小鼠缺血性脑损伤模型急性期海马SIRT3与自噬相关蛋白表达水平的相关性分析

摘要 目的：观察小鼠缺血性脑损伤模型急性期海马区域沉默信息因子3(SIRT3)和自噬相关蛋白的表达,并探讨两者的相关性。方法：选择C57BL/6J小鼠,采用大脑中动脉阻塞(MCAO)法建立缺血性脑损伤模型,并将小鼠随机分为假手术组(sham)和模型组(MCAO)。缺血性脑损伤急性期(6h),采用间接免疫荧光法观察小鼠海马CA1、CA3和DG区域SIRT3的表达,应用蛋白印迹法检测SIRT3、自噬相关蛋白LC3 I/II和Beclin-1的表达,而后用Spearman相关性分析明确SIRT3和LC3-II、Beclin-1表达的相关性。结果：海马各区域SIRT3阳性细胞数量在损伤后明显增多(P<0.05),且SIRT3蛋白表达也相对上调(P<0.05);损伤后自噬相关蛋白LC3-II和Beclin-1表达亦增高(P<0.05);Spearman相关性分析发现SIRT3与自噬相关蛋白LC3-II、Beclin-1表达均呈显著正相关(P<0.05)。结论：小鼠缺血性脑损伤模型急性期海马区域SIRT3和自噬相关蛋白的表达具有显著相关性,SIRT3对海马区域自噬的调节可能有重要作用。     

Neurochemistry,Neuropathology, and Heredity in SAMP8: A Mouse Model of Senescence

The SAMP8 strain spontaneously develops learning and memory deficits with characteristics of aging, and is a good model for studying the mechanism of cognitive dysfunction with age. Oxidative stress occurs systemically in SAMP8 from early on in life and increases with aging. Neuropathological changes such as the deposition of Aβ, hyperphosphorylation of tau, impaired development of dendritic spines, and sponge formation, and neurochemical changes were found in the SAMP8 brain. These changes may be partially mediated by oxidative stress. Oxidative damage is a major factor in neurodegenerative disorders and aging. A decline in the respiratory control ratio suggesting mitochondrial dysfunction was found in the brain of SAMP8. The rise in oxidative stress following mitochondrial dysfunction may trigger neuropathological and neurochemical changes, disrupting the development of neural networks in the brain in SAMP8. Special issue article in Honour of Dr. Akitane Mori.     

虎杖苷对老年小鼠术后认知功能障碍及海马氧化应激和神经炎症的影响

摘要 目的：探讨虎杖苷对老年小鼠术后认知功能障碍及海马氧化应激和神经炎症的影响。方法：C57BL/6J雄性老年小鼠,18月龄,体重24~28 g,随机分为4组：假手术组（Sham组）、术后认知功能障碍组（POCD组）、低剂量虎杖苷组（PD1组）、高剂量虎杖苷组（PD2组）。Sham组老年小鼠仅接受水合氯醛麻醉,不行外科手术;POCD组老年小鼠接受腹部手术以制备POCD老年动物模型;PD1组小鼠制备POCD模型,并于术后即刻、24 h和48 h分别腹腔注射低剂量虎杖苷25 mg/kg;PD2组小鼠制备POCD模型,并于术后即刻、24 h和48 h分别腹腔注射高剂量虎杖苷50 mg/kg。使用Morris水迷宫行为学实验评估老年小鼠术后认知功能,使用Western blot法检测术后小鼠海马Nrf2、HO-1、HMGB1、Iba-1蛋白水平,检测海马活性氧（ROS）、丙二醛（MDA）、超氧化物歧化酶（SOD）含量以反映术后海马氧化应激水平。结果：（1）与Sham组比较,POCD组老年小鼠术后逃离潜伏期显著增加（P<0.05）,穿越平台次数和目标象限停留时间下降（P<0.05）,海马Nrf2和HO-1蛋白表达水平明显降低（P<0.05）,氧化应激产物ROS和MDA含量增加（P<0.05）,抗氧化酶SOD活性降低（P<0.05）,Iba-1和HMGB1蛋白表达水平增加（P<0.05）;（2）与POCD组比较,PD1组和PD2组小鼠术后逃离潜伏期缩短（P<0.05）,穿越平台次数和目标象限停留时间增多（P<0.05）,海马Nrf2和HO-1蛋白表达水平升高（P<0.05）,ROS和MDA含量减少（P<0.05）,SOD活性增加（P<0.05）,Iba-1和HMGB1蛋白表达减少（P<0.05）。结论：虎杖苷可减轻老年小鼠术后认知功能损伤,缓解术后海马氧化应激和神经炎症,其机制可能与促进Nrf2/HO-1信号通路有关。     

