Influence of perinatal transient hypoxia on mRNA expression of neurogenic differentiation in rats

目的:观察短暂性缺氧后鼠脑神经源性分化因子(NeuroD)表达量的变化,探讨其在神经系统再生中的可能作用.方法:通过延迟剖宫产术建立胎鼠宫内窘迫模型,原位杂交技术检测不同时间海马结构NeuroD mRNA表达量的变化.结果:原位杂交结果显示,模型组无论在齿状回颗粒下层(SGZ)还是CA1区,NeuroD在P13、P20、P27 d的表达量与对照组相比均增高.结论:短暂性缺氧后诱导NeuroD mRNA增高,可能参与了神经系统的再生.     

神经源性分化因子在新生大鼠短暂脑缺氧中的表达

目的 观察新生大鼠短暂性缺氧后脑神经源性分化因子(NeuroD)表达的变化.方法 新生10～24h的SD大鼠短暂置于100%氮气环境中,建立缺氧窒息模型.33只新生大鼠随机分为对照组、缺氧10min组、缺氧20min组,采用免疫荧光法和Western blotting检测NeuroD表达的变化.结果 在大脑皮质区和海马齿状回可见NeuroD免疫荧光染色阳性细胞;Western blotting分析显示,随缺氧时间的延长,NeuroD的表达量明显增加,缺氧10min组与对照组、缺氧20min组与缺氧10min组比较,差异有统计学意义(P<0.01).结论 短暂缺氧可引起NeuroD表达量的增加,表达部位主要在大脑皮质、海马等神经干细胞/神经前体细胞聚集区.     

短暂性缺氧后海马神经元神经源性分化因子表达增高

目的 观察短暂性缺氧后神经源性分化因子(NeuroD)表达量的变化,探讨其在神经系统再生中的可能作用.方法 体外培养海马神经元,经神经元特异性烯醇化酶(NSE)免疫组织化学方法、尼氏体染色法鉴定.将培养5d的神经元置于三气培养箱(37℃、94%N2、5%CO2、1%O2)内缺氧培养.RT-PCR检测缺氧3h和6h后神经...     

RT-PCR法检测神经源性分化因子mRNA在大鼠脑发育过程中的表达

目的 观察大鼠不同发育时期脑内神经源性分化因子（NeuroD）mRNA表达量的变化,探讨其对神经细胞的分化和成熟发挥的作用。方法 提取不同发育阶段大鼠不同脑组织中的mRNA,RT-PCR法反转录扩增NeuroD和β-actin,利用凝胶成像系统检测其相对表达量。 结果 NeuroD在受孕7.5d（E7.5）时出现表达,在E10.5和E21.5时分别达到两次高峰,平均吸光度值为20437.88±598.28和14482.23±1134.49,表达水平显著高于其他各组（P<0.01）,NeuroD/β-actin比值在E12.5、E18.5和E21.5分别为1.59±0.09、1.61±0.07和1.70±0.11,显著高于其他时间段（P<0.01）。 结论 在大鼠脑发育过程中,NeuroD mRNA的表达呈明显时间特异性,在E10.5左右大鼠各原始脑泡的形成过程中,NeuroD-mRNA明显增高;在胚胎发育的晚期,脑组织形态进一步完善和成熟的过程中NeuroD达到第二次表达高峰,与鼠脑的发育过程相符。推测NeuroD对鼠脑的早期神经细胞分化以及晚期细胞的成熟均起一定作用。     

小鼠诱导性多能干细胞移植治疗宫内缺氧新生鼠脑损伤

目的:研究小鼠诱导性多能干细胞(iPSCs)移植对宫内缺氧新生鼠脑损伤的治疗作用及机制。方法:于孕14 d开始孕鼠置于动物缺氧培养箱中,制作胎鼠宫内缺氧模型。孕鼠分娩后新生鼠经侧脑室注入BrdU标记的iPSCs悬液移植组和PBS缺氧组,对照组不治疗,H-E染色观察脑组织形态学变化、八臂迷宫测试动物记忆功能、免疫组织化学检测脑BrdU阳性细胞和神经颗粒素(Ng)和神经元烯醇化酶(NSE)的表达、原位杂交检测血管内皮生长因子mRNA(VEGF mRNA)的表达。结果:移植组小鼠的学习记忆能力和脑皮质区细胞结构较缺氧组均明显改善;脑切片检出BrdU阳性细胞存在,缺氧组Ng和NSE阳性细胞表达较对照组显著减少,而VEGF mRNA的表达较对照组显著增加;移植组Ng、NSE和VEGF mRNA表达均较缺氧组显著增加。结论:小鼠iPSCs移植于宫内缺氧的新生小鼠脑中,可减轻缺氧神经元的损伤和提高学习能力,其机制可能通过上调VEGF mRNA和Ng的表达有关。     

