15-HETE影响大鼠脑动脉平滑肌细胞内钙离子浓度的机制

目的 观察15-羟化二十烷四烯酸(15-HETE)对脑动脉平滑肌细胞内钙离子浓度([Ca2+]i)的影响,进一步探讨15-HETE引起[Ca2+]i变化的钙来源,从而明确15-HETE引起脑动脉平滑肌收缩的机制. 方法 酶法分离大鼠脑动脉平滑肌细胞,分为15-HETE组与对照组,15-HETE组添加15-HETE处理;对照组正常培养,不做其他处理.激光共聚焦技术测定15-HETE对[Ca2+]i的影响;进一步通过阻断外钙内流和耗竭内钙,探明15-HETE引起钙动员的来源;应用血管环技术从功能上判定细胞外钙对15-HETE引起的颈内动脉环收缩有无影响. 结果 15-HETE组与对照组相比,[Ca2+]i明显增加,差异有统计学意义(P＜0.05);预先加入硝苯地平、镧离子及改用无钙液阻断外钙内流后,15-HETE组[Ca2+]i仍明显高于对照组,差异有统计学意义(P＜0.05);而预先加入咖啡因耗竭细胞内钙后,15-HETE组[Ca2+]i较对照组差异无统计学意义(P＞0.05);采用无钙液去除细胞外钙后,15-HETE引起的血管环张力增加与有钙液中比较差异无统计学意义(P＞0.05). 结论 15-HETE可通过促使内钙释放而使[Ca2+]i增加,进而引起大鼠脑动脉平滑肌收缩.     

脑微血管内皮细胞缺氧模型转化生长因子β1表达与脑溢安的干预

背景：临床及动物实验证实脑溢安能促进脑出血急性期血肿吸收及神经功能恢复，提高生活质量。 目的：观察脑溢安血清对体外缺氧培养的大鼠脑微血管内皮细胞转化生长因子β1 mRNA表达的影响。 方法：用分离培养的大鼠脑微血管内皮细胞移入厌氧培养箱培养18 h建立缺氧损伤模型，并随机分为正常组、模型组、正常血清组及含脑溢安血清组。正常组为同一批正常培养的脑微血管内皮细胞；模型组将正常培养的细胞放入厌氧培养箱内培养18 h；正常血清对照组细胞在培养液中加5%正常血清后，放入厌氧培养箱中培养18 h；脑溢安血清组细胞在培养液中加5%脑溢安血清后，放入厌氧培养箱内培养18 h。 结果与结论：缺氧培养使脑微血管内皮细胞存活数量减少，其表达的转化生长因子β1 mRNA增强，而脑溢安血清能明显增加脑微血管内皮细胞存活数量，降低转化生长因子β1 mRNA表达水平(P < 0.05)。说明脑溢安血清下调转化生长因子β1mRNA的表达可能是其抑制脑微血管内皮细胞的凋亡对缺氧损伤脑微血管内皮细胞的保护作用机制之一。     

低氧预处理中Kv1.4和Kv4.2对海马和皮质的保护作用及其机制的研究

目的观察低氧预处理中Kv1.4和Kv4.2对海马和皮质的保护作用及机制。方法将35只雄性Wister大鼠随机分成低氧预处理组、严重低氧组和空白组。采用低压氧舱造模,低氧预处理组先置于低压氧舱4000米,每天2 h,连续3 d后再置于7000米,3 h;严重低氧组置于低压氧舱7000米,3 h;空白组不给予低氧处理。造模成功后于0 h、3 h、24 h 3个时间点,分别取材海马和皮质,采用Q-PCR的方法分别检测各组Wister大鼠海马和皮质Kv1.4和Kv4.2的基因表达,采用统计软件进行数据分析。结果 (1)低氧预处理组、严重低氧组大鼠海马中KV1.4和Kv4.2、大鼠皮质中Kv4.2的mRNA表达,在3 h、24 h均较空白组高,大鼠皮质中Kv1.4的mRNA表达在24 h高于空白组,有显著性差异(P<0.01),说明低氧时出现脑损害。(2)低氧预处理组大鼠海马和皮质中Kv1.4和Kv4.2的mRNA表达,与严重低氧组比较有下降,尤其是24 h明显降低,有显著性差异(P<0.01),说明低氧预处理过程可抑制Kv1.4和Kv4.2的mRNA过度表达,具有脑保护作用。结论低氧预处理对脑的保护作用可能与抑制Kv1.4和Kv4.2的mRNA表达有关。     

