Mononuclear cell proliferation and hyperplasia during Wallerian degeneration in the visual system of the goldfish in the presence or absence of regenerating optic axons

Patterns of proliferation and changes in non-neuronal cell number in the visual system of the goldfish have been quantitatively examined during optic axon regeneration after an optic nerve crush (ONC). In addition, in order to examine the effect of the regenerating axons on cellular responses in the visual pathways, we did a similar analysis of animals with the right eye removed (ER). Finally, we used double labeling protocols to demonstrate that the proliferating cells that we were counting were mostly phagocytic cells of the mononuclear lineage. In animals with an ONC, we observed an early burst of proliferation that peaked between 7 and 14 days after surgery in all parts of the visual system. In the optic tract, there was also a secondary rise that peaked at 21 days. Levels of proliferation returned to normal by 32 days postoperative in the tract and tectum, while they remained somewhat elevated in the optic nerve for at least 93 days. The total number of non-neuronal cells in the visual paths also rose to peak values between 7 and 14 days after ONC surgery. In the optic tract and tectum, the values fell rapidly after this time, while in the optic nerve, there was a secondary peak at 32 days after which values remained elevated for the duration of the experiment. As compared to animals with an ONC, enucleation resulted in elevated proliferation and hyperplasia at early postoperative intervals. However, because these differences occurred when axons had not yet regenerated into the affected structures, these data do not provide strong evidence for a direct effect of regenerating optic axons on the early cellular responses during Wallerian degeneration in the goldfish. In addition, in the tectum, there was an early increment in cell number that was not associated with elevated levels of proliferation. We believe that this increment represents immigration of resident microglia from other regions of the brain.     

应用神经轴突示踪技术研究人类视觉神经通路胚胎发育

目的 :应用神经轴突示踪方法 ,研究人类视觉神经通路在胚胎期 12w时的发育情况。方法 :取 12w胎龄的胚胎 ,浸泡在 4 %的甲醛缓冲液中固定后 ,分别在视束、上丘臂和视皮质下植入DiI染料 (DiI,1,1’ dioctadecyl 3,3,3’ ,3’ tetramethylin docarbocyanineperchlorate)。于室温下放置 4w ,待DiI晶体充分扩散后 ,根据神经轴突方向切片 ,通过共焦激光扫描显微镜观察并记录结果。结果 :大体标本观察可见到 ,视网膜神经纤维投射已到达外侧膝状体、上丘。切片观察发现 :外侧膝状体处尚未出现分层现象 ;上丘处 ,来自视网膜的神经纤维位于上丘臂的背侧 ;视皮质下已存在板层结构 ,板层内神经元呈多种形态。结论 :通过神经轴突示踪技术发现 ,12w时视网膜的神经纤维投射已经到达外侧膝状体和上丘 ,视皮质下存在板层结构。这说明人类视觉神经通路发育存在“等待期”。     

腹部手术影响小肠平滑肌细胞收缩的细胞内信号转导机制研究

目的:探讨腹部手术操作对小肠平滑肌细胞收缩功能的影响及细胞内信号转导途径。方法:采用Wistar大鼠制作腹部手术动物模型,酶解法获取小肠游离环行平滑肌细胞,检测平滑肌细胞在乙酰胆碱作用下的收缩反应和胞浆游离钙离子浓度([Ca2 ]i)的变化,然后将细胞分别与一氧化氮合酶抑制剂L-NNA、钙活化的K 通道阻滞剂A-pamin、蛋白激酶A抑制剂H89和蛋白激酶G抑制剂KT5823等一同孵育,重复以上实验过程。测定小肠环行平滑肌细胞培养24 h后培养基中一氧化氮(NO)的含量。结果:培养24 h后,实验组小肠环行平滑肌细胞产生的NO量明显高于对照组(P<0.05),L-NNA可明显降低实验组NO的产量,而对照组NO产量没有明显影响;L-NNA和KT5823可明显提高实验组小肠环行平滑肌细胞的收缩能力及乙酰胆碱刺激的[Ca2 ]i升高的水平(P<0.05),而Apamin和H89没有明显的影响。结论:腹部手术操作导致小肠肌层大量生成NO,激活鸟苷酸环化酶产生cGMP,通过PKG途径降低细胞内[Ca2 ]i的水平,抑制小肠平滑肌细胞的收缩能力。     

