retardation, renal dysfunction, and dermatological abnormalities. The TSC1 gene was recently identified and has 23 exons, spanning 45 kb of genomic DNA, and encoding an 8.6 kb mRNA. After screening all 21 coding exons in our collection of 225 unrelated patients, only 29 small mutations were detected, suggesting that TSC1 mutations are under-represented among TSC patients. Almost all TSC1 mutations were small changes leading to a truncated protein, except for a splice site mutation and two in frame deletions in exon 7 and exon 15. No clear difference was observed in the clinical phenotype of patients with an in frame deletion or a frameshift or nonsense mutation. We found the disease causing mutation in 13% of our unrelated set of TSC patients, with more than half of the mutations clustered in exons 15 and 17, and no obvious under-representation of mutations among sporadic cases. In conclusion, we find no support for a genotype-phenotype correlation for the group of TSC1 patients compared to the overall population of TSC patients.     

低氧预适应上调大鼠海马神经元和星形胶质细胞在急性缺氧时的葡萄糖转运蛋白的活性和基因表达

目的 研究低氧预适应对海马神经元和星形胶质细胞在急性缺氧暴露时的葡萄糖转运蛋白的活性和基因表达的影响。方法 培养大鼠海马神经元和星形胶质细胞,每天间歇暴露于低氧混合气体（1% O2、10% CO2、89% N2）20min,连续6d。最后1次低氧暴露24h后,将细胞暴露于无氧混合气体（10% CO2、90% N2）6h,然后立即检测[3H]-2-脱氧葡萄糖（2-DG）的摄取率、GLUT1和GLUT3的mRNA水平及神经元的存活率。结果 低氧预适应上调急性缺氧时神经元和星形胶质细胞的2-DG吸收率、星形胶质细胞GLUT1 mRNA的表达及神经元GLUT1和GLUT3 mRNA的表达,提高神经元存活率,这一作用可被细胞松弛素B消除。结论 低氧预适应上调海马神经元和星形胶质细胞在急性缺氧时的葡萄糖摄取率和葡萄糖转运蛋白的基因表达。