束缚应激上调小鼠脾脏淋巴细胞钾离子通道(Kv1.3)的表达

目的观察束缚应激条件下小鼠脾脏细胞Kv1.3表达的变化。方法采用RT-PCR、Western blot等分子生物学及免疫组化实验方法,分别从基因和蛋白质水平揭示束缚应激对小鼠脾脏细胞Kv1.3表达的调节。结果束缚应激16 h后小鼠脾脏细胞Kv1.3 mRNA和蛋白质表达水平明显上调(P<0.05)。但是,相同束缚应激条件下小鼠脑组织Kv1.3表达无明显变化(P>0.05)。结论束缚应激状态下小鼠脾脏细胞Kv1.3 mR-NA和蛋白质表达水平呈明显组织特异性上调,提示束缚应激条件下免疫功能的调节可能与钾通道有关。这为进一步研究神经内分泌系统调节淋巴细胞功能的机制提供了新的思路。     

钾通道Kv1.2与致痫大鼠发病相关性的研究

目的通过检测戊四唑(PTZ)致痫大鼠钾通道Kv1.2蛋白表达,探讨钾通道Kv1.2与癫痫发病的相关性。方法 40只SD大鼠分成实验组30只和正常对照组10只。实验组30只大鼠通过腹腔注射PTZ建立全身强直阵挛发作大鼠癫痫模型,取成功致痫鼠24只均分成3组,分别于致痫后3个时间段(1h、24h、48h)取脑组织。用免疫组化法和Western blot法检测大鼠钾通道Kv1.2蛋白。结果实验组大鼠海马区钾通道Kv1.2蛋白表达水平在致痫后3个时间段(1h、24h、48h)均明显低于对照组(P0.05)。实验组大鼠海马区钾通道Kv1.2蛋白表达水平在致痫后3个时间段(1h、24h、48h)之间无显著性差异(P0.05)。结论大鼠海马区钾通道Kv1.2表达的减少与全身强直阵挛发作大鼠癫痫发病密切相关。     

电压门控钾通道Kv1.1在戊四唑致痫大鼠中的表达

目的通过检测戊四唑(PTZ)致痫大鼠钾通道Kv1.1蛋白表达,探讨钾通道Kv1.1与癫痫发病的相关性。方法40只SD大鼠分成实验组30只和正常对照组10只。实验组30只大鼠通过腹腔注射PTZ建立全身强直-阵挛发作大鼠癫痫模型,取成功致痫鼠24只均分成3组,分别于致痫后3个时间段(1h、24h、48h)取脑组织。用免疫组化法和Westernblot法检测大鼠钾通道Kv1.1蛋白。结果实验组大鼠海马区钾通道Kv1.1蛋白表达水平在致痫后3个时间段(1h、24h、48h)均明显低于对照组(P0.05),但3个时间段之间钾通道Kv1.1蛋白表达水平差异无统计学意义(P0.05)。结论大鼠海马区钾通道Kv1.1表达的减少与全身强直阵挛发作大鼠癫痫发病密切相关。     

