动物,新生

摘要： 目的 探究妊娠期铅(Pb)、镉(Cd)暴露对新生大鼠海马脑源性神经营养因子(BDNF)及锌转移体7(ZnT7)蛋白表达的影响.方法 妊娠SD大鼠分为4组:对照组(NC组,饮用蒸馏水)、Pb组(300 mg/L)、Cd组(10 mg/L)及Pb+Cd组(300 mg/L Pb+10 mg/L Cd),每组3只.采用饮水染毒,干预21 d.采集各组21 d龄新生大鼠的血液及海马组织样本,PE700Z型原子吸收光谱仪测定血液及海马组织Pb、Cd水平;水迷宫实验测定新生大鼠认知能力;蛋白免疫印迹(Western blot)法检测海马组织中BDNF及ZnT7蛋白水平;Pear-son法分析Pb+Cd组新生大鼠海马组织中BDNF、ZnT7蛋白水平与血液、海马中Pb、Cd水平的相关性.结果 与NC组比较,Pb组、Cd组及Pb+Cd组新生大鼠逃避潜伏期、进入盲端次数、海马组织中ZnT7蛋白水平显著增加,BDNF水平显著降低(P<0.05),其中以Pb+Cd组变化最明显.Pb+Cd组新生大鼠海马组织中BD-NF水平与血液及海马组织Pb、Cd水平均呈负相关(P<0.05),ZnT7蛋白水平与血液及海马组织Pb、Cd水平均呈正相关(P<0.05).结论 妊娠期Pb、Cd暴露可协同降低新生大鼠认知能力,可能与海马BDNF表达降低,ZnT7蛋白表达升高有关.

摘要： 新生儿缺氧缺血性脑病(HIE)是围生期窒息导致的缺氧缺血性脑损伤(HIBD),是导致新生儿急性死亡和远期神经系统疾病的主要原因.对于HIE迄今尚无根治性治疗措施,临床以对症支持治疗为主,疗效有限,而且不能促进受损神经修复和再生.间充质干细胞移植(MSCT)作为治疗新生儿HIE的新策略,在HIBD动物病理模型研究中,具有明显神经保护作用,其作用机制较为复杂,可能通过分泌细胞外囊泡(ECV)、调节免疫、促进神经元修复与再生、抗细胞凋亡及抗氧化等机制发挥作用,从而达到改善预后的目的 .笔者拟就间充质干细胞(MSC)对HIBD动物病理模型的神经保护作用可能机制的最新研究进展进行阐述.

摘要： 目的 评价下丘脑芳香化酶在七氟烷麻醉致新生大鼠癫痫波中的作用.方法 清洁级健康新生SD大鼠30只,雌雄各半,5日龄,体重10～ 15 g,采用随机数字表法分为3组(n=10):对照组(C组)、七氟烷组(S组)和芳香化酶抑制剂福美司坦+七氟烷组(F组).监测新生大鼠皮层脑电图(EEG),30 min后F组皮下注射福美司坦2 mg/kg,C组和S组皮下注射生理盐水.皮下给药后30 min时S组和F组吸入6％七氟烷麻醉诱导3min,随后调整为2.1％麻醉维持57 min.记录七氟烷麻醉期间癫痫波总持续时间、单次持续时间和发作次数;EEG记录结束后剖腹,左心室穿刺取血行血气分析及ELISA法检测皮质酮水平;取脑组织,冰块上迅速分离下丘脑,采用PCR法检测下丘脑芳香化酶mRNA、Na+-K+-2Cl-共同转运体1 (NKCC1) mRNA和Na+-K+共同转运体2(KCC2) mRNA的表达.结果 C组未见癫痫波.与C组比较,S组和F组皮层癫痫波总持续时间和单次持续时间延长,发作次数增加,血清皮质酮浓度升高,下丘脑芳香化酶mRNA表达上调,NKCC1/KCC2 mRNA比值升高(P＜0.05);与S组比较,F组皮层癫痫波总持续时间和单次持续时间缩短,发作次数减少,血清皮质酮浓度降低,下丘脑芳香化酶mRNA表达下调,NKCC1/KCC2 mRNA比值下降(P＜0.05).结论 下丘脑芳香化酶表达上调参与了七氟烷麻醉致新生大鼠癫痫波发生的过程.

摘要： 目的 观察艾灸对缺氧缺血性脑病新生小鼠脑组织炎症细胞浸润和CD11b表达的影响.方法 出生7 d新生ICR小鼠,随机分为3组:假手术组(n=20)、模型组(n=24)和艾灸组(n=20).假手术组只做颈部手术,不闭合颈总动脉,不缺氧;模型组行颈总动脉闭合术,并给予缺氧处理;艾灸组在模型组的基础上给予艾灸治疗,每日1次,35 min/次.采用TTC染色检测小鼠脑梗死的面积;HE染色观察小鼠脑组织形态结构和炎症细胞浸润;组织免疫荧光染色检测脑组织CD11b表达.结果 与假手术组相比,模型组小鼠的脑梗死面积增大,患侧脑组织大量细胞坏死脱落,并伴有大量炎症细胞浸润,CD11b阳性细胞数明显增多;与模型组相比,艾灸组小鼠的脑梗死面积缩小,患侧脑组织细胞排列较致密、整齐,炎症细胞较少,CD11b阳性细胞数明显减少.结论 艾灸具有减轻新生小鼠缺氧缺血性脑损伤的作用,这可能与其减少脑组织小胶质细胞浸润、减轻炎症反应有关.

