ERK1/2在高温致神经管畸形神经上皮细胞中的表达变化

目的 探讨高温致神经管畸形(NTDs)作用的分子机制,为防治NTDs的发生提供理论依据.方法 在高温致金黄地鼠NTDs模型的基础上,应用免疫荧光染色技术,观察NTDs发生过程中p-ERK1/2在鼠胚神经上皮细胞中的表达变化.结果 对照组和实验组孕鼠在高温水浴处理后16、24h,p-ERK1/2免疫阳性产物分布于鼠胚神经上皮细胞和周围间充质细胞的胞浆中;水浴后36、60h,p-ERK1/2表达部位出现了由细胞浆向细胞核的转移;高温处理后,p-ERK1/2在实验组各期胚胎神经上皮细胞内的表达均比对照组减弱.结论 ERK1/2参与胚胎神经管的发育过程,其表达降低在高温致神经管畸形的发生中起重要作用.     

热休克蛋白在高温致神经管畸形中的表达

目的：检测热休克蛋白在高温致神经管畸形中的表达状况,以探讨高温致神经管畸形的机制。方法：在高温致金黄地鼠神经管畸形的动物模型上,利用免疫组织化学(SABC法)方法,检测高温致神经管畸形中,热休克蛋白(HSP70和HSP90)在神经上皮细胞及周围间充质细胞中的表达状况;同时利用地高辛标记的寡核苷酸探针进行原位杂交,检测HSP70 mRNA和HSP90 mRNA在神经上皮细胞及周围间充质细胞中的转录状况。结果：高温处理后2h,鼠胚神经上皮细胞及周围间充质细胞HSP70、HSP90的表达与正常对照组相比明显增强,8h和16h的表达达到高峰,24h后与对照组水平一致。原位杂交结果显示,高温处理后2h神经上皮细胞及周围间充质细胞中出现HSP70 mRNA及HSP90mRNA杂交阳性信号,8h阳性信号最强,16h后阳性信号减弱,至24h后未见阳性信号。结论：高温可引起神经上皮细胞及其周围间充质细胞HSP70和HSP90应激性表达,这可能是胚胎受到高温作用后发生的一种保护性反应。     

大豆Lea5基因的克隆与表达分析

为了解高温处理促进大豆幼胚萌发成苗的分子生物学机制,采用经典的基因表达差异显示技术,分析大豆品种日本晴高温处理的幼胚与对照组材料的差异表达基因。对其中一个差异表达基因K6进行了测序和比对分析,结果表明该基因序列与大豆Williams 82基因组中的Lea5基因相似度高达99%,可以确定为Lea5基因。Blastp比对分析结果表明大豆Lea5蛋白为LEA-3亚家族成员。利用蛋白质分析软件分析了Lea5基因推测的蛋白质结构的特点,结果表明该蛋白质由113个氨基酸组成,相对分子量为12.283 k D,等电点高达10.12。该蛋白质不形成典型的二级结构。RT-PCR分析表明该基因表达具有组成型特点,在大豆幼胚发育过程中稳定表达,在大豆的根、胚轴和叶片等器官中均有表达。     

生长抑素基因在不同性别和年龄黑线仓鼠哈氏腺的差异表达

哈氏腺作为“视网膜-松果体轴”的候补结构,介导了体外光信号与体内神经内分泌调控过程,具有多样性和复杂的生理功能,并受神经和体液因素的双重调控。生长抑素（somatostatin, SS）是调控动物生长发育的主要因子之一,检测SS基因在黑线仓鼠哈氏腺的差异表达模式,为光信号调控动物的生长发育机制提供理论依据。以野生黑线仓鼠（Cricetulus barabensis）为研究对象,测量了不同性别和年龄个体哈氏腺的形态学指标,克隆了哈氏腺SS cDNA序列,实时荧光定量PCR方法检测了哈氏腺中SS基因的表达变化。结果显示：（1）黑线仓鼠体重无性别差异,随年龄增长而增加;哈氏腺的长度和重量也随年龄增长而增加;调整体重影响后,哈氏腺的重量和长度没有年龄之间的差异,但哈氏腺重量存在性别间差异（P<0.05）。（2）克隆到黑线仓鼠哈氏腺SS cDNA序列489 bp ,并对序列结构进行分析,黑线仓鼠哈氏腺SS cDNA具有典型分泌蛋白cDNA的特征,并在进化上高度保守;（3）检测到SS mRNA在黑线仓鼠哈氏腺中均有表达,表达量存在年龄和性别差异（P<0.05）;（4）随着个体性成熟和衰老,黑线仓鼠哈氏腺中SS mRNA的相对表达量逐渐降低,其表达量分别与哈氏腺长度和重量的发育变化负相关（r=-0.557,P=0.022;r=-0.806,P＜0.001）。结果表明,黑线仓鼠哈氏腺重量和SS表达量均表现出两性异形,可能是对不同繁殖策略的适应;SS基因作为负调控因子参与野生黑线仓鼠哈氏腺的生长发育过程。     

