Notch1信号系统在氟西汀上调大鼠海马神经再生中的作用

目的 了解慢性氟西汀干预正常大鼠所导致的海马神经再生上调与Notch1信号系统功能改变的关系.方法 应用大鼠腹腔注射氟西汀建立在体模型,分为对照组、14 d干预组、28 d干预组(n=12),采用免疫组化、real time PCR和Western blot,测定大鼠海马神经干细胞的增殖、存活和分化以及Notch1信号通路各个因子(NICD、Hes1、Hes5、Jag1)的基因及蛋白表达水平的改变.结果 ①与对照组(2919.50±188.80)比较,14 d氟西汀干预组(3706.50±228.04)、28 d氟西汀干预组(4334.33±217.48)海马齿状回神经干细胞增殖明显增加(P<0.001);与对照组(2404.50±148.77)相比,Flu干预28 d组(3273.16±156.68)海马齿状回神经干细胞存活明显增加(P<0.001);与对照组比较,氟西汀干预组NeuN/BrdU、GFAP/BrdU比例无明显差异(P＞0.05).②与对照组[NICDmRNA (0.30±0.03),Hes1mRNA (0.53±0.03),Hes5mRNA (0.21±0.02),Jag1mRNA(1.04±0.07)]比较,氟西汀(Flu)干预14d组[NICDmRNA (0.45±0.05),Hes1mRNA (0.65±0.06),Hes5mRNA (0.31±0.06),Jag1mRNA(2.46±0.39)]和Flu干预28 d组[NICDmRNA (0.42±0.03),Hes1mRNA (0.85±0.06),Hes5mRNA (0.39±0.02),Jag1mRNA(3.21±0.34)]Notch1信号通路各因子基因水平均明显升高(P<0.01或P<0.001).③与对照组[NICD(2.36±0.17),Hes1(1.09±0.25),Jag1(2.33±0.31)]比较,Flu干预14 d组[NICD(3.20±0.25),Jag1(2.86±0.25)]和Flu干预28 d组[NICD(3.40±0.19),Hes1(1.43±0.13),Jag1(3.35±0.14)]NICD、Hes1、Jag1蛋白水平明显升高,差异有统计学意义(P<0.01或P<0.001).与对照组Hes5比较,Flu干预14 d组Hes5和Flu干预28 d Hes5蛋白水平无改变,差异无统计学意义(P＞0.05).结论 氟西汀促进大鼠海马齿状回神经干细胞的增殖和存活,但对分化无影响;同时,海马Notch信号功能激活,提示Notch1信号系统可能参与氟西汀介导的大鼠海马神经再生上调.     

首乌益智胶囊对Notch通路的调控作用的研究

目的探讨首乌益智胶囊对Notch通路的调控作用。方法采用永久性双侧颈总动脉结扎法建立慢性脑缺血认知障碍大鼠模型,随机分为首乌益智胶囊组、脑复康组、模型组、假手术组,每组20只大鼠。首乌益智胶囊组大鼠给予首乌益智胶囊(1 ml/100 g),脑复康组给予脑复康(1 ml/100 g),模型组及假手术组给予等量的生理盐水灌胃,共28 d。电镜下观察海马超微结构;实时定量PCR法检测海马Notch1、Hes3及Stat3 mRNA表达。结果与假手术组比较,模型组、脑复康组和首乌益智胶囊组大鼠海马Notch1、Hes3及Stat3 mRNA表达显著升高(P0.05~0.01)。与模型组比较,脑复康组及首乌益智胶囊组大鼠海马Notch1、Hes3及Stat3 mRNA表达显著升高(P0.05~0.01)。与脑复康组比较,首乌益智胶囊组大鼠海马Notch1、Hes3及Stat3 mRNA表达显著升高(均P0.05)。结论首乌益智胶囊可以上调Notch1、Stat3、Hes3基因的表达,从而减轻慢性脑缺血对神经元的损伤,维持神经细胞结构与功能的完整。     