Subcellular compartmentalization of a potassium channel (Kv1.4): preferential distribution in dendrites and dendritic spines of neurons in the dorsal cochlear nucleus

Voltage-dependent ion channels have specific patterns of distribution along the neuronal plasma membrane of dendrites, cell bodies and axons, which need to be unravelled in order to understand their contribution to neuronal excitability and firing patterns. We have investigated the subcellular compartmentalization of Kv1.4, a transient, fast-inactivating potassium channel, in fusiform cells and related interneurons of the rat dorsal cochlear nucleus. A polyclonal antibody which binds to a region near the N-terminus domain of a Kv1.4 channel was raised in rabbits. Using a high-resolution combination of immunocytochemical methods, Kv1.4 was localized mainly in the apical dendritic trunks and cell bodies of fusiform cells, as well as in dendrites and cell bodies of interneurons of the dorsal cochlear nucleus, likely cartwheel cells. Quantitative immunogold immunocytochemistry revealed a pronounced distal to proximal gradient in the dendrosomatic distribution of Kv1. 4. In plasma membrane localizations, Kv1.4 was preferentially present in dendritic spines, either in the spine neck or in perisynaptic locations, always away from the postsynaptic density. These findings indicate that Kv1.4 is largely distributed in dendritic compartments of fusiform and cartwheel cells of the dorsal cochlear nucleus. Its preferential localization in dendritic spines, where granule cell axons make powerful excitatory synapses, suggests a role for this voltage-dependent ion channel in the regulation of dendritic excitability and excitatory inputs.     

谷氨酰胺∶6-磷酸-果糖酰基转移酶抑制剂细胞筛选模型的建立

目的建立己糖胺途径的关键酶谷氨酰胺∶6-磷酸-果糖酰基转移酶(glutamine∶fructose-6-phosphate amidotransferase,GFAT)抑制剂的细胞筛选模型。方法用改进的GDH法测定GFAT的活性。以速效胰岛素依赖的葡萄糖摄取观察细胞对胰岛素的反应性；用长效胰岛素诱导HIRc细胞，形成己糖胺途径过度活跃和胰岛素抵抗的细胞模型，并应用该细胞模型和GDH法筛选GFAT抑制剂。结果用25 nmol·L^-1长效胰岛素刺激HIRc细胞36 h，可明显激活己糖胺途径，使GFAT活性上升47%；同时产生胰岛素抵抗，使速效胰岛素依赖的葡萄糖摄取能力降低21%。Azaserine可明显抑制该模型中GFAT的活性。结论长效胰岛素既可过度激活HIRc细胞己糖胺途径，又可使其产生胰岛素抵抗。该模型可用于筛选GFAT抑制剂。     

