肺腺癌和癌旁组织蛋白质表达谱差异的研究

通过比较肺癌患者癌组织和癌旁组织的二维凝胶电泳图谱(two-dimensional electrophoresis,2DE),以寻找肺癌相关蛋白、肺癌患者癌组织和癌旁组织,采用固相pH梯度(immobilized pH gradient,IPG)2DE分离总蛋白质,凝胶经银染显后,ImageMaster 2D图像分析软件进行比较分析、识别差异表达的蛋白质并进行质谱鉴定获取肺癌相关蛋白信息。结果分析得到肺癌组织蛋白点709个,癌旁组织蛋白点722个,选择表达量差异达3倍以上的40个,进行胶内酶解,基质辅助电离解析飞行时间质谱(MALDI TOF MS)获得肽质量指纹图谱(PMF),搜寻蛋白质数据库,得到7个肺癌标志物的候选蛋白,但这些差异蛋白点在肺癌发生发展中的作用需要进一步研究。     

大白鼠舌周核至小脑的纤维投射

大白鼠38只,用微电泳方法把HRP分别导入到小脑皮层各叶和深部各核团,观察舌周核中的标记细胞。结果表明,舌下神经前置核、Roller核均向小脑发出投射纤维。但没有见到闰核有纤维到小脑。舌下神经前置核及Roller核的纤维终止于小脑Ⅳ-Ⅵ小叶蚓部皮层、背侧旁絮叶、旁正中小叶、脚Ⅰ、顶核、间位核和齿状核。没有发现脚Ⅱ有舌周核的终止纤维。本文对舌周核一小脑纤维的起源、终止和舌周核的功能进行了讨论。     

家兔小脑至蓝斑的纤维投射

本实验用成年家兔25只,分三组进行实验:第一组吸去家兔一侧小脑半球或损毁一侧齿状核,或仅损毁部分半球皮质;第二组吸去小脑蚓叶大部分,或损毁顶核,或仅损毁蚓叶皮质;第三组吸去或损毁一侧小脑旁绒球。术后用Fink—Heimer法染色,追踪小脑至蓝斑的纤维投射。第一组实验中由小脑齿状核来的大量溃变纤维进入同侧结合臂,其中一些溃变纤维由结合臂内侧分出,行向前内方穿过三叉神经中脑核后,进入并终止于同侧蓝斑核及蓝斑下核。小脑半球皮质的溃变纤维不进入蓝斑区。第二组实验显示,小脑蚓叶(顶核或皮质)的溃变纤维除投射至小脑中央核及脑干其它部位外,左右两侧备有少量溃变纤维进入同侧臂旁核小细胞部,极少量溃变纤维进入结合臂腹侧部,前行至脑桥吻段,经过三叉神经中脑核而终止于蓝斑核及蓝班下核。第三组实验,由旁绒球来的少量溃变纤维,经同侧臂旁核小细胞部而至蓝斑核。溃变纤维较少,在胞体周围可见终末前溃变。     

增生性瘢痕与正常皮肤的蛋白质组学研究

目的 比较增生性瘢痕与正常皮肤组织的蛋白质组表达差异,筛选出增生性瘢痕的特异蛋白质.方法 以中山大学附属第一医院烧伤外科2010年1月至5月8例增生性瘢痕患者的增生性瘢痕组织和3例整形外科患者正常躯干皮肤为研究对象,运用蛋白质组学技术、双向凝胶电泳对比分析增生性瘢痕组织和正常皮肤组织中蛋白表达差异,选择差异蛋白点进行MALDI - TOF/TOF质谱分析和生物学信息分析.结果 获得重复性较好的双向电泳荧光染色图谱,增生性瘢痕和正常皮肤组织凝胶电泳图谱中平均蛋白点分别为2975和3053,对其中表达差异超过4倍的蛋白点进行质谱分析和数据库检索,共鉴定出24种蛋白质,其中上调蛋白有11种,下调蛋白有13种.结论 成功地建立增生性瘢痕的双向电泳荧光染色图谱,鉴定出增生性瘢痕组织与正常皮肤组织间的差异蛋白质表达.     

