新生大鼠小脑内发育相关基因的筛选

为筛选脑发育相关的基因 ,应用新发展的抑制消减杂交技术 ,以新生大鼠小脑mRNA逆转录cDNA作为测试者 (tester) ,成年大鼠小脑mRNA逆转录cDNA作为驱动者 (driver) ,进行消减杂交 ,克隆制备成新生大鼠小脑高表达的cDNA文库。挑选出 5 0个重组质粒 ,测序得到 37个不同基因片段序列。再次用反Northern杂交确认有意义的差异表达基因。同时将测序结果与GenBank注册序列进行同源性比较 ,2 0个大鼠中首次测得的基因表达序列标签 (EST)已被GenBank收录 (BG6 95 72 6 -BG6 95 74 5 )。     

发育相关基因ndrg2在胎儿小脑发育早期的表达

目的为探索新基因ndrg2在胎儿小脑组织中表达分布的特点,进一步研究ndrg2功能提供实验依据。方法收集引产4、5个月胎脑组织标本,分别进行组织石蜡切片和总RNA提取,应用免疫组化方法和逆转录酶-多聚酶链反应(RT-PCR)技术检测ndrg2在蛋白质及mRNA表达水平变化规律。结果免疫组化结果表明,在上述胎儿小脑脑区神经元胞浆均有ndrg2蛋白不同程度的表达。RT-PCR结果显示,ndrg2mRNA在正常胎小脑均有表达,5个月胎儿小脑表达高于4个月胎儿小脑。结论随胎龄增加,ndrg2表达水平增高。     

抑郁模型大鼠小脑的比较蛋白组学初步研究

目的 通过抑郁模型大鼠小脑的差异蛋白组学分析,探讨小脑在抑郁症病理生理过程中的作用.方法 建立慢性不可预见性温和应激(CUMS)抑郁大鼠模型,并应用同位素标记相对和绝对定量技术结合多维液相色谱-串联质谱分析CUMS组和对照组大鼠小脑差异蛋白质.随后对结果采用DAVID生物信息数据库进行分析.结果 与对照组大鼠比较,CUMS组大鼠糖水偏好值明显降低,差异有统计学意义(P＜0.05).对两组样品鉴定,共得到差异蛋白55个,其中在CUMS组中表达下调的有18个,表达上调的有37个.这些蛋白主要参与神经递质代谢、糖酵解、ATP合成利用、脂质代谢、氨基酸代谢等生物过程.结论 本研究所发现的差异蛋白可能是抑郁症小脑功能异常的神经生物学基础,对深入探讨小脑在抑郁症发病机制中的作用提供了重要的线索.     

SD大鼠小脑共济失调检测仪的研制及行为学研究

目的 自制大鼠小脑共济失调检测仪,研究检测方法并评价其有效性、科学性.方法 自制大鼠共济失调检测仪;采用鼠脑立体定位仪,微量注射海人藻酸(KA)损毁SD大鼠小脑各部.观察大鼠共济失调及行为学变化,病理学证实小脑各损害部位.结果 行为学表现为小脑受损后肢体行动困难、笨拙,行走不稳,躯体向左右侧倾倒或打转,自己难以翻正,反应迟钝,胆小,紧张等表现; 病理学证实KA注射位点准确,注射部位的神经元已大部分坏死、脱失;SD大鼠各部位损害有不同的共济失调表现及行为学表现;自制大鼠小脑共济失调检测仪能定性定量地反映SD大鼠的共济运动及小脑受损后的共济失调.结论 自制共济失调检测仪可用于SD大鼠小脑共济失调的定性和定量检测,SD大鼠左、右小脑和蚓部损害后有不同的共济失调表现及行为学异常.     