宫内窘迫后胎鼠肾脏转化生长因子β1表达变化的研究

目的研究宫内窘迫后胎鼠肾脏转化生长因子β1(TGF-β1)的动态变化,探索TGF-β1在围产期窒息后肾损伤发生发展中的可能作用.方法通过钳夹孕21d大鼠供应子宫的血管制成胎鼠宫内不同程度缺血缺氧模型和再灌注不同时间模型.采用Western杂交技术检测胎肾组织TGF-β1蛋白表达变化;同时观察肾组织形态学改变.结果假手术组肾组织TGF-β1蛋白表达较弱,在缺血15min再灌注过程中,肾组织TGF-β1蛋白表达呈逐渐增加趋势,15h明显增加(P<0.05),30h达最高水平(P<0.01).组织学改变:缺血15min再灌注15h病理改变最严重,此后恢复,至30h时肾小管仅余有轻微空泡变性.结论宫内急性缺血缺氧后再灌注过程中,胎肾组织病理学改变呈进行性加重一段时间后逐渐恢复;而肾组织TGF-β1产物持续增加,提示TGF-β1可能参与肾损伤的修复.     

当归对宫内缺氧新生大鼠神经元的影响及机制

目的:探讨宫内缺氧对新生大鼠神经元分化的影响及当归的调控作用.方法:将孕SD大鼠随机分为对照组、缺氧组和当归治疗组.于孕14d开始将当归组与缺氧组孕鼠置于低张氧浓度三气培养箱中,制作胎鼠宫内缺氧模型.3组孕鼠分娩当日取脑组织行神经元烯醇化酶(NSE)和血管内皮生长因子(VEGF)原位杂交,DAB显色.结果:缺氧组新生鼠NSE阳性细胞表达较对照组减少,而VEGF mRNA的表达较对照组增加;当归组NSE mRNA和VEGF mRNA阳性细胞表达较缺氧组增加.结论:当归注射液对宫内缺氧新生大鼠脑组织具有保护作用,其作用机制可能是通过上调VEGF mRNA的表达而改善缺氧环境所致.     

大鼠急性脊髓损伤后NeuroD的时空表达

目的:探讨神经源性分化因子NeuroD在大鼠急性脊髓损伤过程中的表达及意义。方法:以改良Allen's法建立急性脊髓损伤(SCI)大鼠模型,以假手术组为对照,应用免疫组织化学、半定量RT-PCR、免疫印迹分别检测脊髓损伤后不同时间点NeuroD的表达变化。结果:NeuroD特异性表达于脊髓灰质,与对照组相比,急性脊髓损伤后3 d,NeuroD mRNA的相对表达量明显增加,5 d达到峰值,7 d后回落;Neurod的蛋白相对表达量则在急性脊髓损伤5 d后明显升高,持续至第7天,14 d明显回落。结论:急性脊髓损伤可上调NeuroD表达水平,提示NeuroD参与了急性脊髓损伤后的病理过程。     

围产期缺氧缺血性脑损伤时基膜与TPA变化的相关性

为了探讨缺氧缺血后基膜与脑组织纤溶酶原激活剂变化的相关性。本实验通过“延迟剖宫产术”致胎鼠宫内窘迫 ;实验分空白对照组、缺氧缺血 15 min组和缺氧缺血 3 0 min组。上述三组分别取额、顶叶 ,透射电镜下观察血脑屏障的变化 ,每组各取8例测试脑组织纤溶酶原激活剂的活性。结果显示 :缺氧缺血 15 min时部分星形胶质细胞足板肿胀、血管周隙扩张 ;缺氧缺血 3 0min,基膜样物质减少 ,神经元显著肿胀。缺氧缺血 15 min、3 0 min,组织纤溶酶原激活剂活性升高 ,并且随缺氧缺血时间的延长 ,组织纤溶酶原激活剂呈增高趋势 ,经 t检验 ,P<0 .0 1。以上结果表明 :缺氧缺血后脑组织纤溶酶原激活剂活性增高 ,引起毛细血管基膜降解 ,打开血脑屏障 ,导致脑水肿。组织纤溶酶原激活剂是脑缺氧缺血引起神经元不可逆损伤的一个重要媒介     