化学致癌物DNP致人胚鼻咽上皮细胞转化相关基因的鉴定

为了探讨DNP致癌的分子机理,鉴定出化学致癌物二亚硝基哌嗪(DNP)致人胚鼻咽上皮细胞转化相关的基因及其活化方式．采用DNA共转染、裸鼠致瘤性试验、Southern杂交、PCR测序和序列同源性比较分析等,对DNP转化的人胚鼻咽上皮细胞株HENE—DNP进行研究．经过两轮DNA共转染和裸鼠致瘤性实验．Southern杂交表明,裸鼠肿瘤DNA中均含有人特异性高度重复序列Alu．用人Ha-ras、Ki-ras及N-ras癌基因特异性引物对裸鼠肿瘤DNA进行PCR扩增,仅能扩增出人Ha-ras基因相应的片段．Southem杂交进一步证实．裸鼠肿瘤DNA中存在与人Ha-ras基因片段大小一致的杂交带．RT-PCR产物测序,并将测序结果与GenBank进行序列同源性比较分析,发现裸鼠肿瘤中人Ha-ras基因cDNA第26位密码子第2位碱基发生了T→C的转换,编码的氨基酸由亮氨酸相应地变换成丝氨酸．化学致癌物DNP致人胚鼻咽上皮细胞转化相关的基因是Ha-ras,原癌基因c-Ha-ras激活可能是DNP转化人胚鼻咽上皮细胞的分子机制之一。     

甲氨蝶呤对早期神经胚基因表达的影响

目的:利用甲氨蝶呤(methotrexate,MTX)干预孕鼠,探讨MTX对早期神经胚基因表达的影响。方法:用MTX(4.5 mg/kg体重)干预孕鼠,通过NimbleGene表达谱芯片、Real time-PCR及免疫组化等方法进行差异表达基因的筛选和验证。结果:MTX处理后神经管畸形(NTDs)发生率为32.1%。表达谱芯片筛选出166个差异表达基因,其中4个凋亡相关基因(Endog,Trp53,Casp3,Bax)均表现为上调(fold change1.5,P0.05),3个增殖相关基因(Ptch1,Pla2g4a,Foxg1)均表现为下调(fold change0.67,P0.05);NTDs胚胎神经上皮Caspase-3表达显著升高(P0.05),phospho-histone H3(pH3)表达显著降低(P0.05)。结论:MTX影响了早期神经胚的基因表达,尤其是引起了凋亡、增殖相关基因表达的异常,这可能在叶酸缺乏引起NTDs发生的相关机制之一。     

人胚与鼠胚神经干细胞体外培养的差异

为比较人胚与鼠胚神经干细胞体外培养的差异。实验采用具有丝裂原作用的细胞生长因子。结合无血清细胞培养技术从人胚和鼠胚皮层分离神经干细胞。在连续传代过程中观察其体外培养特性,免疫荧光染色检测Nestin抗原和分化后特异性成熟神经细胞抗原的表达,并用流式细胞仪检测神经干细胞分化情况。结果表明：（1）使用单一生长因子即可从鼠胚皮层分离神经干细胞,但在人胚却需同时使用多种生长因子,协同使用bFGF,EGF和LIF是人胚神经干细胞体外培养的较佳条件;（2）鼠胚皮层神经干细胞在连续传代过程中增殖速度快于人胚,其Nestin阳性率和BrdU标记的阳性率亦高于人胚,表明其增殖能力明显高于人胚,（3）人胚神经干细胞较鼠胚更易分化为神经元。     

热中性区临界温度对黑线仓鼠哺乳期能量收支的影响

本研究测定了热中性区上、下临界点温度(25℃和32.5℃)下黑线仓鼠Cricetulus barabensis哺乳期能量收支、血清催乳素水平和褐色脂肪组织(BAT)解偶联蛋白1(UCP_1)基因的mRNA表达等。结果发现,32.5℃组静息代谢率、热传导率、摄入能和泌乳能量支出,以及胎仔质量均显著低于25℃组,32.5℃组BAT UCP_1 mRNA表达显著下调。血清催乳素水平、下丘脑神经肽Y和刺鼠相关蛋白基因的mRNA表达的组间差异未达到显著水平。结果表明,热中性区上临界点温度显著抑制哺乳期黑线仓鼠的最大能量收支,支持"热耗散限制假说",催乳素介导的神经内分泌调节机制可能未参与高温下黑线仓鼠哺乳期最大能量收支的热耗散限制。     