钾通道Kv4.2、Kv1.4在硫化氢后处理对大鼠短暂全脑缺血神经保护中的作用研究

目的研究硫氢化钠(sodium hydrosulfide,Na HS)后处理对短暂全脑缺血大鼠海马中钾通道Kv4.2和Kv1.4 mRNA表达变化的影响及其脑保护作用,从而探讨Na HS对大鼠短暂全脑缺血神经保护作用的机制。方法用4VO方法建立大鼠短暂性全脑缺血(transient global cerebral ischemia,t GCI)模型,大鼠被随机分配到3组,分别为:假手术组(sham)、t GCI组、Na HS后处理组。Na HS后处理组为t GCI之后1 d,给予大鼠腹腔注射Na HS 24μmmol/kg或者180μmmol/kg。通过尼氏染色与Neu N免疫染色确定海马神经元的死亡,通过RT-PCR方法检测海马组织Kv4.2和Kv1.4mRNA水平的表达变化。结果 (1)与t GCI组比较,在t GCI之后1 d给予24μmol/kg Na HS后处理使海马CA1区存活细胞数目显著增加,而高剂量的Na HS(180μmol/kg)后处理对t GCI大鼠海马CA1区则无明显的保护作用。(2)在Re 26 h和Re 48 h,海马组织中Kv4.2、Kv1.4的mRNA表达水平均明显低于假手术组(P<0.05)。在Re 26 h+Na HS组,kv4.2(1.24±0.08)和kv1.4(1.11±0.07)的mRNA表达水平均分别高于Re 26 h组的kv4.2(0.75±0.04)和kv1.4(0.79±0.06),差异均有显著性(P<0.05)。结论外源性Na HS可能通过上调大鼠t GCI后海马区Kv4.2和Kv1.4 mRNA的表达,从而导致膜电位超极化,降低神经元兴奋性和氧耗,继而保护神经元免受脑缺血损伤。     

脑缺氧缺血后新生大鼠bcl-2 mRNA的表达

目的　探讨新生大鼠脑缺氧缺血后bcl 2mRNA表达的变化及其与脑缺氧缺血所致细胞凋亡的关系。方法　通过建立新生大鼠缺氧缺血性脑病动物模型 ,应用快速竞争性RT PCR技术对缺氧缺血后不同时间点的实验侧大脑组织中bcl 2mRNA的表达进行半定量分析 ,并在相同缺血基础上观察缺氧 1 5h、2 .5h和 3 5h对bcl 2mRNA表达的影响。结果　缺氧缺血后 ,新生大鼠脑bcl 2mRNA的表达自缺氧结束后 6h开始明显增强 ,1 2h达高峰 ,2 4h回落至较低水平 ,至 72h已基本检测不到。随着缺氧时间的延长即缺氧程度的加重 ,bcl 2mRNA的表达有增强趋势 ,但在缺氧 1 5h组和 2 5h组之间无显著性差异 ,而缺氧 1 5h组或 2 5h组与缺氧 3 5h组之间均具显著性差异 (P <0 0 1 ) ,后者表达更强。结论　缺氧缺血可诱导新生大鼠脑bcl 2mRNA的表达。在一定范围内 ,缺氧越严重 ,其表达越强。但由于严重损伤时蛋白质的合成常受到抑制 ,因此在判断bcl 2mRNA与凋亡的关系时必须结合其产物bcl 2蛋白的合成情况进行分析。     

银杏叶提取物对脑动脉粥样硬化大鼠血清TG、TC和HO-1水平的影响

目的 探讨银杏叶提取物对脑动脉粥样硬化大鼠血清甘油三酯(Triglyceride,TG)、总胆固醇(Total cholesterol,TC)和血红素氧合酶-1(Heme oxygenase-1,HO-1)水平的影响。方法 30只SD健康雄性大鼠,20只大鼠建立老龄脑动脉粥样硬化模型,分为假手术组、模型组和银杏叶组; 检测血清TG、TC、高密度脂蛋白胆固醇(High density lipoprotein cholesterol,HDL-C)、HO-1水平、动脉硬化指数(Arteriosclerosis index,AI); 采用MTT检测细胞增殖能力,流式细胞仪检测脑动脉平滑肌细胞凋亡,Western blot法检测Caspase-3、Bcl-2、Bax蛋白水平。结果 与模型组比较,银杏叶组脑动脉斑块面积、新生内膜面积、内膜厚度降低,管腔面积升高(P<0.05); 与模型组比较,银杏叶组TG、HDL-C水平、AI降低,TC、HO-1水平升高(P<0.05); 与模型组比较,银杏叶组24、48、72 h细胞凋亡率降低(P<0.05),银杏叶组24 h细胞凋亡率低于48和72 h细胞凋亡率(P<0.05); 与模型组比较,银杏叶组48、72 h细胞增殖率升高(P<0.05),银杏叶组24h细胞增殖率低于48和72 h细胞增殖率(P<0.05); 与模型组比较,银杏叶组Caspase-3、Bax水平降低,Bcl-2水平升高(P<0.05)。结论 银杏叶提取物能够改善脑动脉粥样硬化大鼠血清TG、TC、HO-1水平,并通过下调Caspase-3、Bax蛋白表达,上调Bcl-2表达来促进脑动脉平滑肌细胞增殖,抑制脑动脉平滑肌细胞凋亡。     

Viral gene transfer of dominant-negative Kv4 construct suppresses an O2-sensitive K+ current in chemoreceptor cells.