摘要： Objective To analyze the role of intestinal fatty acid-binding protein (I-FABP) expression in a neonatal rat model of necrotizing enterocolitis (NEC).Methods A total of 24 newborn rats were randomly divided into two groups:control group (n=6) and NEC group (n=18).Rats in the NEC group were fed with formula and experienced hypoxia,reoxygenation,cold stress and sequentially Lipopolysaccharide (10 mg/kg) lavage for three consecutive days to establish NEC model,after which were respectively sacrificed on day 1,2 and 3 (six for each day).Those in the control group were all sacrificed on day 3.Ileocecal tissues were collected for morphological and histological analysis.I-FABP expression was detected using Western blot and immunohistochemistry (IHC).One-way analysis of variance,LSD-t test,Kruskal-Wallis H test,Mann-Whitney U test and Pearson's correlation analysis were used for statistical analysis.Results The NEC model (intestinal pathological score ≥ 2) was established successfully without causing death.Compared with the control group,the NEC group showed less body weight gain [M (P25-P75):1.00 (0.48-1.35) vs 1.74 (1.62-1.86),1.25 (0.75-1.40) vs 2.61 (2.53-2.99),1.35 (0.88-1.48) vs 3.60 (3.48-3.73);Z=-2.898,-2.903,-2.892;all P＜0.05] and higher intestinal pathological scores [(2 (2-3),3 (2-3),4 (3-4) vs 0 (0-1);all P＜0.05] on day 1,2 and 3.The intestinal pathological score on day 3 was significantly higher than that on day 2 and day 1 (both P＜0.05).Expression of I-FABP and the number of I-FABP positive enterocytes in the NEC model group were increased compared with those in the control group [Western blot:0.179 (0.179-0.186),0.231 (0.211-0.245),0.202 (0.192-0.225) vs 0.091 (0.086-0.093);IHC:59 (55-60),80 (83-86),80 (84-88) vs 44 (39-47);all P＜0.05].Moreover,the expression of I-FABP protein and the number of I-FABP positive enterocytes on day 2 and day 3 were significantly higher than those on day 1 (all P＜0.05).I-FABP expression was positively associated with intestinal pathological score (Western blot:r=0.932,95％CI:0.872-0.969;IHC:r=0.709,95％CI:0.484-0.872).Conclusions I-FABP is an efficient marker for NEC and correlates with the severity of intestinal injury.%目的 观察新生大鼠肠道组织中肠型脂肪酸结合蛋白(intestinal fatty acidbinding protein,I-FABP)的表达情况,探讨I-FABP与新生儿坏死性小肠结肠炎(necrotizing enterocolitis,NEC)的相关性. 方法 24只2日龄无特定病原体级新生Sprague-Dawley大鼠随机分为空白对照组(6只)和NEC模型组(18只).通过连续3d人工喂养+缺氧复氧+冷刺激+脂多糖灌胃(10 mg/kg)建立NEC模型,分别在造模成功后第1、2、3天随机选取6只大鼠空腹处死;空白对照组于NEC模型组造模成功后第3天空腹处死.处死后取大鼠回盲部肠管行组织病理评分,以及采用蛋白质印迹技术及免疫组织化学法检测回盲部肠组织I-FABP蛋白表达情况.采用单因素方差分析、LSD-t检验、Kruskal-Wallis H秩和检验、Mann-Whitney U检验,以及Pearson相关分析进行统计学分析. 结果NEC模型组大鼠全部顺利完成造模,造模过程中并无死亡,且肠组织病理学评分均≥2分,造模成功.NEC模型组造模后第1、2、3天体重增加[M(P25～P75)]均低于空白对照组[分别为1.00(0.48～1.35)与1.74(1.62～1.86)g,1.25(0.75～1.40)与2.61 (2.53～2.99)g,1.35 (0.88～1.48)与3.60(3.48～3.73)g,Z值分别为-2.898、-2.903及-2.892,P值均＜ 0.05],但肠组织病理学评分均高于空白对照组[分别为2(2～3)、3(2～3)、4(3～4)与0(0～1)分,P值均＜ 0.05],而NEC模型组造模后第3天大鼠肠组织病理评分显著高于造模后第1及第2天(P值均＜ 0.05).NEC模型组造模后第1、2、3天大鼠肠组织I-FABP表达水平及I-FABP阳性细胞数均高于空白对照组[I-FABP表达水平:0.179 (0.179～0.186)、0.231 (0.211～0.245)、0.202(0.192～0.225)与0.091(0.086～0.093);I-FABP阳性细胞数:59 (55～60)、80(83～86)、80(84～88)与44(39～47)个;P值均＜0.05],与造模后第1天相比,NEC模型组造模后第2及3天大鼠肠组织I-FABP表达水平及I-FABP阳性细胞数均较高(P值均＜0.05).蛋白质印迹技术与免疫组织化学法检测肠道组织I-FABP表达水平与肠道组织病理评分均呈正相关(r=0.932,95％CI:0.872～0.969;r=0.709,95％CI:0.484～0.872). 结论 I-FABP与NEC肠组织损伤程度存在相关性.