Further separating and identifying of differentially expressed proteins analyzed by SELDI

目的 探讨以SELDI蛋白质芯片筛选后的细胞差异表达蛋白的分离和鉴定方法及其意义.方法 以经体外培养及10 μmol/L褪黑素药物干预的内皮祖细胞差异表达蛋白质为研究对象,分别采用Tricine-SDS-PAGE和双向凝胶电泳方法进行差异蛋白分离及结果比较,以FT-MS二级质谱鉴定.结果 经Tricine-SDS-PAGE分离后,选取分子量为53 ku左右的趋势性差异表达蛋白进行FT-MS分析,质谱鉴定为微管蛋白-α3(吻合度评分为87).经双向凝胶电泳分离后,于凝胶近酸性端、分子质量在72 ～95 ku之间区域,选取蛋白表达量较高的一点,质谱鉴定其为人热休克蛋白90-α(吻合度评分为91).结论 Tricine-SDS-PAGE对于大致35～62 ku范围的蛋白分离较清晰、重复性好且样品用量少,2-DE对最低上样量有较严格要求,但它能提供分子质量、等电点双重参数来更准确定位所感兴趣蛋白点,且对大分子质量蛋白的分离效果更佳,经质谱鉴定结果理想.     

大鼠脊髓源神经干细胞与运动神经元的差异蛋白质组学

目的 探讨大鼠脊髓源神经干细胞与运动神经元的差异蛋白质组学,寻找出重要的差异蛋白质.方法 采用双向电泳分离两种细胞的蛋白质,用DeCyder软件分析蛋白表达差异,并采用质谱(HPLC-ESI-MS/MS)进行鉴定.结果 脊髓源神经干细胞与运动神经元蛋白质凝胶分析获得1 300余个清晰的蛋白质斑点,在比国产分析的87个差异点中,初步鉴定出44个差异表达蛋白,其中24个在神经干细胞高表达,20个在运动神经元高表达.结论 脊髓源神经干细胞与运动神经元有各自特定的蛋白质表达,某些差异蛋白可能在神经干细胞向运动神经元分化中发挥关键作用.     

蛋白质组学技术鉴定吉兰-巴雷综合征患者血清急性期反应蛋白的表达

背景：吉兰-巴雷综合征患者血液中存在与发病有关的抗体、补体和细胞因子,以蛋白质组学技术分离鉴定这些标志蛋白,可为寻找新的药物靶标提供依据。 目的：以蛋白质组技术比较吉兰-巴雷综合征患者与正常对照组血清的差异表达蛋白。 方法：采集确诊的吉兰-巴雷综合征患者和正常者血清各30例,提取血清蛋白质以固相pH梯度等电聚焦为第一向,SDS-PAGE垂直电泳为第二向进行双向电泳,图象分析软件Imagemaster 2D分析电泳图谱,MALDI-TOF/TOF串联质谱鉴定差异表达蛋白。 结果与结论：在吉兰-巴雷综合征患者与正常者中24种蛋白质的表达量显著不同,其中α-2-巨球蛋白,血浆铜蓝蛋白,血清淀粉样P物质,丛生蛋白,抗糜蛋白酶,触珠蛋白,血红素蛋白,α-1-抗胰蛋白酶,血清转铁蛋白等9种蛋白质被鉴定为急性期反应蛋白。结果说明吉兰-巴雷综合征患者血清中急性期反应蛋白表达量发生了明显变化,加深了对吉兰-巴雷综合征发病分子机制的理解。     

辣根过氧化物酶逆行标记鸡小脑向海马结构的直接投射

目的:观察鸡是否存在小脑向海马的直接投射.方法:将30%辣根过氧化物酶水溶液注射到鸡一侧端脑的海马结构,逆行追踪双侧端脑和小脑向海马结构的传入纤维联系.结果:在小脑,第Ⅰ～Ⅵ叶、Ⅹ叶和Ⅸ叶的前半叶皮质中的蒲氏细胞被标记.在端脑,双侧的外侧隔核、海马结构的旁海马内侧区(APHm)均有多量神经元被标记,初步表明禽类海马结构在端脑的传入联系类似于哺乳类.结论:鸡小脑蒲氏细胞存在向端脑海马结构的直接投射.     

SELDI蛋白质芯片筛选差异蛋白分子的二级鉴定

 目的 探讨以SELDI蛋白质芯片筛选后的细胞差异表达蛋白的分离和鉴定方法及其意义。 方法 以经体外培养及10μmol/L褪黑素药物干预的内皮祖细胞差异表达蛋白质为研究对象,分别采用Tricine-SDS-PAGE和双向凝胶电泳方法进行差异蛋白分离及结果比较,以FT-MS二级质谱鉴定。 结果 经Tricine-SDS-PAGE分离后,选取分子量为53ku左右的趋势性差异表达蛋白进行FT-MS分析,质谱鉴定为微管蛋白-α3（吻合度评分为87）。经双向凝胶电泳分离后,于凝胶近酸性端、分子量在72～95ku之间区域,选取蛋白表达量较高的一点,质谱鉴定其为人热休克蛋白90-α（吻合度评分为91）。结论Tricine-SDS-PAGE对于大致35～62ku范围的蛋白分离较清晰、重复性好且样品用量少,2-DE对最低上样量有较严格要求,但它能提供分子量、等电点双重参数来更准确定位所感兴趣蛋白点,且对大分子量蛋白的分离效果更佳,经质谱鉴定结果理想。     