Notch信号通路在大鼠局灶性脑缺血再灌注损伤恢复期的表达变化

目的探讨局灶性脑缺血再灌注损伤恢复期Notch信号通路的表达变化。方法构建SD大鼠大脑中动脉模型(MCAO)。将大鼠分为:对照组(12只),急性期组(24只),恢复期组(24只);急性期组分为3 d和7 d两个亚组,恢复期组分为15d和30d两个亚组。采用RT-PCR和Western blot测定Notch信号分子的mRNA和蛋白的表达变化。结果与对照组大鼠相比,脑缺血再灌注后第3天Delta-like 1、Delta-like 3、Delta-like 4和Jagged 1的mRNA表达水平明显升高(P 0. 05);第30天的表达水平显著低于对照组(P 0. 05)。Notch 1和Notch 2的mRNA表达水平在脑缺血再灌注后第3天显著升高(P 0. 05);第30天,Notch 1(P 0. 05)、Notch 2(P 0. 05)、Notch 3(P 0. 05)和Notch 4(P 0. 01)的mRNA表达水平均显著低于对照组。脑缺血再灌注后第3天Hes-1(P 0. 01)和Hey-1(P 0. 05)的表达水平较对照组均明显升高;第30天,Hes-1和Hey-1的表达水平低于对照组(P 0. 05)。NICD1和NICD2在大脑局灶性缺血后第3天半暗带区的表达水平显著上升(P 0. 05);第30天的表达水平显著下降(P 0. 05)。结论大鼠脑缺血再灌注损伤恢复期可能通过抑制Notch信号通路的表达,起到神经保护作用。     

立体定向移植人骨髓间充质干细胞对局灶性脑缺血大鼠海马区α-微管蛋白表达的影响

目的 探讨立体定向移植人骨髓间充质干细胞(hMSCs)治疗对局灶性脑缺血大鼠海马区α-微管蛋白表达的影响.方法 体外培养、扩增hMSCs;100只大鼠随机分为正常对照(NC)组、假手术组、缺血再灌注(IR)组、假移植组、移植组,每组再分为1 d、3 d、7 d、28 d 4个时间点;制作大鼠局灶性脑缺血90 min再灌注1 h的IR模型,制模成功后移植组大鼠立即进行立体定向hMSCs移植,移植后相应时间点应用免疫组化及免疫荧光染色,观察各组大鼠海马区α-微管蛋白表达.结果 NC组、假手术组大鼠各时间点海马区α-微管蛋白表达的差异无统计学意义;与之相比,IR组、假移植组各时间点及移植组移植后1 d、3 d时表达明显下降(均P＜0.01);但移植组各时间点海马区α-微管蛋白表达明显高于IR组及假移植组(均P＜0.01),移植7～28 d时与NC组比较,差异无统计学意义.结论 局灶性脑缺血大鼠海马区α-微管蛋白表达减少;hMSCs移植促进α-微管蛋白表达增加,这可能是其减轻脑缺血后神经损伤的机制之一.     

大鼠局灶性脑缺血预处理后XBP-1表达的变化

目的 观察缺血预处理对大鼠脑缺血再灌注后X盒结合蛋白1(XBP-1)mRNA及其蛋白表达的影响.方法 SD大鼠60只,随机分为假手术组(SO)、脑缺血再灌注组(MCAO)、脑缺血预处理组(BIP)3组,每组按照再缺血后12h、1d、2d、3d4个时间点分为4个亚组.采用二次线栓法制备大鼠局灶性脑缺血预处理模型,用实时荧光定量PCR和Western blot法观察再缺血后各个时间点XBP-1 mRNA及其蛋白的表达变化.结果 MCAO组XBP-1 mRNA及其蛋白表达均于缺血再灌注后12h开始明显上升,24h达高峰(P＜0.01),随再灌注时间延长其表达逐渐下降,但仍保持较高表达水平(P ＜0.01);BIP组较MCAO组XBP-1 mRNA及其蛋白表达明显升高(P＜0.05,P＜0.01).结论 脑缺血预处理可能通过诱导XBP-1表达发挥其神经保护作用.     

Notch通路对缺氧诱导N9小胶质细胞释放炎症因子的调节作用

目的 应用γ-分泌酶抑制剂(DAPT)抑制Notch信号通路活化,探讨Notch信号通路在缺氧诱导小胶质细胞释放炎症因子中的作用. 方法 将体外培养的N9小胶质细胞分4组:常氧组、缺氧组、常氧+ DAPT组和缺氧+DAPT组.常氧+DAPT组与缺氧+DAPT加入10 μmol/LDAPT处理12h,常氧组和缺氧组加入等量溶剂,缺氧组、缺氧+10 μmol/L DAPT组同时进行缺氧处理12 h (3％O2).提取各组细胞mRNA及蛋白,实时定量PCR检测各组细胞白介素(IL)-6、IL-1β、肿瘤坏死因子-α(TNF-α) mRNA水平,Western blotting检测Notch信号通路中Notch1胞内段(N1ICD)、Hes1、Hey1蛋白水平,并运用ELISA法检测各组细胞IL-6、IL-1β、TNF-α分泌水平. 结果 DAPT对炎性因子IL-6、IL-1β、TNF-α mRNA及蛋白分泌具有抑制作用,并随着浓度的增加,抑制作用增大.分组处理12h后,缺氧组炎性因子IL-6、IL-1β、TNF-α mRNA及蛋白分泌水平较常氧组明显升高,Notch通路信号分子N1ICD、Hes1、Hey1水平升高,差异有统计学意义(P＜0.05);与缺氧组比较,缺氧+DAPT组炎性因子IL-6、IL-1β、TNF-α mRNA及蛋白分泌水平降低,Notch通路信号分子N1ICD、Hes1、Hey1水平降低,差异有统计学意义(P＜0.05);常氧+DAPT组与常氧组比较,上述指标差异均无统计学意义(P＞0.05). 结论 Notch信号通路参与调控缺氧诱导的小胶质细胞炎症介质的释放.     