摘要： Objective To evaluate the effects of penehyclidine hydrochloride on the nuclear factor erythroid 2-related factor 2/antioxidant responsive element (Nrf2/ARE) signaling pathway during endotoxin-induced acute lung injury(ALI) in neonate rats.Methods Forty 7-day-old Wistar rats weighing 12-18 g were randomly divided into 4 groups (n=10) using a random number table:normal saline group(NS group),acute lung injury(ALI group),penehyclidine hydroehloride group(PHC group) and penehyclidine hydrochloride + Nrf2 siRNA plasmid group(PNS group).The ALI model was induced with intraperitoneal endotoxin (5.0 mg/kg) in groups ALI,PHC and PNS.In groups PHC and PNS,penehyclidine hydrochloride (2.0 mg/kg) was injected intraperitoneally at 1 h before ALI respectively,while the equal volume of normal saline was administered in groups NS and ALI.The animal of PNS group were inhaled adenovirus packaging of Nrf2-siRNA three times (one time a day) before modeling.At 4 h after endotoxin injection,the rats were sacrificed.The lungs were collected to determine the wet/dry(W/D) lung weight ratio.The expression of Nrf2 and heme oxygen and enzyme 1(HO-1) were determined by Western blotting,contents of tumor necrosis factor-alpha(TNF-α),interleukinl0 (IL-10)were determined by enzyme-linked immunosorbent assay(ELISA).The cell apoptosis were determined by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TUNEL),and the apoptotic index was calculated.Results The W/D ratio in NS,ALI,PHC and PNS groups were(4.2±0.1),(9.6±0.7),(6.5±0.6),(8.3± 1.3)respectively.The apoptotic index were(3.7±0.5)％,(31.5±3.2)％,(17.6±4.2)％,(28.1 ±3.5)％respectively.The contents of TNF-c were(10.3± 1.6),(98.5±8.5),(68.5 ±6.7),(89.9±8.5)pg/ml respectively.The contents of IL-10 were (7.9±0.6),(102.8±9.3),(72.5±5.8),(97.7±9.1)pg/ml respectively.The expression of Nrf2 were (23.2±7.6),(79.8± 13.0),(155.5± 16.7),(12.0±3.3)respectively.The expression of HO-1 were(31.7 ± 8.6),(90.8 ± 10.3),(147.6 ± 22.5),(61.4 ± 9.7) respectively.There were statistically significant differences among different groups(F=86.013,154.897,328.810,374.198,333.965,125.274,all P＜0.05).Compared with group NS,the W/D ratio,apoptotic index and the contents of TNF-α,IL-10 increased,the expression of Nrf2 and HO-1 up-regulated in group ALI and group PHC(all P＜0.05).Compared with group ALI,the W/D ratio,apoptotic index and the contents of TNF-α,IL-10 decreased,the expression of Nrf2 and HO-1 up-regulated in group PHC (all P＜0.05).Compared with group ALI,no significant differences were found in the W/ D ratio,apoptotic index and the contents of TNF-α,IL-10 in group PNS(all P＞0.05),while the expression of Nrf2 and HO-1 down-regulated in group PNS(all P＜0.05).Compared with group PHC,the W/D ratio,apoptotic index and the contents of TNF-α,IL-10 increased,the expression of Nrf2 and HO-1 down-regulated in group PNS(all P＜0.05).Conclusion Nrf2/ARE signaling pathway is involved in the reduction of ALI by penehyclidine hydrochloride in neonate rats.%目的 评价盐酸戊乙奎醚对新生大鼠内毒素急性肺损伤(ALI)时肺组织核因子E2相关因子-2 (Nrf2)/抗氧化反应元件(ARE)信号通路的影响.方法 清洁级健康雄性Wistar大鼠40只、体质量12～ 18 g,7日龄.采用随机数字表法分为4组(n=10):对照组(NS组)、急性肺损伤组(ALI组)、盐酸戊乙奎醚组(PHC组)和盐酸戊乙奎醚+Nrf2-siRNA质粒组(PNS组).腹腔注射内毒素5.0 mg/kg制备ALI模型.PHC组和PNS组于内毒素注射前1h腹腔注射盐酸戊乙奎醚2.0 mg/kg,NS组和ALI组给予等容量生理盐水.PNS组新生大鼠进行模型制备前3d,吸入采用腺病毒包装的Nrf2-siRNA液3次(1次/d).于注射内毒素4h时处死大鼠取肺组织标本,计算肺组织湿/干重比(W/D),采用免疫印迹法(Western blotting)测定Nrf2和血红素氧合酶-1(HO-1)的表达,采用酶联免疫吸附测定法(ELISA)测定肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)的含量;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)计数凋亡细胞,计算细胞凋亡指数(AI).结果 NS组、ALI组、PHC组、PNS组W/D分别为(4.2±0.1)、(9.6±0.7)、(6.5±0.6)、(8.3±1.3),AI分别为(3.7±0.5)％、(31.5±3.2)％、(17.6±4.2)％、(28.1±3.5)％,TNF-α分别为(10.3±1.6)、(98.5±8.5)、(68.5±6.7)、(89.9±8.5)pg/ml,IL-10分别为(7.9±0.6)、(102.8±9.3)、(72.5±5.8)、(97.7±9.1) pg/ml,Nrf2分别为(23.2±7.6)、(79.8±13.0)、(155.5±16.7)、(12.0±3.3),HO-1分别为(31.7±8.6)、(90.8±10.3)、(147.6±22.5)、(61.4±9.7),差异均有统计学意义(F=86.013、154.897、328.810、374.198、333.965、125.274,均P＜0.05);与NS组相比较,ALI组和PHC组肺组织W/D、AI和TNF-α、IL-10含量升高,Nrf2和HO-1的表达上调(均P＜0.05);与ALI组相比较,PHC组肺组织W/D、AI和TNF-α、IL-10含量下降,Nrf2和HO-1的表达上调(均P＜0.05);与ALI组相比较,PNS组肺组织W/D、AI和TNF-α、IL-10含量差异均无统计学意义(均P＞0.05),Nrf2和HO-1的表达下调(均P＜0.05);与PHC组相比较,PNS组肺W/D、AI和TNF-α、IL-10含量升高,Nrf2和HO-1表达下调(均P＜0.05).结论 Nff2/ARE信号通路参与了盐酸戊乙奎醚减轻新生大鼠内毒素性ALI.

摘要： 目的 探讨新生大鼠哺乳期间断吸入七氟烷对老年期行为学及海马中载脂蛋白E (ApoE)的影响.方法 健康7日龄SD大鼠48只,随机分为七氟烷哺乳期组(Sev1组)、七氟烷老年期组(Sev2组)及哺乳期对照组(Con1组)、老年期对照组(Con2组),共4组,每组12只.Sev1、2组于生后7、14和21 d分别吸入2.6％七氟烷2h,Con1、2组吸入空氧混合气体.哺乳期组大鼠于生后31 d、老年期组大鼠于生后20个月行Morris水迷宫实验;实验结束后取海马组织,荧光定量聚合酶链反应(PCR)测ApoE mRNA表达,免疫组织化学测ApoE、ApoE4表达.结果 定位航行实验中,各组大鼠逃避潜伏期均逐渐缩短,但Sev1组与Con1组比较差异无统计学意义(P＞0.05),而Sev2组与Con2组比较,第3天逃避潜伏期延长(P＜0.05);空间探索实验中,Sev1组与Con1组比较、Sev2组与Con2组比较差异均无统计学意义(P＞0.05);Sev1组与Con1组比较、Sev2组与Con2组比较,海马ApoE mRNA表达均上调(P＜0.05);Sev1组相对Con1组大鼠海马CAl区和CA3区ApoE表达上调(P＜0.05),DG区ApoE表达差异无统计学意义(P＞0.05);Sev2组相对Con2组海马各分区ApoE表达差异无统计学意义(P＞0.05);Sev1组与Con1组比较、Sev2组与Con2组比较,海马各分区ApoE4表达差异均无统计学意义(P＞0.05).结论 哺乳期大鼠间断吸入2.6％七氟烷麻醉后,对大鼠海马ApoE有短暂影响,但并不造成其老年期海马ApoE异常及明显行为学改变.