Coxsackievirus B3-Induced Chronic Myocarditis in Mouse: Use of Whole Blood Culture to Study the Activation of TNFα-Producing Cells

The pathogenesis of CVB3-induced chronic myocarditis remains unknown. Activated monocytes and macrophages may maintain ongoing inflammation during a persistent CVB3 infection and possibly represent the major mechanism leading to chronic myocarditis. We decided to study the activation status of cells by studying TNFα secretion in vitro using whole blood culture in CVB3-induced murine chronic myocarditis. Seven DBA/2 +/+ mice and 18 NMRI nu/nu mice were inoculated intraperitoneally with 5 × 10⁵ pfu of CVB3, and mice were mock-infected. Thirty-one days post-infection, all mice were sacrificed, blood samples were obtained from the heart, and the heart was removed. Enteroviral genomic detection by RT-PCR, virus isolation and histological analysis of heart samples were performed. Heparinized whole blood (25 μl) was cultured for 4 hr and 24 hr in sterile 96 well-plate containing 225 μl RPMI in the presence or the absence of activators (LPS + PHA). The TNFα levels in the whole blood from mock-infected DBA/2 (n = 4) and NMRI nu/nu mice (n = 5) were not different. A moderate increase of TNFα was observed in three out of five DBA/2 mice with negative CVB3 that had no histological abnormalities in myocardium. An increased level of TNFα was found in the sole DBA/2 mouse with positive CVB3 detection and chronic myocarditis. An increased level of TNFα was found in one out of nine NMRI nu/nu mice with positive CVB3 detection and chronic myocarditis and in one out of seven mice with positive CVB3 detection exempt of lesions in myocardium. In other infected mice, the level of TNFα was normal. Enteroviral genome was not detected in the blood from infected mice at 31 days post-infection. The increased TNFα level in some mice may be designed for a beneficial inflammatory and immune response, however, an exaggerated release may be associated with an adverse effect. The normal TNFα level in whole blood cultures from mice with chronic myocarditis does not exclude enhanced cytokine production at infected loci such as myocardial tissue. This is the first report to use whole blood cultures to study the production of cytokines in virus-induced disease in a small animal model.     

小鼠Smad3基因的克隆及其在小鼠组织中的表达

采用PCR获得的Smad3cDNA片段作为探针筛选小鼠脑cDNA文库 .克隆了小鼠全长的Smad3基因 .对小鼠Smad3基因的全编码区进行了序列测定 .结果表明 ,小鼠SMAD3与人SMAD3氨基酸同源性高达 99% .与小鼠Smad2基因相比 ,碱基同源性高达 91 8% .Northern杂交显示 ,Smad3基因在小鼠胚胎发育和各成体器官中普遍表达 .原位杂交显示 ,Smad3基因表达在小鼠胚胎期E16 5d的软骨、骨髓和皮肤角质细胞中     

PI3K/AKT/FOXO信号通路介导的自噬在糖尿病认知功能障碍中的作用

糖尿病作为一种高血糖为主要特征的代谢性疾病,会引起中枢神经系统损伤,造成脑组织结构和功能改变,进而导致认知功能障碍.目前,糖尿病对认知功能障碍的影响及相关调控机制已成为国内外研究的热点和难点.磷酸肌醇3激酶/蛋白激酶B/叉头样转录因子(PI3 K/AKT/FOXO)通路是自噬的重要上游调控机制.本文概述了PI3 K/A...     