凋亡基因在缺氧缺血新生大鼠脑组织中的动态变化及银杏叶提取物的保护效应

目的 探讨银杏叶提取物(EGb)对新生大鼠缺氧缺血性脑损伤(HIBD)的保护作用。方法 夹闭妊娠大鼠子宫血管，制成HIBD新生鼠模型，治疗组给予腹腔注射EGb，在生后不同时间比较两组仔鼠脑组织凋亡基因Bcl-2、Bax的变化及神经细胞的凋亡情况。结果缺氧缺血后随再灌注时间的延长，脑组织中Bcl-2与Pax的比例下降，同时凋亡细胞数增加，表明Bcl-2、Bax两者比值的降低促进凋亡的发生。EGb治疗后Bcl-2表达增加显著，同时观察到治疗组的凋亡细胞数量减少。结论 EGb对HIBD有保护作用。     

大鼠代谢型谷氨酸受体1亚型基因片段的克隆及其在小脑的表达

目的：克隆大鼠代谢型谷氨酸受体1亚型（mGluR1)基因特异片段，制备cDNA探针。方法：从Wistar大鼠小脑中提取总RNA，以RT－PCR方法得到预期的599bp条带，将这一片段克隆到pGEM-T easy载体上，经酶切鉴定正确后送测序。将重组质粒经限制性内切酶酶切制备成线性模板，通过体外转录的方法 合成地高辛标记的mGluR1cRNA正义及反义探针。取成年Wistar大鼠小脑组织进行原位杂交实验，以检测探针的可靠性。结果：测序证实用RT－PCR的方法获得了mGluR1基因特异片段，成功地构建了pGEM-TmGluR1重组质粒。根据斑点杂交实验结果计算出正义、反义探针浓度分别为10ng/μl及30ng/μl。原位杂交实验的结果显示，用mGluR1反义探针进行杂交的阳性信号主要分布在大鼠小脑蒲肯野氏细胞胞浆，用正义探针杂交无阳性信号。结论：本实验克隆了mGluR1基因特异片段，并制备了cRNA探针，并用大鼠小脑进行的原位杂交实验显示，此探针灵敏度高，特异性好。     

脊髓小脑共济失调7型一个家系的临床和基因分析

目的总结脊髓小脑共济失调7型（SCA7）的临床表现，开展基因检测。方法对1个表现为视力下降、辨色力异常和行走不稳的家系完成家系调查及体格检查，部分成员行视网膜形态学及电生理检查；19名家系成员及12名健康对照者行SCA7突变基因PCR，测序仪直接检测三核苷酸胞嘧啶-腺嘌呤-鸟嘌呤（CAG）重复数目。结果6例成员存在小脑性共济失调、视力下降和辨色力异常，眼底示黄斑及视网膜周边色素异常，视网膜电图波形熄灭，震荡电位幅值和光闪视觉诱发电位振幅明显下降；正常等位基因CAG重复数目为8～25次，该6例异常等位基因CAG重复数目为50～97次，诊断为SCA7患者；1例无异常临床表现的成员CAG重复数目分别为18次和56次，后者超出正常范围，诊断为未到发病年龄的症状前患者。结论SCA7患者的临床表现具有异质性，CAG重复数目检测可以为基因诊断和症状前诊断提供依据。     

垂体腺苷酸环化酶激活肽增加新生大鼠小脑颗粒细胞钙通道电流

应用膜片箝"全细胞"电流记录方法，研究了垂体腺苷酸环化酶激活肽（PACAP）对新生大鼠小脑颗粒细胞高电压激活（HVA）的钙电流作用。实验结果发现：PACAP－38、PACAP－27能明显增加小脑颗粒细胞钙电流的幅值，两者增加钙内流的效应无显著差异，并且都具有脱敏现象；而出于同一家族的血管活性肠肽（VIP）对此电流却没有任何影响。当记录电极内加入GTPγS后，PACAP增加钙电流的效应成为不可逆；而细胞内液加入GDPβS或预先用百日咳毒素孵育细胞12h后，PACAP的钙通道激活作用完全消失。此外，细胞内应用cAMP和磷酸二酯酶抑制剂3－isobutyl－1－methylxanthine（IBMX），既不影响钙通道本身的活动，也不修饰PACAP增加颗粒细胞钙通道电流的效应。我们的结果提示：新生大鼠小脑颗粒细胞上存在着具有功能意义的PACAP受体，该受体被PACAP－38和PACAP－27激活后可增加细胞膜上钙通道的开放，促进钙内流。PACAP的这种作用与百日咳毒素敏感的G蛋白偶联，但并不依赖腺苷酸环化酶参与的第二信使系统。     