大鼠局灶性脑缺血预处理后激活转录因子4mRNA及其蛋白表达的变化

目的 观察缺血预处理对大鼠脑缺血再灌注后激活转录因子4 (ATF4) mRNA及其蛋白表达的影响.方法 SD大鼠120只,随机分为假手术组(SO)、脑缺血再灌注组(MCAO)、脑缺血预处理组(BIP)3组,每组按照再缺血后12h、ld、2d、3d4个时间点分为4个亚组.采用二次线栓法制备大鼠局灶性脑缺血预处理模型,用原位杂交法和Western blot法观察再缺血后各个时间点ATF4 mRNA及其蛋白表达的变化.结果 MCAO组12hATF4mRNA及其蛋白表达均达高峰(P＜0.01),随再灌注时间延长其表达逐渐下降;BIP组较MCAO组ATF4 mRNA及其蛋白表达均明显降低(P＜0.05,P＜0.01).结论脑缺血预处理可能通过抑制ATF4表达发挥其神经保护作用.     

抑郁状态下大鼠海马Notch1信号系统的改变

目的 了解大鼠抑郁模型中海马神经重塑障碍与Notch1信号系统功能改变的关系.方法 54只大鼠随机分为CUMS 14 d组、CUMS 28 d组和对照组,前两组接受慢性不可预知温和应激和孤养(chronic unpredictable mild stress,CUMS)14 d和28 d建立抑郁模型.采用免疫组化、免疫荧光、RT-PCR和Western blot 法.测定大鼠海马神经干细胞的增殖、存活和分化以及Notch1信号通路各个因子的基因及蛋白表达水平的改变.结果 与对照组比较,CUMS 14 d组和CUMS 28 d组大鼠海马神经干细胞增殖与存活明显减少(P<0.001).CUMS 28 d组大鼠海马神经干细胞分化NeuN/BrdU、GFAP/BrdU比例无明显差异(P>0.05).与对照组比较.CUMS 14 d组和CUMS 28 d组Notch1信号通路各因子(NICD、Hes1、Hes5和Jag1)基因表达和蛋白水平明显降低(P<0.01).结论 抑郁大鼠海马齿状回神经干细胞增殖和存活受到抑制,但分化无改变;同时,大鼠海马Notch1功能下调.提示Notch1信号系统可能与抑郁症海马神经再生障碍有关.     

脑卒中后抑郁大鼠海马齿状回5-羟色胺1A受体的表达

目的 观察脑卒中后抑郁(PSD)大鼠海马齿状回5-羟色胺1A(5-HT1A)受体的表达.方法 将SD雄性大鼠分为正常对照组、卒中组、应激抑郁对照组和PSD组,每组6只.应用左侧大脑中动脉阻塞(MCAO)联合不可预见的慢性温和应激(CMS)刺激及孤养法建立PSD大鼠模型,采用荧光实时定量聚合酶链反应和Western印迹法检测并比较各组大鼠CMS第19天和第28天齿状回5-HT1A受体(mRNA)和蛋白表达水平.结果 (1)CMS第19天,PSD组5-HT1A受体mRNA表达(O.012±0.001)低于正常对照组(0.361±0.010)和卒中组(0.039±0.002;P＜0.001);其5-HT1A受体蛋白表达(0.400±0.030)低于正常组(1.320±0.060)和卒中组(0.610±0.060;均P＜0.001).(2)CMS第28天,PSD组5-HT1A受体mRNA(0.013±0.001)低于正常对照组(0.336±0.011)、卒中组(0.063±0.006;均P＜0.001);其5-HT1A受体蛋白表达(0.080±0.020)低于正常组(0.620 ±0.030)、卒中组(0.260±0.040)和应激抑郁组(0.320±0.020;均P＜0.001).结论 PsD大鼠海马齿状回5-HT1A受体表达水平降低,此改变可能是PSD发病的分子机制之一.     