三七总皂苷改善宫内缺氧新生鼠脑损伤及学习记忆功能

目的:制作胎鼠宫内缺氧模型,探讨三七总皂苷对宫内缺氧新生大鼠突触重建及学习记忆功能的影响。方法:孕14 d开始将孕鼠置于动物缺氧培养箱中,制作胎鼠宫内缺氧模型。新生鼠经腹腔分别注入三七总皂苷和生理盐水;正常对照组不治疗。免疫荧光法检测脑微管相关蛋白-2(MAP-2)、胶质纤维酸性蛋白(GFAP)、突触素(SYN)和突触分化诱导基因1(SynDIG1)的表达。Morris水迷宫实验检测各组大鼠的学习记忆功能。结果:Morris水迷宫结果表明,与对照组相比,模型组大鼠学习记忆能力明显下降,经三七总皂苷治疗后学习记忆能力明显改善。缺氧后新生鼠脑MAP-2、SYN和SynDIG1表达明显减少,GFAP表达明显增多;治疗组MAP-2、SYN、SynDIG1表达明显高于模型组,GFAP表达明显低于模型组。结论:三七总皂苷可减轻缺氧缺血后神经元的损伤,减弱GFAP的表达,促进突触的重建,改善宫内缺氧大鼠学习记忆功能。     

短暂缺氧对新生鼠脑细胞凋亡和神经再生的影响

Objective To investigate effects of transient hypoxia on the expression level of neurogenic differentiation factor (NeuroD), apoptosis and neurogenesis in the rat brain.Methods The model was established as previously described by Grojean, newborn rats were transiently exposed to 100% N2. Ninety-six animals were randomly divided into normal control group and 20min hypoxia-treated group. Rats were sacrificed at day 13, 20 and 27 post-hypoxia, and the expression levels of NeuroD and BrdU in the brain were analyzed by immunofluorescence staining and immunohistochemistry. Alternatively, the rats were sacrificed at day 6 and 20 post-hypoxia, and apoptosis in the brain was analysed with TUNEL staining. Results Immunofluorescence/immunohistochemisty analysis showed that the expression of NeuroD and BrdU significantly increased at day 13 and 27 in the 20min hypoxia-treaded group compared to that in the control group (P0.05), with the highest expression level at day 20 (P0.01). TUNEL positive cells also significantly increased at day 6 in the 20min hypoxia-treaded group, while no difference was seen between the hypoxia-treated and the control group at day 20. Conclusion The transient neuronal loss induced by birth hypoxia may lead to cell proliferation, neuron differentiation and also may trigger neurogenesis, which may in turn participate in brain repai     

短暂缺氧对3日龄SD大鼠海马胰岛素受体和β-catenin及NeuroD1表达的影响

目的:探讨短暂缺氧对3日龄SD大鼠海马胰岛素受体和β-catenin及NeuroD1表达的影响.方法:将3日龄SD大鼠置于8％O2+92％N2混合气体中2 h,建立缺氧性脑损伤模型(模型组),对照组为将3日龄SD大鼠置于缺氧箱2 h,不通入混合气体.2组大鼠分别于缺氧后2、4、24?h断头取脑,采用免疫印迹和免疫荧光法...     

Urocortin调控Bcl-2家族与大鼠缺氧/复氧心肌细胞凋亡

目的观察神经肽Urocortin对大鼠缺氧/复氧心肌细胞Bcl-2及相关基因mRNA表达和细胞凋亡的影响。方法培养新生大鼠的心肌细胞并缺氧/复氧处理,用RT-PCR检测Bcl-2、Bax、Bcl-xL、Bad mRNA表达,TUNEL法检测细胞凋亡。结果Urocortin治疗组Bcl-2的mRNA表达与缺氧/复氧组比较明显增加(P<0.05),Bax的表达明显下降(P<0.05),Bcl-xL、Bad mRNA表达无明显改变(P>0.05)。Urocortin治疗组凋亡细胞率与缺氧/复氧组比较减少(P<0.05)。结论Urocortin调节心肌细胞Bcl-2家族基因转录下凋促凋亡基因Bax表达,上调抑凋亡基因Bcl-2使Bcl-2/Bax比值增加。这可能是Urocortin抗大鼠缺氧/复氧心肌细胞凋亡的机制之一。     