稳定表达小鼠CD40L的NIH3T3细胞系的建立及其在B细胞培养和激活中的应用

该研究构建小鼠CD40L真核表达重组质粒pcDNA3.1-mCD40L,通过电转法将重组质粒转至NIH3T3细胞中。利用G418对转染后细胞进行压力筛选,获得稳定转染细胞株。提取稳定转染细胞株RNA,通过RT-PCR法检测Neo基因的mRNA表达情况。分离稳定转染细胞上清,利用ELISA法检测小鼠CD40L蛋白水平的表达情况。RT-PCR结果显示,Neo基因能够在稳定转染细胞中表达,ELISA结果显示,获得的稳定转染细胞株NIH3T3-mCD40L细胞上清中CD40L的表达量高达1.286 ng/mL。进一步活性研究表明,该细胞系能够在体外与IL-2和IL-21共同作用培养B细胞至14天,并刺激B细胞产生特异性抗体。该细胞系的成功构建,为利用体外B细胞分离培养和活化法分离特异性单克隆抗体奠定了良好的基础。     

Stimulatory Effects of Exogenous Thyroid Hormone and Growth Hormone on sn-Glycerol-3-Phosphate Dehydrogenase Activity in the Genetic-Hypothyroid Mutant Mouse Cerebellum: Restricted to the Second 20 Days of Postnatal Life

We recently reported that by postnatal day 40 the activity of sn-glycerol-3-phosphate dehydrogenase (GPDH) was significantly depressed in the cerebellum of genetic-hypothyroid mutant mice. This mutant mouse-GPDH combination was used in the present study to define the critical time period during which thyroid hormone (T4) and growth hormone (GH) are essential for maturation of Bergmann glial cells. Our findings are that (a) induction of GPDH activity in the Bergmann glial cell is dependent on T4, (b) T4 is most effective when administered during the second 20 days of postnatal life, (c) the effect of GH on GPDH activity is complementary to or synergistic with that of T4, and (d) Bergmann glial cells and radial glial fibers of the mutant mice contain immunoreactive GPDH following various hormonal treatments. These results suggest that T4 is indispensable for the maturation of Bergmann glial cells.     

Glial Cell Markers in the Reeler Mutant Mouse: A Biochemical and Immunohistological Study

The glial cell contents of S100 protein, 2',3'-cyclic AMP, 3'-phosphohydrolase (CNP), isoenzyme II of carbonic anhydrase (CAII) and butyrylcholinesterase (BuChE) were biochemically determined in the cerebellum and cerebrum of the reeler mutant mouse. Astrocytes and oligodendrocytes, shown by this study, contain abnormal amounts of these components. The CAII concentration was significantly increased in the particulate fraction of the reeler cerebellum and cerebrum (by 50% and 89%, respectively). The BuChE specific activity was greatly increased in the reeler, by 120% for cerebellum and by 40% in cerebrum. In contrast, the S100 protein concentration was reduced in the reeler cerebellum by 40% and by 25% in cerebrum, while the CNP specific activity increased by 30% in the reeler cerebellum. In addition, the glial cell distribution was studied by immunohistological techniques with antibodies directed against S100 protein, glial fibrillary acidic protein (GFA) and CAII. Apparently the density of glial cells is not significantly affected. However, the Golgi epithelial cells were usually abnormally placed and their Bergmann fibres were less well developed.     

Cerebroside and Sulfatide Biosynthesis in the Brain of Snell Dwarf Mouse: Effects of Thyroxine and Growth Hormone in the Early Postnatal Period

Snell dwarf mice (dw/dw) and normal mice (+/?) were injected with thyroxine (T₄) (1 μg/animal, four injections) and growth hormone (GH) (20 μg/animal, four injections) from the 5th to the 15th day of life. In the untreated dw/dw mouse brain, the specific activities of UDP-galactose:ceramide galactosyltransferase (CGalT), PAPS:cerebroside sulfotransferase (CST), and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) were decreased by 28, 25, and 37%, respectively, compared with the control untreated +/? mice. The major effect of T₄ was an increase of the brain CNP in the +/? mice (+40%) and dw/dw mice (+111%). The treatment with T₄ also brought to normal the level of CGalT in dw/dw brain; a somewhat less marked effect on CST was observed. The treatment with GH had a great stimulatory effect on CNP: the specific activity of this enzyme increased by 40 and 69% in +/? and dw/dw mouse brain, respectively. On the contrary, no effect of GH on the CGalT activity was observe in this study. Our results suggest that T₄ and GH may have both independent and complementary actions on the myelin-associated enzymes during the early postnatal period of brain development.     