Wnt3a信号通路通过JMJD6的表观遗传修饰参与神经病理性疼痛

目的：探讨Wnt3a通过Jumonji C结构域6( Jumonji C domain 6,JMJD6)的表观遗传修饰在神经病理性疼痛 中发挥作用的机制。方法：将SD大鼠分为4组：Sham组,慢性缩窄性损伤(chronic constriction injury,CCI)组,CCI+阴性慢病毒表达载体(LV-NC)组;CCI+慢病毒过表达载体(LV-JMJD6)组。构建SD大鼠坐骨神经CCI模型和JMJD6慢病毒 表达载体。CCI术后第3天通过鞘内导管给药,按照分组分别给予生理盐水和含慢病毒的试剂(病毒滴度1×108 TU/mL) 各20 μL。监测大鼠的机械缩足阈值(paw withdrawal mechanical threshold,PWMT)和热缩足潜伏期(paw withdrawal thermal latency,PWTL),并运用蛋白质印迹法检测脊髓水平Wnt3a及NR2B蛋白的表达变化,免疫共沉淀检测JMJD6与Wnt3a之间是否存在直接相互作用。结果：与Sham组相比,CCI术后各组大鼠的PWMT明显降低和PWTL明显缩短(P<0.05)。与CCI组和CCI+LV-NC组相比,CCI+LV-JMJD6组的PWMT在术后第10和14天明显升高,PWTL在术后第14 天明显延长(P<0.05)。CCI术后第14天,CCI组及CCI+LV-NC组Wnt3a和NR2B蛋白表达水平较Sham组明显升高,鞘内注 射慢病毒载体后, CCI+LV-JMJD6组的Wnt3a和NR2B蛋白表达水平较CCI+LV-NC组降低(P<0.05)。免疫共沉淀结果显示Wnt3a与JMJD6之间无直接相互作用。结论：Wnt3a参与调节神经病理性疼痛,其作用可能与JMJD6的表观遗传修饰相关,两者可能通过间接相互作用进行调节。     

Association of PTEN gene methylation with genetic alterations in the phosphatidylinositol 3-kinase/AKT signaling pathway in thyroid tumors

BACKGROUND: The phosphatidylinositol 3-kinase (PI3K)/AKT pathway plays an important role in thyroid tumorigenesis and progression. Genetic alterations, particularly PIK3CA amplification and mutations and ras mutations, are the major cause of aberrant activation of this pathway in thyroid tumors. Epigenetic silencing of the PTEN gene, a negative regulator of the PI3K/AKT pathway, also occurs in thyroid tumors, but its relationship with genetic alterations in this pathway is unclear. METHODS: By using quantitative methylation-specific polymerase chain reaction, the authors examined PTEN methylation and its relationship with genetic alterations in the PI3K/AKT pathway in various types of thyroid tumors. RESULTS: The authors found PTEN methylation to become progressively higher from benign thyroid adenoma to follicular thyroid cancer and to aggressive anaplastic thyroid cancer, which harbored activating genetic alterations in the PI3K/AKT pathway correspondingly with a progressively higher prevalence. The association of PTEN methylation was seen with both overall genetic alterations and individual genetic alterations, particularly PIK3CA alterations and ras mutations, in the PI3K/AKT pathway within each of the 3 types of thyroid tumors. In contrast, no such relationship was observed for the tumor suppressor gene RASSF1A. CONCLUSIONS: The authors found an interesting association of PTEN methylation with the activating genetic alterations in the PI3K/AKT pathway in thyroid tumors. This finding is consistent with a model in which aberrant methylation and hence silencing of the PTEN gene, which coexists with activating genetic alterations of the PI3K/AKT pathway, may enhance the signaling of this pathway aberrantly activated by genetic alterations and hence contribute to the progression of thyroid tumors. Cancer 2008.     

Malignant ascites protect against TRAIL-induced apoptosis by activating the PI3K/Akt pathway in human ovarian carcinoma cells

Ascites are commonly found in ovarian cancer patients with advanced disease and are rich in cellular components and growth-promoting factors. The purpose of this study was to assess the effect of malignant ascites on TRAIL-induced apoptosis. We demonstrate that malignant ascites obtained from women with advanced ovarian cancer protect tumor cells from TRAIL- and FasL-induced apoptosis but not against cisplatin-induced apoptosis. This antiapoptotic effect was consistently found among different malignant ascites while nonmalignant peritoneal fluids or conditioned medium from TRAIL-resistant cells failed to protect tumor cells against TRAIL killing. Malignant ascites strongly inhibits TRAIL-induced caspase-3 activation and PARP cleavage. Furthermore, ascites activate PI3K and its downstream target Akt and increases c-FLIP(S) protein levels without affecting ERK phosphorylation status. The antiapoptotic effect of malignant ascites is abrogated by the inhibition of PI3K with LY294002, by a specific inhibitor of Akt and by Akt siRNA. We further show that the pro-survival effect of ascites can be suppressed by down-regulation of c-FLIP(S). Our data indicate that malignant effusions protect against TRAIL-induced apoptosis by activating the PI3K/Akt pathway. These findings demonstrate that the tumor microenvironment may contribute to the resistance of ovarian cancer cells to death receptor-induced apoptosis.     