鸟结核分枝杆菌感染巨噬细胞后的外泌体刺激巨噬细胞产生的细胞因子及其蛋白质表达改变

目的 探讨鸟结核分枝杆菌(M.avium)感染巨噬细胞后分泌的外泌体[(+)外泌体]刺激巨噬细胞后产生的分子免疫机制及其差异蛋白质成分研究.方法 冷冻高速离心法收集经鸟分枝结核杆菌感染及未感染的巨噬细胞上清中的外泌体,然后利用外泌体刺激巨噬细胞;ELISA方法检测经外泌体刺激巨噬细胞后细胞上清中IFN-γ、TNF-α的含量;流式细胞术从蛋白水平检测巨噬细胞受外泌体刺激后CD80、CD86蛋白表达情况;2-DE MALDI-TOF/TOF MS技术分析鉴定巨噬细胞受M.avium感染后分泌的外泌体中的差异蛋白.结果 IFN-γ、TNF-α在(+)外泌体刺激巨噬细胞后培养上清中含量增加;CD80、CD86蛋白在巨噬细胞受(+)外泌体刺激后表达上调.外泌体经2-DE MALDI TOF/TOF MS分析获得18个差异蛋白,且质谱成功鉴定12个.结论 (+)外泌体促进巨噬细胞TNF-α、IFN-γ的分泌从而增强巨噬细胞的炎症反应,并且可促进巨噬细胞CD80、CD86蛋白表达增强;筛选出差异蛋白的功能与细胞骨架结构、蛋白质合成与加工,炎症反应密切相关.     

Physiology of morphologically identified cells in the posterior caudal lobe of the mormyrid cerebellum

The cerebellum of the mormyrid fish consists of three major divisions: the valvula, the central lobes, and the caudal lobes. Several studies have focused on the central lobes and the valvula, but little is known about the caudal lobes. The mormyrid caudal lobe includes anterior and posterior components. The anterior caudal lobe is associated with the lateral line and eighth nerve end organs, whereas the posterior caudal lobe is associated with the electrosensory system. The present study examines the physiology and pharmacology of morphologically identified Purkinje cells and efferent cells in an in vitro slice preparation of the posterior caudal lobe. We found that the Purkinje cells in the posterior caudal lobe can be classified into three subtypes based on both their morphology and on their physiological responses to intracellular current injection and to synaptic inputs from parallel fibers and climbing fibers. Similarities and differences between the physiology of the caudal lobe and that of other regions of the mormyrid cerebellum and the mammalian cerebellum are discussed.     

Regional cerebellar choline acetyltransferase activity following peduncular lesions

Summary The activity of choline acetyltransferase (ChAT) in the cerebellar lobules of the rat was determined after bilateral destruction of either the superior, middle, or inferior cerebellar peduncles. A significant, 40–60% decrease in ChAT activity occurred in all subdivisions of the cerebellum (anterior lobe, posterior lobe hemisphere, posterior lobe vermis/nodulus and flocculus/paraflocculus) following bilateral inferior peduncle lesions. In contrast, bilateral lesions of the superior or middle peduncles did not result in significant reductions of enzyme activity. These findings indicate that the cholinergic afferents to the cerebellum are contained predominantly in the inferior peduncle, from which they emerge to innervate all of the gross subdivisions of the cerebellum.Supported by NINCDS post doctoral fellowship No. 1F32NS06399-02     

The origin of cerebellar-induced inhibition of Deiters neurones III. Localization of the inhibitory zone

Summary By recording intracellularly from Deiters neurones of cats, there was a survey of those cerebellar areas that, when stimulated, produced inhibitory postsynaptic potentials (IPSPs) monosynaptically in Deiters neurones. The monosynaptic inhibitory area expanded longitudinally mainly along the ipsilateral vermal cortex of the anterior lobe. The ipsilateral cortex of the posterior lobe was also effective in inhibiting Deiters neurones though less prominently than the anterior lobe. The inhibitory fibers could be stimulated in the white matter of the cerebellum, predominantly in the ipsilateral side at rostral regions of nuclei fastigii and interpositus. It was further shown that the monosynaptic inhibition from the anterior and posterior lobes occurs chiefly in the dorsal portion of Deiters nucleus. Since in both the cerebellum and Deiters nucleus the spatial pattern of distribution of the inhibitory fibers conforms to that of the corticovestibular fibers as histologically defined, the experimental findings are in accord with the hypothesis that the cerebellar Purkinje cells are inhibitory in nature.     