摘要： 目的 探讨不同环境微生物暴露对生命早期小鼠肠道菌群定植的影响. 方法 选取无特殊病原体6～8周BALB/c小鼠24只(雌16只,雄8只),雌雄2:1合笼,受孕后按子代不同时期(胎儿期、哺乳期、儿童期)的饲养环境分为4组.A组:胎儿期、哺乳期、儿童期均于清洁环境中饲养;B组:胎儿期、哺乳期于清洁环境中饲养,儿童期于普通环境中饲养;C组:胎儿期于清洁环境中饲养,哺乳期、儿童期于普通环境中饲养;D组:胎儿期、哺乳期、儿童期均于普通环境中饲养.采集小鼠生后3周末和5周末粪便样本,提取样本细菌总DNA,采用Illumina MiSeq高通量测序仪对细菌的16S rDNA V4区进行测序及生物信息学分析.采用Kruskal-Wallis及Dunn-Bonferroni检验进行统计学分析. 结果 1、生后3周末:(1)物种组成分析:①门水平:4组间厚壁菌门、疣微菌门、变形菌门和放线菌门相对丰度比较,差异均有统计学意义(P值均＜ 0.05);A和B组厚壁菌门相对丰度分别低于C和D组[30.876(23.448～41.218)×10-2、3.317(1.116～4.641)×10-2与71.936(53.587～86.713)×10-2、79.105(56.305～82.736)×10-2],而疣微菌门、变形菌门相对丰度高于C和D组[疣微菌门:17.249(9.748～35.106)×10-2、58.883(0.017～6.047)×10-2与0.152(0.066～1.890)×10-2、0.003(0.000～0.016)×10-2;变形菌门:12.640(0.336～15.070)×10-2、3.653(3.362～4.596)×10-2与0.219(0.134～0.325)×10-2、0.124(0.116～0.165)×10-2],差异均有统计学意义(P值均＜0.05或0.01);②属水平:4组间乳酸杆菌属、阿克曼菌属、拟杆菌属等相对丰度比较差异均有统计学意义(P值均＜ 0.01);A和B组乳酸杆菌属相对丰度低于C和D组[19.283(8.618～31.541)×10-2、0.339(0.264～22.278)×10-2与58.414(34.874～71.942)×10-2、66.007(55.141～76.940)×10-2],而阿克曼菌属、拟杆菌属、克雷伯杆菌属相对丰度高于C和D组[阿克曼菌属:17.247 (9.748～35.106)×10-2、58.883(0.017～60.475)×10 2与0.152(0.066～1.890)×10-2、0.003(0.000～0.017)×10-2;拟杆菌属:3.978(0.683～25.171)×10-2、8.216(6.023～9.946)×10-2与0.141 (0.061～0.281)×10-2、0.568(0.149～1.455)×10-2;克雷伯杆菌属:0.209 (0.050～8.888)×10-2、1.402(0.865～1.692)×10-2与0.003(0.000～0.039)×101、0.000(0.000～0.001)×10-2],差异均有统计学意义(P值均＜0.05或＜0.01).(2)α多样性分析:4组间操作分类单位(operational taxonomic unit,OTU)数量、Chaol指数比较差异有统计学意义(P值均＜0.05),Shannon指数差异无统计学意义(P值＞0.05);A和B组OTU数量低于D组[246 (221～348)和257 (209～280)与387 (324～478),P值分别为0.045和0.0082].2、生后5周末:(1)物种组成分析:①门水平:4组间厚壁菌门、疣微菌门、变形菌门相对丰度比较差异有统计学意义(P值均＜0.05或0.01);A组厚壁菌门相对丰度低于B、C和D组[13.765 (64.181～24.238)×10-2与48.912(37.280～59.466)×10-2、86.065(50.149～89.856)×10 1、53.847(31.946～72.936)×10-2],而疣微菌门相对丰度高于B、C和D组[58.089(22.459～61.285)×10-2与0.001 (0.000～0.005)×10-2、0.0000 (0.000～0.001)×10-2、0.003(0.000～0.006)×10-2],差异均有统计学意义(P值均＜0.05或0.01);②属水平:4组间乳酸杆菌属、阿克曼菌属相对丰度比较差异均有统计学意义(P值均＜ 0.01).A组乳酸杆菌属相对丰度低于B、C和D组[1.755 (0.805～8.833)×10-2与26.391(17.550～37.265)×10-2、70.688 (45.713～77.953)×10-2、28.675(15.660～57.224)×10-2],而阿克曼菌属相对丰度高于B、C和D组[58.089(22.460～61.285)×10-2与0.000(0.000～0.006)×10-2、0.000(0.000～o.001)×101、0.003(0.000～0.006)×10-2],差异均有统计学意义(P值均＜0.05或0.01).(2)α多样性分析:4组间OTU数量、Chao1指数、Shannon指数比较差异均有统计学意义(P值均＜0.05或0.01);A组OTU数量低于B、C、D组[268 (241～410)与438 (380～516)、562 (533～588)、546(473～599)],B组OTU数量、Chao1指数、Shannon指数均低于C和D组[OTU数量:438(380～516)与562 (533～588)、546 (473～599);Chao1指数:1 033 (883～1 181)与1 285 (1 220～1 338)、1 328 (1 155～1 516);Shannon指数:3.85 (3.25～4.50)与4.28 (3.30～5.11)、4.17(3.62～4.38)],差异有统计学意义(P值均＜0.05或0.01). 结论 不同环境微生物暴露会影响生命早期小鼠肠道菌群的多样性与结构;环境清洁度越高,肠道菌群多样性越低,菌群结构组成也有一定差异;哺乳期可能是肠道菌群定植的重要“时间窗”.%Objective To investigate the influences of exposure to different environmental microbes on early-life gut microbiota colonization in mice.Methods Male (n=8) and female (n=16) adult specific pathogen free (SPF) BALB/c mice were caged together at a ratio of 2:l.After conception,the mice were divided into four groups according to the environments where the offsprings were reared at three different periods (fetal period,breastfeeding period and childhood).Group A:Offsprings were kept in a SPF environment throughout the study;group B:SPF environment during fetal and breastfeeding periods,and then ordinary environment during childhood;group C:SPF environment during fetal period,and then ordinary environment during breastfeeding period and childhood;group D:ordinary environment all the time.Fecal samples were collected at the end of week 3 and 5.Total bacterial DNA was extracted from each sample and analyzed by high throughput analysis.Kruskal-Wallis and Dunn-Bonferroni test were applied for statistical anaysis.Results 1.At the end of three weeks:(1) Diversity:① Phylum level:There were significant differences in the abundance of Firmicutes,Verrucomicrobia,Proteobacteria and Actinobacteria among the four group (all P＜0.01).Compared with group C and D,group A and B showed significantly decreased abundance of Firmicutes [30.876(23.448-41.218)× 10-2,3.317(1.116-4.641) 10-2 vs 71.936(53.587-86.713)× 10-2,79.105(56.305-82.736)× 10-2],but increased abundance of Verrucomicrobia and Proteobacteria [Verrucomicrobia:17.249(9.748-35.106)× 10-2,58.883(0.017-6.047)× 10-2 vs 0.152(0.066-1.890)× 10-2,0.003(0.000-0.016)× 10-2;Proteobacteria:12.640(0.336-15.070)× 10-2,3.653(3.362-4.5955)× 10-2 vs 0.219(0.134-0.325)× 10-2,0.124(0.116-0.165) × 10-2,all P＜0.05 or 0.01].② Genus level:There were significant differences in the abundance of Lactobacillus,Akkermansia and Bacteroides among the four groups (all P＜0.01).Compared with group C and D,group A and B showed significantly decreased abundance of Lactobacillus [19.283(8.618-31.541)× 10-2,0.339(0.264-22.278) × 10-2 vs 58.414(34.874-71.942)× 10-2,66.007(55.141-76.940)× 10-2],but increased abundance of Akkermansia,Bacteroides and Klebsiella [Akkermansia:17.247(9.748-35.106)× 10-2,58.883(0.017-60.475)× 10-2 vs 0.152(0.066-1.890)× 10-2,0.003(0.000-0.017)× 10-2;Bacteroides:3.978(0.683-25.171)× 10-2,8.216(6.023-9.946)× 10-2 vs 0.141(0.061-0.281)× 10-2,0.568(0.149-1.455)× 10-2;Klebsiella:0.209(0.050-8.888)× 10-2,1.402(0.865-1.692)× 10-2 vs 0.003(0.000-0.039) 10-2,0.000(0.000 0.001)× 10-2,all P＜0.05 or 0.01].(2) Alpha diversity:Significant differences were found in operational taxonomic unit (OTU) and Chaol index (P＜0.05),but not in Shannon index among the four groups (P＞0.05).The OTUs of group A and B were significantly lower than that of group D [246(221-348),257(209-280) vs 387(324-478),P=0.045 and 0.008,respectively].2.At the end of five weeks:(1) Diversity:① Phylum level:There were significant differences in the abundance of Firmicutes,Verrucomicrobia and Proteobacteria among the four groups (P＜0.05 or 0.01).The abundance of Firmicutes in gut microbiota in group A was lower than that in group B,C and D [13.765(64.181-24.238)× 10-2 vs 48.912(37.280-59.466)× 10-2,86.065(50.149-89.856) × 10-2,53.847(31.946-72.936) × 10-2],while that of Verrucomicrobia was higher [58.089(22.459-61.285)× 10-2 vs 0.001(0.000-0.005)× 10-2,0.000(0.000-0.001)× 10-2,0.003(0.000-0.006)× 10-2],all P＜0.05 or 0.01.② Genus level:There were significant differences in the abundance of Lactobacillus and Akkermansia among the four groups (P＜0.01).The abundance of Lactobacillus in gut microbiota in group A was lower than that in group B,C and D[1.755(0.805-8.833)× 10-2 vs 26.391(17.550-37.265)× 10-2,70.688(45.713-77.953) × 10-2,28.675 (15.660-57.224) × 10-2],while that of Akkermansia was higher [58.089(22.460-61.285)× 10-2 vs 0.000(0.000-0.006)× 10-2,0.000(0.000-0.001)× 10-2,0.003(0.000-0.006)× 10-2,all P＜0.05 or 0.01].(2) Alpha diversity:There were significant differences in OTU,Chaol and Shannon index among the four groups (P＜0.05 or 0.01).The OTU of group A was lower than that of group B,C and D [268(241-410) vs 438(380-516),562(533-588),546(473-599)],and the OTU,Chaol and Shannon index of group B were all lower than those of group C and D [OTU:438(380-516) vs 562(533-588),546(473-599);Chaol index:1 033(883-1 181) vs 1 285(1 220-1 338),1 328(1 155-1 516);Shannon index:3.85(3.25-4.50) vs 4.28(3.30-5.11),4.17(3.62-4.38),all P＜0.05 or 0.01].Conclusions Early-life exposure to different environments has an obvious impact on the diversity and composition of intestinal microbiota in mice.The less clean the living environment is,the more diverse the gut microflora will be.Furthermore,the window of opportunity for gut microbiota colonization seems to be related to breastfeeding period.