单眼视觉剥夺大鼠视皮质基因表达差异的研究

本实验利用差异显示反转录ＰＣＲ（ＤＤＲＴ－ＰＣＲ）技术分析单眼视觉剥夺大鼠左、右侧视皮质基因差异表达情况,并通过反Northern杂交去除假阳性筛选差异表达基因片段,最终获得３个差异表达基因片段。通过克隆、测序和同源性比较发现,其中一个片段５'端的１６６个碱基与人ＫＩＡＡ０５４１蛋白的mRNA编码区的１６６bp区段的８３％的同源性。本研究表明单眼视沉剥夺导致视皮质基因表达的改变。     

利用Affymetrix芯片筛选全脑缺血大鼠海马差异表达基因

目的 筛选出全脑缺血大鼠海马差异表达基因.方法 建立大鼠体外循环模型,并将大鼠按随机数字表法分成两组:对照组(n=3),建立体外循环5min后取大鼠海马组织;实验组(n=3),停循环全脑缺血5 min后取大鼠海马组织.采用Affymetrix大鼠全基因组芯片检测两组大鼠海马基因表达的变化,获取差异表达基因.结果 筛选出差异表达基因共55个(上调30个,下调25个),其中28个基因表达差异有统计学意义(P＜0.05).结论 筛选出的全脑缺血大鼠海马差异表达基因对阐明全脑缺血性损伤的分子生物学机制有重要意义.     

Differential effects of ethanol on c-jun N-terminal kinase, 14-3-3 proteins,and Bax in postnatal day 4 and postnatal day 7 rat cerebellum

These studies investigated ethanol effects on upstream cellular elements and interactions which contribute to Bax-related apoptosis in neonatal rat cerebellum at ages of peak ethanol sensitivity (postnatal day 4 [P4]), compared to later ages of relative resistance (P7). Analyses were made of basal levels of the pro-apoptotic c-jun N-terminal kinase (JNK), Bax, and the 14-3-3 anchoring proteins, as well as the responsiveness of these substances to ethanol at P4 versus P7. Dimerization of Bax with 14-3-3 was also investigated at the two ages following ethanol treatment, a process which sequesters Bax in the cytosol, thus inhibiting its mitochondrial translocation and disruption of the mitochondrial membrane potential. Cultured cerebellar granule cells were used to examine the protective potential of JNK inhibition on ethanol-mediated cell death. Basal levels of JNK were significantly higher at P4 than P7, but no differences in the other proteins were found. Activated JNK, and cytosolic and mitochondrially-translocated Bax were increased in P4 but not P7 animals following ethanol exposure, while protective 14-3-3 proteins were increased only at P7. Ethanol treatment resulted in decreases in Bax:14-3-3 heterodimers at P4, but not at P7. Inhibition of JNK activity in vitro provided partial protection against ethanol neurotoxicity. Thus, differential temporal vulnerability to ethanol in this CNS region correlates with differences in both levels of apoptosis-related substances (e.g., JNK), and differential cellular responsiveness, favoring apoptosis at the most sensitive age and survival at the resistant age. The upstream elements contributing to this vulnerability can be targets for future therapeutic strategies.     