Hypoxia initiates the neurosecretory response of the carotid body (CB) by inhibiting one or more potassium channels in the chemoreceptor cells. Oxygen-sensitive K(+) channels were first described in rabbit CB chemoreceptor cells, in which a transient outward K(+) current was reported to be reversibly inhibited by hypoxia. Although progress has been made to characterize this current with electrophysiological and pharmacological tools, no attempts have been made to identify which Kv channel proteins are expressed in rabbit CB chemoreceptor cells and to determine their contribution to the native O(2)-sensitive K(+) current. To probe the molecular identity of this current, we have used dominant-negative constructs to block the expression of functional Kv channels of the Shaker (Kv1.xDN) or the Shal (Kv4.xDN) subfamilies, because members of these two subfamilies contribute to the transient outward K(+) currents in other preparations. Delivery of the constructs into chemoreceptor cells has been achieved with adenoviruses that enabled ecdysone-inducible expression of the dominant-negative constructs and reporter genes in polycistronic vectors. In voltage-clamp experiments, we found that, whereas adenoviral infections of chemoreceptor cells with Kv1.xDN did not modify the O(2)-sensitive K(+) current, infections with Kv4.xDN suppressed the transient outward current in a time-dependent manner, significantly depolarized the cells, and abolished the depolarization induced by hypoxia. Our work demonstrate that genes of the Shal K(+) channels underlie the transient outward, O(2)-sensitive, K(+) current of rabbit CB chemoreceptor cells and that this current contributes to the cell depolarization in response to low pO(2).     

15d-PGJ2对糖尿病大鼠脑缺血再灌注损伤小胶质细胞活化及神经细胞凋亡的影响

目的 观察PPAR-γ激动剂15d-PGJ2对糖尿病脑缺血再灌注大鼠脑缺血再灌注损伤小胶质细胞活化及神经细胞凋亡的影响。方法 成年SD大鼠80只,随机分为4组:(1)假手术组;(2)正常血糖脑缺血组;(3)糖尿病脑缺血组;(4)糖尿病脑缺血+15d-PGJ2干预组。采用链脲佐菌素诱导糖尿病,应用改良的Zea-Longa法制作大鼠大脑中动脉闭塞再灌注模型。糖尿病脑缺血组+15d-PGJ2干预组在成功制备糖尿病大鼠模型后给予15d-PGJ2 200 μg·kg^-1·d^-1腹腔注射21 d后应用改良的Zea-Longa法制作大鼠大脑中动脉闭塞再灌注模型,再灌注后3 h腹腔注射15d-PGJ2 400 μg·kg^-1,以后6 d每天给予15d-PGJ2 200μg·kg^-1·d^-1腹腔注射。每组分别于24 h、7 d各处死一批大鼠,并随机分为2组:一组行免疫组化法检测小胶质细胞CD68的表达水平及ELISA检测TNF-α与IL-1β水平,另一组用TUNEL法原位标记DNA片段检测凋亡细胞计数。结果 正常血糖脑缺血组、糖尿病脑缺血组、糖尿病脑缺血+15d-PGJ2干预组与假手术组比较,再灌注24 h、再灌注7 d CD68阳性面积、TNF-α与IL-1β水平、神经细胞凋亡率均明显增加(P<0.05); 糖尿病脑缺血组在再灌注24 h、再灌注7 d CD68阳性面积、TNF-α与IL-1β水平、神经细胞凋亡率明显高于正常血糖脑缺血组(P<0.05); 糖尿病脑缺血+15d-PGJ2干预组再灌注24 h、再灌注7 d CD68阳性面积、TNF-α与IL-1β水平、神经细胞凋亡率低于未干预组(P<0.05)。结论 糖尿病脑缺血组与正常血糖脑缺血组相比较CD68阳性面积更大、TNF-α与IL-1β水平更高及神经细胞凋亡率更高; 15d-PGJ2可减少糖尿病脑缺血大鼠小胶质细胞激活、减少炎症因子分泌、降低神经细胞凋亡率。     