植物体细胞胚发生过程中基因表达的研究进展

植物体细胞胚胎发生是一个复杂的发育过程,研究者们通过分析植物体细胞胚发生过程中的基因表达或胚性组织和非胚性组织中基因的差异表达,获得了在体细胞胚发生过程不同时期表达的基因,并分析了这些基因在胚胎发生途径中可能的作用。综述了在植物体细胞胚发生过程中细胞周期相关基因、胁迫和激素应答相关基因、信号转导相关基因、晚期胚胎丰富蛋白基因及与体细胞胚发生相关的胞外蛋白基因表达的研究进展。     

EB病毒潜伏膜蛋白1诱导人鼻咽上皮细胞端粒酶的表达

上皮细胞逃避老化期是细胞永生化过程中一个重要分子事件,端粒酶活性表达是维持人染色体端粒长度、抑制细胞进入老化期的关键因素之一.我们利用最新的端粒酶PCR-ELISA半定量技术,检测永生化早期阶段人胚鼻咽上皮细胞中端粒酶表达的情况,探讨EB病毒在人胚鼻咽上皮细胞永生化过程中的分子机制.结果表明,EB病毒诱导老化前期人胚鼻咽上皮细胞端粒酶表达,从而促使人胚鼻咽上皮细胞逃避老化期、进入永生化早期阶段.此外,我们还首次发现,人胚鼻咽上皮细胞表达端粒酶活性依赖于EB病毒LMP1蛋白的表达水平和LMP1分子的完整性,LMP1可能通过诱导端粒酶活性表达促进人鼻咽上皮细胞永生化.我们的实验为进一步探讨EB病毒诱导人胚鼻咽上皮细胞永生化的作用机制提供了实验基础.     

Thrombospondin-1 is a mediator of the neurotypic differentiation induced by EGF in thymic epithelial cells

Thymic epithelial cell component originates from cranial neural crest as well as from endoderm and ectoderm of the third pharyngeal pouch and branchial cleft. Epidermal growth factor (EGF) has been previously shown to play a crucial role in directing thymic epithelial cells toward a neural-oriented cell fate. To identify genes that are involved in the EGF-induced neurotypic differentiation of the thymic stroma-derived TC-1S cell line, we studied EGF-treated and untreated cells by RNA fingerprinting PCR-based differential screening. We obtained 23 distinct sequences including 18 known genes and 5 sequences previously unreported, which are currently under characterization. Here, we describe the involvement of one of the isolated genes, the thrombospondin-1, as a mediator of the neurotypic differentiation induced by EGF in TC-1S cells. We show that thrombospondin-1 mRNA and protein levels are increased by EGF. Moreover, exogenous thrombospondin-1 is able to enhance the outgrowth of neurite-like processes as well as the expression of neurofilaments and neural cell adhesion molecule in TC-1S cells. These observations suggest that the up-regulation of thrombospondin-1 synthesis induced by EGF contributes to the differentiation choice of thymic epithelial cells toward a neural fate, reminiscent of their neural crest origin.     

Expression of EphA8‐Fc in transgenic mouse embryos induces apoptosis of neural epithelial cells during brain development

EphAs and ephrin‐As are expressed in multiple regions of the developing brain and have been implicated in regulating brain size. Here, we report the identification of a novel mechanism in which reverse signaling through ephrin‐As controls neural epithelial cell number in the developing brain. Ectopic expression of EphA8‐Fc in transgenic embryos induced apoptosis of neural epithelial cells, which was accompanied by a dramatic decrease in brain size. The number of ephrin‐A5‐expressing cells was significantly reduced in the brain region where EphA8‐Fc was ectopically expressed. Furthermore, in vitro culture of the dissociated neuroepithelial cells revealed that EphA8‐Fc enhanced apoptotic cell death of the ephrinA5‐expressing cells in a caspase‐dependent manner. Thus, our results suggest that reverse signaling through ephrin‐As is biochemically linked with caspase‐dependent proapoptotic signaling during early brain development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 702–712, 2013     

The effect of hyperthermia on the induction of cell death in brain, testis, and thymus of the adult and developing rat