维甲酸致小鼠胚胎畸形发生过程中维甲酸受体α/β及β-catenin和caspase-3基因的表达变化

流行病学研究显示,在胚胎发育过程中摄入过多维甲酸可致各种发育缺陷,其中神经管畸形最为常见. 因此有必要探明维甲酸致各种发育缺陷的发生机制,以便为各种生长缺陷的预防和治疗提供实验依据. 用 RT-PCR 及蛋白质印迹技术,探测了过量维甲酸对昆明小鼠胚胎神经管中维甲酸受体α/β及β-catenin 和 caspase-3 基因表达的调整. 结果显示,在神经管闭合期过量维甲酸显著降低了维甲酸受体α/β及β-catenin 和 caspase-3 的基因表达,神经管闭合后,维甲酸受体β、β-catenin 及 caspase-3 的基因表达又出现了一个明显的回升过程. 提示,过量维甲酸改变了昆明小鼠胚胎神经管中维甲酸受体α/β及β-catenin 和 caspase-3 基因的正常时间表达模式,这种异常的基因表达模式可能参与了维甲酸致昆明小鼠胚胎畸形的发生机制.     

Kit无义突变致W~(-3Bao)小鼠生殖腺异常及纯合子贫血死亡

W-3Bao是本研究组通过诱变获得、Kit无义突变的白斑小鼠.突变基因杂合子小鼠腹部、四肢肢端及尾尖白化,其部分精曲小管内无精原细胞.突变纯合子小鼠在胚胎后期色泽苍白、个体矮小,于出生前后死亡;血液学检查发现纯合子小鼠血色素极低且红细胞变大:18.5天胚胎的连续切片可见精曲小管轮廓欠清晰,精原细胞分散分布于睾丸间质,未迁入精曲小管:卵巢结构紊乱,无明显的原始卵泡结构;骨髓等器官组织未见显著异常.结论:Kit无义突变不仅导致了W-3Bao杂合子小鼠白斑形成及纯合子小鼠贫血死亡,同时影响生殖腺发育.     

Piwil2 及Stat3、Bcl-2 蛋白在雄性不育小鼠 睾丸组织中表达

本研究主要是探讨Piwil2、Stat3、Bcl-2蛋白在不育的雄性小鼠中的表达量及三者之间的表达位置相关性。取雄性昆明种小鼠60只,随机分为实验组与对照组,每组30只。实验组采用雷公藤多苷药物灌胃造小鼠不育模型28 d;对照组按照同样剂量的生理盐水进行灌胃,持续时间及频次同实验组。造模后将两组雄性小鼠分别与雌性小鼠交配;再处死雄性小鼠取出两组的睾丸组织,采用免疫组织化学染色法和蛋白印迹检测法分别检测样本中Piwil2、Stat3及Bcl-2蛋白的表达状况。将实验组与对照组的检测结果进行比较,观察两组的蛋白表达差异性及三个蛋白表达的相关性。H.E染色显示,实验组小鼠睾丸组织生精小管结构与对照组相比,明显被破坏,精原细胞及初次级精母细胞数量明显减少,结合与雌鼠交配后受精能力明显下降的结果,说明不育造模成功。免疫组化(IHC)染色结果显示,实验组Piwil2、Stat3及Bcl-2蛋白的染色程度及阳性细胞数均明显低于对照组(P 0.01)。Western Blot结果同样显示,三种蛋白在实验组的表达量明显低于对照组(Piwil2蛋白P 0.05,Stat3蛋白P 0.05,Bcl-2蛋白P 0.01)。本研究说明,Piwil2、Stat3及Bcl-2蛋白在雄性不育小鼠中表达量均显著降低,这三个蛋白对小鼠精子生成及小鼠不育的发生起到重要调节作用。     