Effects of ethanol on postnatal cell acquisition in the rat cerebellum

Cell generation and cell survival were investigated in the cerebellum of young rats exposed to 10% v/v ethanol in drinking water throughout gestation and lactation. At 12 days postpartum, cell cycle parameters in the external granular layer showed no significant change from control values, and the cell acquisition rate was unaffected. However, the external granular layer appeared thicker in ethanol-treated than in control 12-day-old animals, and a significant increase in cell death in the internal granular layer was observed. It is suggested that the effect of ethanol on the developing cerebellum may involve reduction of granule cell number consequent to increased granule cell death, and possibly retarded migration from the proliferating precursor cell pool.     

Non-lethal active caspase-3 expression in Bergmann glia of postnatal rat cerebellum

Caspase-3, an apoptotic executor, has been shown in recent years to mediate non-lethal events like cellular proliferation and differentiation, primarily in studies related to non-neural tissue. In central nervous system development, the role of active caspase-3 is still unclear. We provide the first evidence for a potential new role of active (cleaved) caspase-3 in promoting differentiation of Bergmann glia. This study was predicated on the hypothesis that active caspase-3 is important for the differentiation of glia. We addressed the hypothesis through the following specific aims: (1) to establish the expression of active caspase-3 in glia; (2) to determine the developmental phenotype of the active caspase-3-expressing glia; and (3) to confirm that active caspase-3 expression is not mediating an apoptotic event. Through a temporal investigation from postnatal day 8 to 21, we observed that Bergmann glia express active caspase-3 without compromising their survival. Potential apoptotic fate of active caspase-3-positive Bergmann glia were ruled out based on immunohistochemical exclusion of phosphatidylserine exposure (Annexin V), DNA fragmentation (TUNEL), and DNA compaction (TOPRO-3). More than 90% of the active caspase-3-positive cells lacked colabeling for one of the apoptotic markers. Correlative studies using a proliferation marker Ki67 and a differentiation marker brain lipid-binding protein suggest that the expression of active caspase-3 was mostly associated with differentiating rather than proliferating Bergmann glia at all ages. Thus, this study supports the hypothesis that active caspase-3 may be regulating both differentiation of Bergmann glia by allowing the cells to exit the cell cycle and their morphogenesis.     

Histochemical distribution of acid glycosaminoglycans in rat cerebellum during postnatal development: Neuropile

Developmentally regulated modifications of glycosaminoglycans (GAGs) in the central nervous system (CNS) have suggested that also in the CNS, these compounds might participate in morphogenesis and nerve cell differentiation. However very few studies have been reported concerning the regional distribution of these compounds by histochemical techniques. We have used the Alcian Blue staining method also in conjunction with enzymatic digestion and with a technique which allowed the measurement of the degree of GAG sulphatation. The combination of the three techniques showed that during the first week GAGs, presumed to be hyaluronic acid, are localized throughout the neuropile of the entire cerebellum and especially in the medullary region. Sulphated glycans appear later in the medullary region (particularly at the border between the medullary region and the internal granular layer) and in all the layers of cerebellum (in particular around the Purkinje and deep cerebellar nuclei neurons and possibly in the cerebellar glomeruli). Sulphated glycans in the medullary region disappear around the 12th day when myelination starts. The transient presence of glycoproteins in the molecular layer was also detected.     

Developmental fates and migratory pathways of dividing progenitors in the postnatal rat cerebellum