Stressful stimuli can elicit 2 distinct reactive cellular responses, the heat shock (stress) response and the activation of cell death pathways. Most studies on the effects of hyperthermia on the mammalian nervous system have focused on the heat shock response, characterized by the transient induction of Hsps, which play roles in repair and protective mechanisms. This study examines the effect of hyperthermia on the induction of cell death via apoptosis, assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and active caspase 3 cytochemistry, in the adult rat brain, testis, and thymus. Results show that a fever-like increase in temperature triggered apoptosis in dividing cell populations of testis and thymus, but not in mature, postmitotic cells of the adult cerebellum. These differential apoptotic responses did not correlate with whole-tissue levels of Hsp70 induction. We further investigated whether dividing neural cells were more sensitive to heat-induced apoptosis by examining the external granule cell layer of the cerebellum at postnatal day 7 and the neuroepithelial layers of the neocortex and tectum at embryonic day 17. These proliferative neural regions were highly susceptible to hyperthermia-induced apoptosis, suggesting that actively dividing cell populations are more prone to cell death induced by hyperthermia than fully differentiated postmitotic neural cells.     

Developmental regulation of central spindle assembly and cytokinesis during vertebrate embryogenesis

Mitosis and cytokinesis not only ensure the proper segregation of genetic information but also contribute importantly to morphogenesis in embryos. Cytokinesis is controlled by the central spindle, a microtubule-based structure containing numerous microtubule motors and microtubule-binding proteins, including PRC1. We show here that central spindle assembly and function differ dramatically between two related populations of epithelial cells in developing vertebrate embryos examined in vivo. Compared to epidermal cells, early neural epithelial cells undergo exaggerated anaphase chromosome separation, rapid furrowing, and a marked reduction of microtubule density in the spindle midzone. Cytokinesis in normal early neural epithelial cells thus resembles that in cultured vertebrate cells experimentally depleted of PRC1. We find that PRC1 mRNA and protein expression is surprisingly dynamic in early vertebrate embryos and that neural-plate cells contain less PRC1 than do epidermal cells. Expression of excess PRC1 ameliorates both the exaggerated anaphase and reduced midzone microtubule density observed in early neural epithelial cells. These PRC1-mediated modifications to the cytokinetic mechanism may be related to the specialization of the midbody in neural cells. These data suggest that PRC1 is a dose-dependent regulator of the central spindle in vertebrate embryos and demonstrate unexpected plasticity to fundamental mechanisms of cell division.     

Quantification and localization of expression of the retinoic acid receptor-beta and -gamma mRNA isoforms during neurulation in mouse embryos with or without spina bifida

BACKGROUND: Previous studies observed that retinoic acid receptor-gamma (RARgamma) is expressed in the open caudal neuroepithelium but that RARbeta is expressed in the closed neural tube. Furthermore, retinoic acid (RA) induces RARbeta expression, a molecular event associated with neural tube closure, but treatment with RA at the appropriate gestation time causes failure of neural tube closure. Since there are four isoforms of RARbeta, perhaps the isoforms expressed in the closed neural tube and induced by RA are different. To investigate the hypothesis that the switch from RARgamma to RARbeta is mechanistically linked to neural tube closure, this study determined the concentrations and distributions of RARbeta and RARgamma isoforms in mouse embryos with RA-induced neural tube defects and in splotch (Sp) mutant embryos with spina bifida. METHODS: Absolute concentrations of RARbeta and RARgamma isoforms were determined throughout primary neurulation (gestational day 8.5-10.0) in treated or untreated C57BL/6J mouse whole embryos by ribonuclease protection analysis. Treatment consisted of an oral dose of 100 mg/kg of all-trans-RA on gestational day 8.5. Spatial distributions of RARbeta and RARgamma were examined in RA-treated and Sp mutant embryos by in situ hybridization. RESULTS: RARbeta2, gamma1, and gamma2 were expressed in untreated embryos and were induced 4.5-, 1.6-, and 4.0-fold, respectively, 4 hr after treatment with RA. In embryos with RA-induced spina bifida, RARbeta2 was expressed in the closed neural tube while RARgamma1 and RARgamma2 were expressed in the open caudal neuroepithelium. In splotch mice with spina bifida, the boundary between RARbeta and RARgamma did not correspond to the site of neural tube closure. CONCLUSIONS: In RA-treated embryos, the relationship between RARbeta expression in the closed and RARgamma in the open caudal neuroepithelium was not altered. However, in splotch embryos with spina bifida, the juncture between RARbeta and RARgamma expression remained in the same anatomical position in the neuroepithelium irrespective of the neural tube closure status and suggests that the switch from RARgamma to RARbeta expression in the closing caudal neuroepithelium may not be causally linked to neural tube closure in the splotch mutant.