摘要： Objectives To investigate the occipital cortex metabolite alterations in repetitive and severe neonatal hypoglycemia rats treated with sodium pyruvate and to reveal the protective role of sodium pyruvate using high resolution 1H nuclear magnetic resonance spectroscopy.Methods Thirty-six 2-dayold Sprague-Dawley rats were randomly divided into hypoglycemia group and pyruvate group with 18 rats in each group.Rats in both groups received intraperitoneal injections of insulin (40 U/kg body weight) at 2,4 and 6 days of age to induce severe hypoglycemia (blood glucose value ≤ 1.4 mmol/L).In the hypoglycemia group,2.5 hours after insulin injection,intraperitoneal injection of 50％ glucose (2 ml/kg) was administered to terminate hypoglycemia,while in the pyruvate group,50％ glucose (2 ml/kg) and sodium pyruvate solution 2.5 ml/kg (500 mg/kg) were injected.Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay was used to observe the status of injured neurons in six neonatal rats,and metabolite changes in occipital cortex of the other 12 rats were detected by 1H nuclear magnetic resonance spectroscopy.The difference between the two groups was compared by independent-samples t test.Results Neonatal rats of both groups reached severe hypoglycemia level 2.5 hours after insulin injection.Compared with hypoglycemia group,pyruvate group had fewer injured neurons (45±5 vs 113 ± 12,t=0.782,P=0.013) and lower injured index in the occipital cortex (0.15 ± 0.03 vs 0.36 ± 0.06,t=l.143,P=0.020).Pyruvate group showed significant decreases in the concentration of taurine [(13.31 ± 2.06) vs (18.44 ± 3.86) mol/kg,t=8.231],glutamine[(1.50 ± 0.24) vs (2.02 ± 0.40) mol/kg,t=3.137],glutamate[(7.04 ± 0.95) vs (9.40 ± 1.73) mol/kg,t=6.449],aspartate[(1.51 ± 0.28) vs (2.15 ± 0.58) mol/kg,t=2.561] and creatine [(6.37±0.99) vs (8.46± 1.77) mol/kg,t =4.226] in the occipital cortex (all P'＜0.017).Conclusions Simultaneous use of glucose and sodium pyruvate to terminate hypoglycemia in repetitive and severe neonatal hypoglycemia rats can effectively alleviate severe hypoglycemia-induced occipital lobe damage via regulating excitatory amino acid neurotransmitters,energy metabolism and other metabolic pathways.%目的 应用高分辨氢质子磁共振波谱技术研究反复严重低血糖新生大鼠经丙酮酸钠干预治疗对脑损伤的保护作用及其机制. 方法 36只2日龄Sprague-Dawley大鼠随机分为低血糖组和干预组,每组1 8只.2组大鼠2、4、6日龄时腹腔注射胰岛素40 U/kg诱导严重低血糖(血糖≤1.4 mmol/L),低血糖组注射胰岛素后2.5 h腹腔注射50％葡萄糖溶液2 ml/kg终止低血糖,干预组则在注射同等量葡萄糖的同时注射丙酮酸钠溶液2.5 ml/kg(500 mg/kg).7日龄时,每组选取6只大鼠,取枕叶皮层行原位末端转移酶标记技术染色以观察脑细胞的损伤情况;另外12只大鼠的枕叶皮层行离体高分辨氢质子磁共振波谱检查以观察代谢物变化情况.采用两独立样本t检验进行统计学分析. 结果 2、4和6日龄时,低血糖组及干预组新生大鼠在给予胰岛素2.5 h后均达到严重低血糖水平.与低血糖组相比,干预组枕叶皮层内损伤神经元个数减少[(45±5)与(113±12)个,t=0.782,P=0.013],损伤指数下降(0.15±0.03与0.36±0.06,t=1.143,P=0.020).相较低血糖组,干预组大鼠枕叶皮层中牛磺酸[(13.31±2.06)与(18.44±3.86)mol/kg,t=8.231]、谷氨酰胺[(1.50±0.24)与(2.02±0.40) mol/kg,t=3.137]、谷氨酸[(7.04±0.95)与(9.40±1.73) mol/kg,t=6.449]、天冬氨酸[(1.51±0.28) 与(2.15±0.58)mol/kg,t=2.561]和肌酸[(6.37±0.99)与(8.46±1.77) mol/kg,t=4.226]的浓度显著下降,差异均有统计学意义(P'值均＜ 0.017). 结论 反复严重新生期低血糖在给予葡萄糖终止低血糖的同时提供丙酮酸钠可通过调节兴奋性氨基酸类神经递质、能量代谢及其他代谢通路而减轻低血糖所致的枕叶损伤,发挥脑保护作用.