成年大鼠局灶脑缺血/再灌注后海马BDNFmRNA和bFGF mRNA的动态变化研究

目的观察局灶脑缺血/再灌注大鼠海马脑源性神经营养因子(brain derived neurotrophic factor,BDNF)mRNA和碱性成纤维生长因子(basic fibroblast growth factor,b FGF)mRNA的动态表达。方法将108只雄性Wistar大鼠随机分为正常组(NC组)、假手术组(SC组)、模型组(I/R组),每组再于缺血1h后再灌注于1,3,7,14,21,28d六个时间点进行观察,正常组和假手术组于相应时间点同步观察。采用线栓法制备大鼠右侧大脑中动脉局灶脑缺血/再灌注模型。应用原位杂交法检测大鼠缺血再灌注后1,3,7,14,21,28d缺血侧海马BDNF mRNA和b FGF mRNA表达。结果正常海马区可见少量BDNF mRNA和b FGF mRNA的阳性表达。BDNF mRNA阳性反应主要集中于齿状回颗粒细胞以及CA2、CA3中的锥体细胞胞浆中,b FGF mRNA主要位于海马锥体细胞层、CA1、CA2区。局灶脑缺血/再灌注后,I/R组缺血侧海马BDNF mRNA表达增加,主要见于齿状回颗粒细胞层和锥体细胞层。缺血/再灌注后1d时BDNF mRNA阳性反应即有增多,3d时达到高峰(P0.01),阳性产物染色较深,7d后迅速下降(P0.01),28d时接近正常水平。局灶脑缺血/再灌注后,I/R组缺血侧海马可见b FGF mRNA的强阳性表达,1d时开始增加(P0.05),3d即达一小高峰(P0.01),7d开始下降,21d时明显下降,28d时降到正常水平(P0.05)。结论局灶脑缺血/再灌注可上调BDNF mRNA和b FGF mRNA的表达,有利于脑缺血后神经功能恢复,具有神经保护作用。     

Asymmetry in the structural organization of the ganglion layer of the retina in the frog

Silver-impregnated retinal preparations were used to study the distribution density and topographic features of small and large ganglionic cells (GC) of Rana ridibunda and Rana temporaria. For both species the increased density of GC (a streak) stretched higher than the naso-temporal axis passing through the optic disk. Beyond the streak the density of small GC was maximal in the central zone of the retina and decreased towards its periphery. For the upper quadrants of the retina the density of small GC was higher than that for the lower ones by 26% on the average. On the contrary, the density of large GC was higher in the lower part of the retina as compared to the upper one, the difference being more pronounced for R. temporaria. The density of large GC was also asymmetric with respect to the dorso-ventral axis being higher in nasal quadrants than in temporal ones by 40-55%. The highest density of large GC was found in the middle zone of the retina. The found structural asymmetry in the retinal output raster may bear an adaptively ecological meaning and may condition the particularities of the formation of the visually guided prey-catching and avoidance reactions.     

Local nonuniformities in the syncytium of the horizontal cells of the retina in the turtle

Electrical coupling between horizontal cells of the turtle retina was investigated by means of two microelectrodes (current and recording ones) penetrating neighbouring cells at a fixed distance from each other. The morphological coupling was revealed by means of fluorescent dye Lucifer Yellow. The electrical coupling was confirmed between elements of similar type (L1--axonal terminals, or L2--cell bodies, or R/G type cells) and no coupling was found between elements of different types, though L1 and L2 are directly connected through thin axons. In the L1 syncytium the electrical coupling at small (less than or equal to 50 microns) but fixed distances between microelectrodes could differ several times depending on the minimal displacement of microelectrodes. This local nonuniformity of coupling can be explained on the basis of structural nonuniformities in the L1 (axon terminal) network. It is unlikely however that the structural nonuniformities can influence the functional properties of horizontal cell network when the retina is stimulated adequately (by light).     

Inhomogeneities in the propagation of the slow wave in the stomach

The propagation of the slow wave in the stomach and its role in inducing sweeping peristaltic contractions toward the pylorus, essential for a proper digestion and emptying, have been studied for many years. Irregularities in the timing or in the pattern of propagation of the slow wave have been known to induce various gastric malfunctions and, recently, several types of gastric dysrhythmias have been described which could lead to gastric contraction abnormalities. In this study, Du et al. have analyzed the disturbances caused by a simple transmural incision in a human stomach, performed to obtain a biopsy of the muscle, on the propagation pattern of the slow wave. In addition, they show that such an incision may by itself also induce new types of gastric dysrhythmias. These results are important in demonstrating that the function of the stomach can easily be disturbed by such procedures. This mini‐review describes several ways in which inhomogeneities in propagation may affect the conduction pattern of the slow wave, including the genesis of several dysrhythmias, and what is currently known about their impact on gastric contraction and digestion.     