Changes in c-fos mRNA in the neonatal rat brain following hypoxic ischemia

We used quantitative in situ hybridization to study changes in the expression of c-fos following hypoxic-ischemia (H-I) in the neonatal rat brain. 7-day-old rat pups were subjected to a unilateral ligation of the common carotid artery followed by a 2 h 15 min hypoxic period (7.7% O₂ in N₂). This resulted in the expected ipsilateral infarction of cortex, lateral hippocampus, lateral-superior aspects of the striatum and the white matter of the corpus callosum. Brain damage was not seen in the contralateral hemisphere subjected only to hypoxia. c-fos mRNA levels increased in the contralateral hemisphere immediately after the hypoxia and had returned towards normal levels 2 h thereafter. In the ipsilateral hemisphere, the expression of c-fos was delayed but very marked at 2 h. Animals subjected only to hypoxia showed little or no increase in c-fos mRNA. Thus the earliest recorded increase in c-fos after hypoxic ischemia, which occurred on the non-ischemic, contralateral side, may represent a generalized response to a more localized insult.     

辛伐他汀对不同氧供条件下大鼠脑微血管内皮细胞跨内皮电阻及MMP-9表达的影响

目的:观察在常氧、短暂缺氧、持续缺氧及复氧条件下,辛伐他汀对大鼠脑微血管内皮细胞(BMECs)跨内皮细胞电阻(TEER)及基质金属蛋白酶-9(MMP-9)表达的影响。方法:原代分离培养SD大鼠BMECs,随机分为常氧组、短暂缺氧组、持续缺氧组、复氧组,各组均实施辛伐他汀0.1μmol/L、1.0μmol/L、10μmol/L3种浓度预处理。观察各组细胞形态学及增殖活性变化;采用TEER评估各组通透性;免疫细胞荧光化学法测定MMP-9蛋白表达。结果:短暂缺氧、持续缺氧及复氧条件下大鼠BMECs细胞受损逐渐加重,MMP-9表达逐渐增加,TEER及细胞增殖活性逐渐下降。辛伐他汀干预能不同程度提高TEER并抑制MMP-9蛋白的表达,使细胞增殖活性提高。但这种抑制MMP-9的作用对短暂缺氧大鼠BMECs的TEER值及细胞增殖活性无显著改善。结论:辛伐他汀能通过抑制MMP-9蛋白的表达来提高持续缺氧及复氧条件下大鼠BMECs的TEER,而对短暂缺氧后TEER的改善作用不明显。     

Nitric oxide and nitric oxide synthase in the early phase of perinatal asphyxia of the rat.

The role of nitric oxide, a compound involved in neurotransmission and regulation of cerebral blood flow, in cerebral ischemia is still not fully elucidated yet. Although well studied in adult systems of cerebral ischemia/hypoxia, information on nitric oxide in perinatal asphyxia is limited and, in particular, no direct evidence for its generation has been provided. We therefore decided to study nitric oxide generation in brain of asphyctic rat pups by biophysical and biochemical methods. We used a simple, non-invasive rat model resembling the clinical situation in perinatal asphyxia: rat pups delivered by Caesarean section were placed into a water bath at 37 degrees C still in patent membranes for various asphyctic periods (up to 20 min). Brain pH, cerebral blood flow, neuronal nitrix oxide synthase messenger RNA (by northern and dot blot analysis), immunoreactive protein (by western blot analysis) and nitric oxide synthase activity were determined; generation of nitric oxide was evaluated directly by electron paramagnetic resonance spectroscopy. Neuronal nitric oxide synthase messenger RNA activity and nitric oxide generation were unaffected, whereas neuronal nitric oxide synthase-immunoreactive protein of 150,000 mol. wt was decreased and of 136,000 mol. wt was increased with the length of the asphyctic period. This is the first report on direct evidence for the generation of nitric oxide in perinatal asphyxia and we demonstrate that nitric oxide production remains unaffected even by 20 min of asphyxia, at a time-point when cerebral blood flow was increased four-fold and severe acidosis was present. However, it was found that levels of immunoreactive neuronal nitric oxide synthase of 136,000 mol. wt were increased paralleling the length of asphyxia. Levels of the 150,000 mol. wt immunoreactive neuronal nitric oxide synthase protein decreased, suggesting a different regulation pattern. Thus, the present biochemical and biophysical results form the basis for further investigations on nitric oxide in perinatal asphyxia.