To investigate the developmental fates and the migratory pathways of dividing progenitors in both the white matter (WM) and the external granule layer (EGL) in the early postnatal rat cerebellum, a replication-deficient retrovirus carrying the β-galactosidase gene (BAG) was injected into the deep cerebellar tissue or the EGL of postnatal rats to label dividing progenitors. After 1–3 days post-injection (1–3 dpi) of BAG into the deep cerebellar tissue of postnatal day 4/5 (P4/5) rats, labeled immature, unipolar cells were found mainly in the WM. From 4 to 6 dpi, similar cells appeared in the internal granule (IGL), Purkinje cell, and molecular layers, although about half of the labeled cells still resided in the WM and appeared immature. The first morphologically definable Bergmann glia, astrocytes, and oligodendrocytes were also observed. From 14 to 20 dpi, most labeled cells had developed into Bergmann glia, astrocytes, oligodendrocytes, and interneurons in their appropriate layers. When BAG injections were performed at P14, unipolar cells were initially observed, but the majority of these differentiated into myelinating oligodendrocytes in the WM and IGL by 17 dpi. Few immature cells were labeled by injections administered at P20, and these did not develop into mature glia, but into cells with lacy, fine processes, possible representing immature oligodendrocytes. In contrast, BAG-labeled progenitors of EGL produced only granule neurons. Thus, within the first 2 postnatal weeks, dividing progenitors in the WM migrate as immature cells into the cortex before differentiating into a variety of glia and interneurons. The genesis of oligodendrocytes continues through the 2nd postnatal week and largely ceases by P20. EGL cells do not produce glia, but only granule cells. © 1996 Wiley-Liss, Inc.     

Changes in expression of P2X purinoceptors in rat cerebellum during postnatal development

Changes in expression of P2X receptors (P2X1-7) during postnatal development of the rat cerebellum are described. At P3, immunoreactivity (ir) to all the P2X receptors, except for P2X3 receptors, was found in Purkinje cells and deep cerebellar nuclei, P2X5-ir being most prominent. Granular and microglial cells were labeled for P2X5 (weakly) and P2X4 receptors, respectively. At P7, expression of all the P2X receptors (with the exception of P2X3) was up-regulated, P2X5 and P2X6 receptors being most prominent. Scattered P2X receptor-ir in unipolar brush cells in the granular cell layer and P2X1- and P2X7-ir of microglial cells was also present. At P14, the dendritic trees of Purkinje cells were intensely labeled by P2X1-7 receptor antibodies, except for P2X3, while P2X1, P2X4 and P2X7 receptor immunostaining in microglial cells and P2X5 receptor immunostaining in granular cells was up-regulated. At P21, expression of all P2X receptors (except P2X3) was down-regulated in the Purkinje cells and deep cerebellar nuclei; P2X1, P2X4 and P2X7 receptors-ir was present in microglial cells. In contrast, expression of P2X5-ir in granular cells was up-regulated. At P60, expression levels of all the P2X receptors (except P2X3) were similar with those at P21. In double-labeling experiments, almost all the P2X-ir Purkinje cells were immunoreactive for calbindin-D28k, while 60-80% of P2X-ir cells in the granular cell layer were immunoreactive for calretinin. The possible short- and long-term functional significance of the changes in expression of P2X receptors during postnatal development is discussed.     

Differential expression of CPD1 during postnatal development in the mouse cerebellum

Several regulated mRNAs were detected by applying differential display to the mouse cerebellum during postnatal development. One cDNA fragment, referred to as CPD1 (GenBank U89345), was characterized and cloned. Northern blots showed maximum mRNA expression at postnatal day seven (P7). The mRNA encodes a protein of 260 amino acids. In situ RT-PCR showed that CPD1 is expressed mainly in granule cells and faintly in Purkinje cells. Polyclonal rabbit antibodies and oligobodies (oligonucleotide-based synthetic antibodies) revealed a protein of 34 kDa in Western blots. Immunohistochemistry showed not only marked nuclear staining but also mild cytoplasmic localization. Granule cells undergoing active division (P4) showed very little expression of CPD1 protein, which increases from P7 to P17. CPD1, affinity-purified using a chemically synthesized oligobody inhibits the activity of protein phosphatase PP2A but not protein phosphatase PP1. Differentiated PC12 cells also showed nuclear and cytoplasmic localization. Interestingly, maximal cytoplasmic CPD1/PP2A colocalization was observed near cell membrane regions that are far from growing neurites, and on growing cones. These results suggest that CPD1 might have an important role in cerebellar development.