百里醌对犕犌犆-803细胞增殖和凋亡影响机制探讨

目的：百里醌（thymoquinone,TQ）是具多种生物活性的天然生物单体,参与多种肿瘤细胞增殖、凋亡及转移过程。MGC-803细胞株为常见的低分化胃黏液腺癌细胞。本研究探讨 TQ 对胃癌 MGC-803细胞增殖和凋亡的影响及其可能的信号通路。方法以终浓度分别为0、10、25、50、75、100和125μmol／L TQ 处理 MGC-803细胞12、24和36 h, MTT 法检测其对增殖的影响,Hoechst 染色观察细胞凋亡,蛋白质印迹法分析 Bax、Bcl-2、survivin、Cyclin D1、caspase-3、caspase-9、PTEN、AKT、P-AKT、聚腺苷酸二磷酸核糖转移酶（poly ADP-ribose polymerase,PARP）、cleaved caspase-3及cleaved caspase-9的表达,流式细胞仪检测细胞周期及凋亡。结果 MTT 结果显示,10～125μmol／L TQ 抑制胃癌MGC-803细胞,呈现明显的剂量-时间依赖性。流式结果显示,0、25、50和75μmol／L TQ 作用 MGC-803细胞24 h,凋亡率分别为（1．59±1．04）％、（4．26±0．35）％、（8．37±0．67）％和（21．73±1．71）％,F ＝209．76,P ＜0．001;G2／M 期细胞分别为（4．18±1．09）％、（6．81±0．28）％、（8．68±0．15）％和（12．26±1．46）％,F＝80．388,P ＜0．001。蛋白质印迹结果显示,PTEN 信号通路相关蛋白 PTEN 及凋亡相关蛋白 Bax、cleaved caspase-3及 cleaved caspase-9表达上调,抑凋亡相关蛋白 Bcl-2、caspase-3、caspase-9、survivin 和 PARP 表达下降。结论PTEN 信号通路参与 TQ 抑制胃癌 MGC-803细胞增殖、诱导其凋亡的过程。     

Dietary glycemic and insulin scores and colorectal cancer survival by tumor molecular biomarkers

Accumulating evidence suggests that post‐diagnostic insulin levels may influence colorectal cancer (CRC) survival. Yet, no previous study has examined CRC survival in relation to a post‐diagnostic diet rich in foods that increase post‐prandial insulin levels. We hypothesized that glycemic and insulin scores (index or load; derived from food frequency questionnaire data) may be associated with survival from specific CRC subtypes sensitive to the insulin signaling pathway. We prospectively followed 1,160 CRC patients from the Nurses' Health Study (1980–2012) and Health Professionals Follow‐Up Study (1986–2012), resulting in 266 CRC deaths in 10,235 person‐years. CRC subtypes were defined by seven tumor biomarkers (KRAS, BRAF, PIK3CA mutations, and IRS1, IRS2, FASN and CTNNB1 expression) implicated in the insulin signaling pathway. For overall CRC and each subtype, hazard ratio (HR) and 95% confidence interval (95% CI) for an increase of one standard deviation in each of glycemic and insulin scores were estimated using time‐dependent Cox proportional hazards model. We found that insulin scores, but not glycemic scores, were positively associated with CRC mortality (HR = 1.19, 95% CI = 1.02–1.38 for index; HR = 1.23, 95% CI = 1.04–1.47 for load). The significant positive associations appeared more pronounced among PIK3CA wild‐type cases and FASN‐negative cases, with HR ranging from 1.36 to 1.60 across insulin scores. However, we did not observe statistically significant interactions of insulin scores with PIK3CA, FASN, or any other tumor marker (p interaction > 0.12). While additional studies are needed for definitive evidence, a high‐insulinogenic diet after CRC diagnosis may contribute to worse CRC survival.