Expression of bone morphogenetic protein 7 in the cerebral cortex of rats after ischemia-hypoxic injury

BACKGROUND: Some researches demonstrate that exogenous bone morphogenetic protein 7 (BMP-7) can protect ischemic cerebral nerve tissue and promote recovery of motor energy function; however, there is lack of direct evidences of endogenous BMP-7 effect. OBJECTIVE: To observe the expression of endogenous BMP-7 in nerve tissue with ischemic-hypoxic injury and investigate the possible effects on damaged nerve tissue. DESIGN: Observational contrast animal study. SETTING: Department of Anatomy and Histoembryology, Peking University Health Science Center. MATERIALS: The experiment was carried out in the Nerve Researching Laboratory of Anatomy Department, Peking University Health Science Center from October 2006 to March 2007. A total of 25 adult male SD rats weighing 250–300 g and several newborn SD rats were selected from Experimental Animal Center, Peking University Health Science Center. Rabbit-anti-BMP-7 polyclonal antibody was provided by Wuhan Boster Company. METHODS: ① Adult rats were randomly divided into ischemia group (n =10), sham operation group (n = 10) and normal group (n =5). Right external-internal carotid artery occlusion was used to infarct middle cerebral artery of adult rats in the ischemia group so as to copy focal cerebral infarction models. Line cork was inserted in crotch of internal and external carotid artery of adult rats in the sham operation group, while adult rats in the normal group were not given any treatments. ② Cerebral cortex of newborn rats was separated to obtain cell suspension. Cells which were cultured for 10 days were divided into control group and hypoxia/reoxygenation group. And then, cells in the hypoxia/reoxygenation group were cultured in hypoxic incubator for 4 hours and given reoxygenation for 24 hours. MAIN OUTCOME MEASURES: Immunohistochemical method was used to measure expression of BMP-7 in cerebral cortex at 24 hours after ischemia/reperfusion culture and in primary hypoxic culture. RESULTS: ① At 24 hours after cerebral ischemia, expression of BMP-7 in cerebral cortex on ischemic side was stronger than that on non-ischemic side in adult rats; meanwhile, numbers of cell expression were increased. However, expression of BMP-7 was not detected in bilateral cerebral cortex of adult rats in both control group and sham operation group. ② After hypoxia of cerebral cortex in primary culture, positive products of BMP-7 were observed in plasma of neuron, but expression of BMP-7 was not found in normal cerebral cortex. CONCLUSION: Endogenous BMP-7 has protective effects on nerve tissue induced by ischemic-hypoxic injury.     

Immunohistochemical study on the distribution of six members of the Kv1 channel subunits in the rat basal ganglia

The differential expression of specialized voltage-gated potassium (Kv) channel subtypes in the nervous system probably reflects the wide range of functions. Although there have been previous reports in the cellular and subcellular localizations of various Kv mRNAs and proteins, the comprehensive study described here is the first in which the expression of six Kv1 channel subunits have been directly compared in the rat basal ganglia. In the present study, we have found that staining patterns of the six Kv1 channel subunits overlap in some areas of the basal ganglia, but each has a unique pattern of expression. It was noted that Kv 1.4 subunit had a strikingly high level of expression in the globus pallidus compared to the caudate-putamen. This distinct distribution formed the clear demarcations between caudate-putamen and globus pallidus. The dot-like staining pattern of Kv1 subunits was observed through the accumbens nucleus. Strong staining for Kv1.4 was observed in the cerebral peduncle, not in the subthalamic nucleus. In the substantia nigra, immunoreactivity for Kv1.4 subunit was prominent in the pars reticulata of the substantia nigra. The staining intensity for Kv1.2 was high in the pars compacta of the substantia nigra. Our immunohistochemical results may support the notion that the formation of heteromultimeric Kv channels possibly represents an important contribution to the generation of Kv channel diversity in the brain, especially in the basal ganglia.