镉诱导小鼠睾丸细胞凋亡与Caspase-3、Bcl-2和Bax mRNA的表达

一个月大雄性小鼠24只,随机分为6组,用30 μmol/kg CdCl2作用小鼠睾丸不同的时间(3 h、6 h、12 h、18 h、24 h)后,利用DNA电泳、免疫组化和半定量RT-PCR技术,分析生殖细胞凋亡过程中三种关键物质Caspase-3、Bcl-2、Bax蛋白和mRNA的表达量变化.结果显示:1) DNA各组 (除对照组外)均出现不同程度断裂.2)Caspase-3蛋白表达量一直上升,与对照组相比差异极显著;Bax蛋白在12 h前一直上升,与对照组相比差异显著,12 h后又开始下降,且与对照组相比无显著差异;Bcl-2蛋白在下降,与对照组相比差异显著.3)RT-PCR结果显示Caspase-3基因表达量减少;Bax基因表达量逐渐上升;Bcl-2基因表达量波动很大.综上所述,Caspase-3、Bcl-2和Bax三个基因可能参与了镉应激状态下小鼠睾丸组织细胞的凋亡过程.     

胰腺p-STAT3在急性胰腺炎危重演变中的表达变化

目的：检测信号转导与转录激活因子3（STAT3）在不同程度胰腺炎模型小鼠胰腺组织中表达的变化,探讨其在急性胰腺炎危重演变中的作用。方法：48只健康雄性balb/c小鼠随机分为3组（n=16）：对照组（Con）、轻症急性胰腺炎（MAP）组、重症急性胰腺炎（SAP）组。Con组腹腔注射0.9% NaCl;MAP组腹腔注射雨蛙素;SAP组腹腔注射雨蛙素联合脂多糖;分别于造模后2 h、6 h检测血清淀粉酶的活性;分离胰腺、称重,计算胰腺湿重比;检测肺组织髓过氧化物酶（MPO）活性,评估炎细胞浸润肺组织的程度;HE染色切片,光镜下观察胰腺、肺组织病理学改变; Western blot法检测磷酸化STAT3（p-STAT3）的变化。结果：与Con组比较, MAP组和SAP组在各时间点血清淀粉酶活性和胰腺组织湿重比均升高（P<0.05）;肺组织MPO活性显著升高（P<0.05）,且SAP组肺MPO含量明显高于MAP组（P<0.01）。MAP组和SAP组,在造模后2 h,胰腺和肺均可见不同程度的病理学改变; SAP组在造模后2 h胰腺p-STAT3的表达最高,6 h表达有所减弱;MAP组各时间点仅有微量表达;Con组在各时间点为阴性表达。结论：p-STAT3在轻症急性胰腺炎和重症急性胰腺炎模型小鼠胰腺中的表达差异明显,说明重症急性胰腺炎的重症化与STAT3的活化关系密切;抑制STAT3活化将成为阻止急性胰腺炎重症化的靶点之一。     

Pharmacological and Biochemical Characterization of Rat Hippocampal 5-Hydroxytryptamine1A Receptors Solubilized by 3–[3–(Cholamidopropyl)dimethylammonio]-1-Propane Sulfonate (CHAPS)

Rat hippocampal 5-hydroxytryptamine1A (5-HT1A) binding sites were solubilized with a yield of 34% using 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS, 10 mM) as detergent. Kinetic analyses of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) binding indicated that the 5-HT1A sites exhibit the same properties in the soluble form as in the membrane-bound form. Furthermore, a positive correlation (r = 0.988) was found between the respective pIC50 values of a series of agonists and antagonists to inhibit [3H]8-OH-DPAT binding to either soluble or membrane-bound 5-HT1A sites. Gel filtration through Sephacryl S-400 as well as chromatography on wheat germ agglutinin (WGA)-agarose did not affect the modulation by guanine nucleotides (5'-guanylylimidodiphosphate) of [3H]8-OH-DPAT binding which suggests that the 5-HT1A binding subunit is a glycoprotein tightly attached to a G protein even in its soluble form. The [3H]8-OH-DPAT binding material eluted from Sephacryl S-400 had an apparent molecular mass of 155 kilodaltons, as expected from a heterodimer with one binding subunit (approximately 60 kilodaltons) and one G protein (approximately 80 kilodaltons). Marked enrichment in 5-HT1A binding sites relative to other soluble proteins was found in the peak fractions eluted from Sephacryl S-400 (by sixfold) and WGA-agarose (by 26-fold) columns, suggesting that these chromatographic steps might be of interest for the purification of central 5-HT1A receptors.