摘要： Objective To study the effects of discoidin domain receptor 1 (DDR1) mediated phosphorylation of protein Tau on hypoxic-ischemic brain damage (HIBD) in neonatal rats and its possible mechanism.Methods Sixty-four seven-day-old male specific-pathogen-free Wistar rats were randomly divided into four groups with sixteen in each: Sham, HIBD, HIBD with normal saline (HIBD+NS) and HIBD with DDR1 inhibitor (HIBD+DI) groups. A rat model of HIBD was established by subjecting the rats to left common carotid artery ligation, followed by exposing them to hypoxia for two hours. In HIBD+DI group, the inhibitor of DDR1 was immediately injected into lateral cerebroventricles of the rats following modeling. Forty-eight hours after injection, tissues of left cerebral cortex were collected from each rat to evaluate histopathological changes with HE staining. Western-blotting was used to assess the phosphorylation levels of DDR1 and protein Tau. Enzyme-linked immunosorbent assay was performed to detect the concentrations of acetylcholine. Analysis of variance ort test were used for statistical analysis.Results (1) Damages in cerebral cortex: Percentages of abnormal neurons in the rats of HIBD group were higher than those in Sham group [(80.28±4.51)% vs (10.40±2.17)%,t=39.491,P<0.01]. Pyknotic or necrotic neurons in the rats of HIBD+DI group were less than those in HIBD+NS group [(31.91±3.05)% vs (82.01±7.20)%,t=18.123,P<0.01]. (2) Phosphorylation of DDR1 and protein Tau: Levels of phosphorylated DDR1 in the cerebral cortexes of rats in HIBD group were higher than those in Sham group (0.922±0.199 vs 0.095±0.023,t=10.379,P<0.01), and those levels in HIBD+NS group were higher than those in HIBD+DI group (1.200±0.171 vs 0.255±0.111,t=11.901, P<0.01). The phosphorylation of protein Tau was similar to that of DDR1 (0.919±0.228 vs 0.194±0.224 in HIBD and Sham groups,t=7.347; 1.100±0.167 vs 0.291±0.210 in HIBD+NS and HIBD+DI groups,t=9.447;bothP<0.01). (3) Levels of acetylcholine: Levels of acetylcholine in cerebral cortexes of rats in HIBD group were lower than those in Sham group [(3.685±0.472) vs (7.429±0.861) ng/g protein,t=10.781,P<0.01], and that levels in HIBD+DI group were higher than those in HIBD+NS group [(7.058±0.915) vs (2.521±0.723) ng/g protein,t=10.989,P<0.01].Conclusions Activation of DDR1 plays a key role in enhancing the phosphorylation of protein Tau and in reducing the secretion of acetylcholine in cerebral cortexes of rats with HIBD. Inhibitor of DDR1 could protect neonatal rats from HIBD through the decreasing of protein Tau phosphorylation and increasing of acetylcholine release by inhibiting the activation of DDR1.%目的 研究盘状结构域受体1(discoidin domain receptor 1,DDR1)调控微管相关蛋白Tau(简称Tau蛋白)表达及其磷酸化在新生大鼠缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)中的作用及其机制.方法 将64只7日龄无特定病原体级雄性Wistar大鼠随机分为假手术组(Sham组)、HIBD组、生理盐水(normal saline,NS)治疗组(HIBD+NS组)及DDR1抑制剂(DDR1 inhibitor,DI)治疗组(HIBD+DI组),每组16只.通过结扎左侧颈动脉后置于低氧箱内2 h,复制新生大鼠HIBD模型.HIBD+DI组造模后,立即给予DDR1抑制剂侧脑室注射治疗.造模后48 h收集左侧皮层组织,HE染色观察大脑皮层的组织病理学改变;蛋白质印迹法检测皮层组织中DDR1及Tau蛋白的磷酸化水平;酶联免疫吸附试剂盒法检测乙酰胆碱含量.用方差分析和t检验对数据进行统计学分析.结果 (1)皮层损伤情况:与Sham组相比,HIBD组异常神经元所占比例明显增多[(80.28±4.51)%与(10.40±2.17)%,t=39.491,P<0.01].HIBD+DI组动物大脑皮质区核固缩及坏死细胞比例较和HIBD+NS组显著降低[(31.91±3.05)%和(82.01±7.20)%,t=18.123,P<0.01)].(2)DDR1和Tau蛋白磷酸化水平比较:DDR1磷酸化水平方面,HIBD组高于Sham组(0.922±0.199与0.095±0.023,t=10.379,P<0.01),HIBD+NS组高于HIBD+DI组(1.200±0.171与0.255±0.111,t=11.901,P<0.01).Tau磷酸化方面也显示相似的趋势(HIBD组和Sham组分别为,0.194±0.224与0.919±0.228,t=7.347;HIBD+DI组和HIBD+NS组分别为1.100±0.167与0.291±0.210,t=9.447,P值均<0.01).(3)乙酰胆碱含量:HIBD组低于Sham组[(3.685±0.472)与(7.429±0.861)ng/g蛋白,t=10.781,P<0.01].而HIBD+DI组高于HIBD+NS组[(7.058±0.915)与(2.521±0.723)ng/g蛋白,t=10.989,P<0.01].结论 DDR1激活引起HIBD新生大鼠皮层组织中Tau过度磷酸化,进而导致神经递质乙酰胆碱释放减少.抑制DDR1活化能够降低HIBD新生鼠皮层组织中Tau的磷酸化水平,增加乙酰胆碱的释放.DDR1抑制剂对HIBD新生鼠脑损伤具有保护作用.