SCN2b mRNA在快速老化小鼠额叶和海马中的表达及变化

本实验目的在于探讨SCN2bmRNA在快速老化小鼠SAMP8的不同生长发育阶段海马和额叶中的表达及变化。按月龄将SAMP8小鼠分为3组:2周龄组、8月龄组和13月龄组,每组5只;并分别以8月龄和13月龄SAMR1小鼠为对照组,每组5只。运用RT-PCR技术,观察SCN2bmRNA在各组额叶和海马内的表达及变化。结果显示:SCN2bmRNA在各组小鼠海马和额叶中均有表达,但仅额叶内SCN2bmRNA的表达在SAMP8小鼠2周龄组分别与8月龄组和13月龄组的比较有统计学差异(P<0.05)。以上结果提示,SCN2b参与了海马和额叶正常生理功能的维持,其在额叶随月龄增加的表达变化可能与衰老、认知记忆功能的减退有关。     

Collateral projections of neurons from the lower part of the spinal cord to anterior and posterior cerebellar termination areas

Summary The collateral projections of spinocerebellar neurons located in the L 2 to Ca 1 spinal segments in the cat were investigated by retrograde fluorescent double labeling technique. Rhodamine labeled latex microspheres (Rm) and Fast Blue (FB) were used for injections into the cerebellum in 8 cats. Two additional cats, with injections of Fluoro-Gold (FG) combined with Rm were excluded because lipofuchsin autofluorescence obscured the labeling. After injections with one tracer unilaterally in the paramedian lobule and another tracer bilaterally in the anterior lobe, double labeled neurons were found on the side of the paramedian lobule injection in the column of Clarke at L 2–L 4, laminae IV–VI at L 2–L 5 and the dorsolateral nucleus at L 2–L 6. After bilateral injections of one tracer in lobule VIII B and another in the anterior lobe, double labeled neurons were found bilaterally in the column of Clarke at L 2–L 4, laminae IV–VI at L 2–L 5, the medial part of lamina VII at L 6–L 7 and in certain cell groups at sacro-coccygeal levels. Neurons in the lateral part of lamina VII at L 3–L 5 and the ventrolateral nucleus of L 4–L 5 were labeled exclusively from injections in the anterior lobe. The findings indicate that spinocerebellar neurons at lumbar and more caudal levels of the cat spinal cord have different projection patterns in the cerebellum. A certain number of neurons which project to the anterior lobe have divergent axon collaterals supplying also the posterior vermis and/or the paramedian lobule. Other neurons project to the anterior or to the posterior lobe only.Abbreviations CC column of Clarke - DL dorsolateral nucleus - FB Fast Blue - lam. lamina - lat. lateral - med. medial - N. f. fastigial nucleus - p. copul. pars copularis (paramedian lobule) - pfl.d. dorsal paraflocculus - pmd paramedian lobule - p.post pars posterior (paramedian lobule) - pyr. pyramis - Rm Rhodamine labeled latex microspheres - VL ventrolateral nucleus - I–IX (on cerebellar diagrams) cerebellar lobules, according to Larsell (1953) - A, B (following Roman numeral VIII) sublobules VIII A and VIII B - IV–IX (on spinal cord diagrams) laminae according to Rexed (1954) On leave from Capital Institute of Medicine, Beijing, The People's Republic of China     

Topography of choline acetyltransferase-containing neurons in the forebrain of the rat

Following horseradish peroxidase injections in the cortex of the sheep cerebellum (except the ventral part of the anterior lobe, the flocculus and ventral paraflocculus), labeled cells were found in nucleus gracilis, medial cuneate and lateral cuneate. The present findings provide evidence that the projection of the dorsal column nuclei to the cerebellum in the sheep is more extensive than has been reported for the common laboratory animals. Projections to the vermis are bilateral, projections to the hemisphere are mainly ipsilateral.     