Early stages in the formation of the cerebellum in the frog

The formation of the cerebellum was studied during the first 6 months of the tadpole stage of the bullfrog by using standard histological methods and reconstructions from serial horizontal sections. Three major developmental phases were noted in the formation of the cerebellum. (1) During the first 5 weeks of development, the neuroepithelium proliferated and the dorsal mesencephalic plates increased in size. (2) Starting in the sixth week, a patch of neuroepithelium began to differentiate and gave rise to a small population of Purkinje cells. In subsequent weeks, the area of differentiation continued to spread and a Purkinje cell layer became established along the dorsal margin of the cerebellar plate. (3) In the 12th week, the ventrolateral part of the cerebellar plate began to increase in size and generate two populations of small cells. The lateralmost part of the neuroepithelium in this area generated a group of cells that formed an external granular layer that was one cell deep. Cells of this external granular layer migrated inward into the primitive molecular layer, and by the 26th week only a remnant of an external granular layer remained in the cerebellum. The more medially situated part of the neuroepithelium gave rise to another population of small cells that formed a column, which appeared to be continuous with the Purkinje cells, but differed from them in size. It should be noted that full maturation of the cerebellum occurs during metamorphosis, which in this species remains some 2 years away.     

Gradients of histogenesis in the ependymal lining of the third ventricle in the rabbit

Tritiated thymidine autoradiography was used to analyze the site, time of origin, and developmental gradients of the specialized lining of the ependymal surface of the third ventricle. Cells destined to form the ependyma are generated between days 15 and 22 of embryogenesis (gestation: 30 +/- 2 days), the majority of the cells undergoing final division on the 18th day of gestation. Ependymal cells originate in an orderly fashion according to 3 gradients. Two gradients of opposite direction (ventrodorsal and dorsoventral) are found in the parasaggital plane. Both gradients start at the level of the hypothalamic sulcus, progressively departing from this anatomical landmark as histogenesis progresses. A third gradient occurs in the caudorostral axis, such that cells located in caudal regions originate earlier than those located in rostral sectors. Thus, an orderly relationship exists between the time of origin of ependymal cells and their final location within the lining of the ventricular wall. These findings indicate, once again, the topographic nature of the gradients of histogenesis. The histogenic gradients displayed by the ependymal lining of the third ventricle appear strongly related to those exhibited by other diencephalic derivatives. The latter suggests that common factors govern the developmental sequence of all diencephalic derivatives as a function of their relative topographic location, independently of their functional role in the adult.     

Challenges in the Management of the HIV Patient in the Third Decade of AIDS

The epidemic of AIDS has been increasingly recognized as a major health and socioeconomic problem, not only in the United States or Africa, but also the rest of the world. The face of the epidemic has changed. The role that mental health providers play has also significantly grown as the epidemic continues on. Prior to the introduction of Highly Active Anti-Retroviral Therapy (HAART) and other advances in HIV care, the patients faced issues that related to death and dying. These advances brought with them renewed hope and resurrected lives. The patients fought with issues related to living new lives with HIV no longer an imminent death threat. In the third decade of AIDS, the struggles of the post-HAART era continue but bring with it more challenges. Mental health providers need to familiarize themselves with these issues so that they can better help HIV patients cope with this devastating disease.     

Analysis of the habituation-like changes in transmission in the temporodentate pathway of the rat

Habituation-like decrements in extracellular measures of synaptic activation (population EPSP) and cell discharge (population spike) were analyzed in the dentate gyrus of the rat following repetitive low-frequency stimulation of the medial and lateral entorhinal cortex. Stimulation of either subdivision of the entorhinal projection system resulted in comparable habituation-like response decrements with similar stimulation regimens. However, habituating stimulation of one subdivision did not result in decreased responsiveness to stimulation of the other. Repetitive low-frequency stimulation or even a single pulse delivered to either subdivision did, however, result in a potentiation of granule cell discharge in response to stimulation of the other subdivision (a form of heterosynaptic potentiation). This heterosynaptic potentiation of granule cell discharge was not accompanied by any increase in the extracellular EPSP. Comparisons of the relationship between the population EPSP and population spike before and during habituating stimulation revealed changes in cell discharge in response to the habituating stimulus which could not be accounted for by changes in synaptic activation alone. The results suggest that repetitive activation of the temporodentate pathway alters granule cell output as a result of two processes, a habituation-like decrement in synaptic activation, and a potentiation of granule cell discharge as a consequence of prior activation.