摘要： 本发明属于生物医药领域，具体涉及视网膜新生血管疾病动物模型、构建方法及其应用。本发明采用源于M1型活化小胶质细胞的外泌体，将该外泌体注射于动物的眼球组织，诱导产生视网膜新生血管，从而构建视网膜新生血管疾病动物模型，本发明利用M1型活化小胶质细胞的外泌体直接促进新生血管产生，同时又促进原位静息的小胶质细胞活化及富集，促进血管信号而间接促进视网膜新生血管的产生。这种炎症‑新生血管的病理模式符合大多数视网膜新生血管产生的病理过程，使得本发明构建的视网膜新生血管疾病动物模型具有良好的代表性、稳定性和病理持续性，具有极高的推广应用价值。

摘要： 本发明公开了一种在筛选新生抗原的疫苗或药物时评价动物模型的引物和探针，创建了其检测方法，并将其制作成试剂盒，该试剂盒通过检测人源肿瘤细胞来筛选和评价新抗原多肽疫苗、核酸疫苗及新抗原反应性细胞药物等动物模型的规范化和一致性，试剂盒操作简单、灵敏度高，灵敏度可达到单细胞水平。

摘要： 本发明公开了及一种利用基因操作技术构建视网膜新生血管疾病动物模型的方法、培育方法和应用，涉及基因编辑技术领域。本发明公开的构建方法利用基因操作技术使目标动物的血管内皮细胞中的Ctnnd1基因不表达或者其表达受到抑制。通过该方法可以得到视网膜新生血管疾病动物模型，丰富了视网膜新生血管疾病动物模型的种类，为视网膜新生血管疾病的相关研究提供了更多样的模型选择，也有利于促进视网膜新生血管疾病的科学研究和相关治疗药物的开发等。