Regulation of dendrite formation of Purkinje cells by serotonin through serotonin1A and serotonin2A receptors in culture

Serotonergic fibers and receptors appear in the rat cerebellum during early postnatal development. In the present study, we investigated the actions of serotonin (5-HT) and its receptors in the dendrite formation of Purkinje cells in organotypic cultures of anterior and posterior lobes of the cerebellum at postnatal day 7. In anterior lobes after 4 days in vitro (4DIV), the dendritic areas and branchings of Purkinje cells were increased by the treatment of 2 microM 5-HT, but decreased by 20 microM 5-HT. In posterior lobes after 4DIV, the dendritic areas of Purkinje cells were increased by 5-HT (2, 20 and 200 microM). In contrast, 5-HT treatment decreased dendritic areas of Purkinje cells in both anterior and posterior lobes after 7DIV. Next, we determined the actions of specific 5-HT receptors in mediating the effects of 5-HT by treatment with selective 5-HT receptor agonists. In anterior lobes after 4DIV, dendritic areas of Purkinje cells were increased by a 5-HT1A receptor agonist (8-OH-DPAT), whereas decreased by a 5-HT2A receptor agonist (DOI). The present study suggested that the dendrite formation of Purkinje cells is promoted by 5-HT through 5-HT1A receptors, but inhibited by 5-HT through 5-HT2A receptors.     

FALSE RECALL AND FALSE RECOGNITION: AN EXAMINATION OF THE EFFECTS OF SELECTIVE AND COMBINED LESIONS TO THE MEDIAL TEMPORAL LOBE/DIENCEPHALON AND FRONTAL LOBE STRUCTURES

The present experiment examined the false recall and false recognition of nonstudied words that are preceded by a list of strong associates in patients with amnesia due to damage restricted to medial temporal lobe or diencephalic regions (MTL/D amnesics), in nonamnesic patients with damage restricted to the frontal lobes (FL nonamnesics), and in amnesic patients with damage to the frontal lobes in addition to medial temporal lobe damage (FL amnesics). We used the Deese/Roediger and McDermott paradigm (Deese, 1959; Roediger & McDermott, 1995) in which people frequently claim that nonstudied words appeared on a presented list. At immediate free recall, the results taken together showed that MTL/D amnesic patients and FL nonamnesic patients produced higher levels of false memory than did controls. At recognition both types of amnesic patients produced less false recognition than either frontal-lobe patients or controls. The results are consistent with the hypothesis that abnormally high levels of false memories are related to the following three conditions: (1) partial memory for the word lists, (2) the ability to extract the semantic gist of the list, and (3) a deficit in strategic monitoring processes.     

The organization of the human cerebellum estimated by intrinsic functional connectivity

The cerebral cortex communicates with the cerebellum via polysynaptic circuits. Separate regions of the cerebellum are connected to distinct cerebral areas, forming a complex topography. In this study we explored the organization of cerebrocerebellar circuits in the human using resting-state functional connectivity MRI (fcMRI). Data from 1,000 subjects were registered using nonlinear deformation of the cerebellum in combination with surface-based alignment of the cerebral cortex. The foot, hand, and tongue representations were localized in subjects performing movements. fcMRI maps derived from seed regions placed in different parts of the motor body representation yielded the expected inverted map of somatomotor topography in the anterior lobe and the upright map in the posterior lobe. Next, we mapped the complete topography of the cerebellum by estimating the principal cerebral target for each point in the cerebellum in a discovery sample of 500 subjects and replicated the topography in 500 independent subjects. The majority of the human cerebellum maps to association areas. Quantitative analysis of 17 distinct cerebral networks revealed that the extent of the cerebellum dedicated to each network is proportional to the network's extent in the cerebrum with a few exceptions, including primary visual cortex, which is not represented in the cerebellum. Like somatomotor representations, cerebellar regions linked to association cortex have separate anterior and posterior representations that are oriented as mirror images of one another. The orderly topography of the representations suggests that the cerebellum possesses at least two large, homotopic maps of the full cerebrum and possibly a smaller third map.     

Compensatory hyperplasia in the rat liver as a result of cytoplasmic atrophy

A protracted process of hepatocyte atrophy was induced in the anterior lobes of the rat liver by portal vein ligation (PVL), and within 3 days the combination of atrophy in the anterior lobes and compensatory hyperplasia of the posterior lobes had resulted in the anterior lobes comprising only about 20% of the total liver weight. Apart from an initial fall in the total liver weight due to atrophy of the anterior lobes without any corresponding growth reaction in the posterior lobes, the total liver weight remained close to normal throughout the first 3 days. Surprisingly, increases in posterior lobe DNA synthesis after PVL were triggered almost as rapidly (15-18 h post-operatively) as occurs after anterior lobe resection, though subsequently the level of proliferative activity was generally lower than is found after partial hepatectomy. The rapidity of the proliferative response to PVL suggests the proliferative signal is generated by a minimal cytoplasmic deficit and/or the increased rate of portal blood perfusion.