摘要： 本申请提供了一种新生动物神经元新生日期的检测方法，包括：根据怀孕动物的体重配制含5‑乙炔基‑2’脱氧尿嘧啶核苷的溶液，其中溶液中5‑乙炔基‑2’脱氧尿嘧啶核苷的质量为10mg/kg体重‑25mg/kg体重；将溶液注射至怀孕动物体内，记录注射时怀孕动物的怀孕天数；饲养怀孕动物至分娩，得到新生动物；对新生动物进行处理，得到新生动物的脑切片；对脑切片进行染色，根据注射时间和染色的结果，获得新生动物的神经元新生日期。该方法操作简单，新生动物的存活率高，检测结果可信度高，对于研究动物的脑部以及神经元发育过程具有重要的意义。本申请还提供了5‑乙炔基‑2’脱氧尿嘧啶核苷标记胚胎的方法。

摘要： 本发明提供了一种构建视网膜新生血管性疾病动物模型的方法，属于动物模型领域。本发明通过基因修饰的方法成功构建了一种可诱导的血管内皮细胞特异性过表达Twist1的转基因小鼠模型(Tg‑Twist1iEC+小鼠)，该小鼠模型由于内皮细胞中Twist1的过度表达，导致小鼠视网膜血管生成异常和病理性新生血管形成。本发明构建的转基因小鼠模型由于血管内皮细胞中Twist1的过度表达，促进了血管内皮细胞增殖，并破坏了血管内皮细胞极性；该转基因小鼠模型促进了神经胶质活化，VEGFa分泌增加，进而加速病理性血管生成。该模型可以用于进一步研究病理性新生血管相关视网膜疾病的发病机理，筛选预防和/或治疗视网膜新生血管性疾病的药物。

摘要： 本发明涉及一种新生反刍动物肠道干细胞增值试验用肠道清洁装置。该肠道清洁装置包括无菌清洗箱，所述无菌清洗箱的上端设置有开口，开口处设置有箱盖，所述无菌清洗箱的内部设置有用于拉持固定在肠道两侧的第一清洗组件、第二清洗组件，肠道被固定在第一清洗组件、第二清洗组件上，第一清洗组件、第二清洗组件可通过驱动组件进行驱动，从而带动肠道拉伸、收缩，实现高效清理肠道的目的，且本装置适用于体外肠道的清洗，便于将肠道清洗充分后用于实验研究，代替了人工清洗的方式，清理效率高，且保持了肠道的无菌清洗环境，可提高实验的准确性。

摘要： 本发明涉及一种用于新生反刍动物肠道干细胞增值试验的无菌实验箱。该无菌实验箱包括无菌实验箱，无菌实验箱的一侧设置有进出箱，肠道在放置在承载组件中时首先开合进出箱远离无菌实验箱一侧的升降式挡板，然后关闭第一进出口，利用进出箱内部的第二杀菌灯对承载组件上的肠道进行消毒杀菌，避免外部因素或者其他细菌等影响观察数据，当肠道消毒杀菌之后，打开靠近无菌实验箱一侧的升降式挡板，承载组件整体进入进出箱的内部，然后关闭第二进出口，此时，肠道在无菌的无菌实验箱中进行观察，而显微镜的一端位于无菌实验箱的外部，显微镜的另一端位于无菌实验箱的内部，显微镜可转动调节，方便从外部直观性的观察无菌实验箱内部的肠道干细胞。

摘要： 本发明提供了一种角膜新生血管动物模型及其构建方法，该动物模型包括在小鼠的角膜中央切口，并在角膜缘的正前方做潜行隧道，将含血管内皮生长因子的滤纸植入隧道顶端，构建得到角膜新生血管动物模型；在小鼠的角膜中央切口之前，用戊巴比妥钠溶液注射小鼠的腹腔，爱尔凯因滴眼液麻醉小鼠的双眼表面；本发明的滤纸较薄、制作时间短，易于手术过程中植入，造模成功率高，从而增加了造模的可重复性，大大降低了成本的投入；本发明使用滤纸结合血管生长因子植入小鼠角膜层间，诱导小鼠角膜新生血管模型，可用于小鼠角膜，具有造模时间短、成本低、创伤小、且可重复性高的特点，为研究角膜新生血管抑制剂提供稳定、可靠的模型体系。

摘要： 本发明属于生物医药领域，具体涉及视网膜新生血管疾病动物模型、构建方法及其应用。本发明采用源于M1型活化小胶质细胞的外泌体，将该外泌体注射于动物的眼球组织，诱导产生视网膜新生血管，从而构建视网膜新生血管疾病动物模型，本发明利用M1型活化小胶质细胞的外泌体直接促进新生血管产生，同时又促进原位静息的小胶质细胞活化及富集，促进血管信号而间接促进视网膜新生血管的产生。这种炎症‑新生血管的病理模式符合大多数视网膜新生血管产生的病理过程，使得本发明构建的视网膜新生血管疾病动物模型具有良好的代表性、稳定性和病理持续性，具有极高的推广应用价值。

摘要： 本实用新型公开了一种新生动物用独立通风育婴箱，其技术方案要点是：包括箱体，所述箱体的一侧开设有放置槽，所述放置槽的内部活动套设有箱门；安装孔，所述安装孔开设在所述箱体的顶面；通风组件，所述通风组件设置在所述安装孔的内部，用于通风，当育婴箱需要换气时，通过气泵使外界的空气进入箱体的内部，通过过滤层对空气中的灰尘进行过滤，避免灰尘进入箱体的内部，当气泵工作时，通过开启负离子发生器产生负离子，通过负离子与空气中的细菌结合后，使细菌产生结构的改变或能量的转移，导致细菌死亡，使空气中细菌迅速减，同时负离子可与空气中的尘埃结合沉淀，达到出尘的目的，避免空气中的细菌进入箱体的内部，影响幼崽的健康。