基因间长非编码RNA:T-UCRs转录活性的验证

目的选择小鼠大脑皮层胚胎发育阶段转录的一类非编码RNA,即基因间区的T-UCRs作为研究对象,探究小鼠基因组上基因间超保守区域的转录活性。方法 1)利用UCSC数据库(NCBI37/mm9)通过生物信息学预测筛选在小鼠14.5 d胎脑中可能表达的基因间UCR;2)Coding Potential Assessment Tool(CPAT)和Coding Potential Caculator(CPC)对筛选结果进行编码能力预测;3)RT-PCR对筛选结果进行验证;4)Real-time PCR分析T-uc.62在不同组织中的表达差异以及在小鼠不同发育阶段脑组织中的表达变化。结果 1)通过UCSC网站上的RNA-seq数据库筛选得到16个可能在小鼠E14.5脑组织中表达的基因间UCRs;2)通过CPAT和CPC分析,16个UCRs均不具有蛋白编码能力,提示可能是非编码RNA;3)经过RT-PCR验证发现有15个是可以表达的;4)以T-uc.62为例,其主要在小鼠发育阶段的脑组织中高表达,而在小鼠的成年组织中低表达甚至不表达。结论基因间超保守区域可以转录生成长非编码RNA,其中,T-uc.62主要在小鼠发育阶段的脑组织中高表达;其可能在大脑发育过程中发挥重要功能。     

三胸家族核心成员DPY30与ASH2L的表达相关性分析

目的探究三胸家族(TrxG)核心成员DPY30与ASH2L表达是否具有相关性。方法 Northern blot检测DPY30与ASH2L在多种人体组织中的表达及佛波醇肉豆蔻酸酯(PMA)诱导人慢性髓源白血病(K562)细胞分化过程中的DPY30与ASH2L表达变化;Western blot与RT-qPCR分别检测ASH2L条件性敲除小鼠(ASH2L-cKO)大脑皮质TrxG成员的蛋白及TrxG核心成员RNA表达情况;利用IGV软件可视化分析小鼠大脑皮质ChIP-seq结果中ASH2L、H3K4me3在ASH2L、DPY30基因染色质上的分布情况。结果 DPY30与ASH2L的表达在人体各组织中分布有一定相似性;DPY30与ASH2L在PMA诱导K562细胞分化过程中的表达均逐渐减少;ASH2L-cKO小鼠大脑皮质中DPY30蛋白水平减少,DPY30的RNA水平改变无统计学意义。结论 DPY30与ASH2L的表达具有相关性,ASH2L可能调控了DPY30蛋白水平。     

CD226基因敲除减轻小鼠失血性休克急性肺损伤

目的探讨内皮细胞特异性敲除CD226(CD226 CKO)对小鼠失血性休克急性肺损伤的影响及其机制。方法雄性野生型(WT)和CD226 CKO小鼠随机分为假手术组和失血性休克组,假手术组只进行心脏穿刺不抽血,失血性休克组抽取30%的总血量。HE染色观察肺脏病变情况,免疫荧光组织化学染色检测肺组织CD31、 CD226和脾脏CD3、 CD226的表达和分布;采用RNA干涉技术(RNAi)敲除人脐静脉内皮细胞(HUVEC)的CD226,厌氧产气袋培养法建立细胞缺氧模型; Western blot法检测小鼠肺组织和HUVEC中Bcl2的蛋白表达; JC-1线粒体膜电位染色检测细胞早期凋亡。结果在失血性休克模型中,与WT小鼠相比, CD226 CKO小鼠急性肺损伤明显减轻,肺组织中Bcl2表达水平升高;体外缺氧细胞学模型同样发现,与siRNA阴性对照(siNC)组细胞相比,特异性敲低CD226的细胞Bcl2蛋白水平增加,细胞凋亡减少。结论敲除CD226基因减轻小鼠失血性休克的急性肺损伤,与增加血管内皮细胞Bcl2的表达、减少细胞凋亡密切相关。     

与小鼠小脑发育相关的一种长非编码RNA Gm2694的鉴定

目的鉴定与小鼠小脑发育相关的长非编码RNA,并验证其各剪接变异体的存在。方法用芯片技术分析小鼠小脑发育不同阶段RNA的表达差异;用实时定量PCR技术对芯片结果加以验证;针对目的 RNA,验证其各剪接变异体的存在及在小鼠小脑中的表达量。结果 1)长非编码RNA Gm2694在小鼠小脑发育过程中呈动态变化趋势;2)其在小鼠小脑中特异性表达;3)在小鼠小脑中检测到Gm2694有8种表达水平各不相同的剪接变异体,其中除Ensembl数据库预测的7个之外,发现了1个新的表达于小脑的剪接变异体。结论 Gm2694是一种在小鼠小脑特异性表达的长非编码RNA,存在8种表达量各不相同的剪接变异体。     

自闭症相关Foxp1突变体Y435X的功能研究

目的:对小鼠Foxp1 Y435X突变体(蛋白序列比对小鼠Y435X等同于人类Y439X)的表达和功能进行研究,探究Y435X突变体与自闭症谱系障碍发病机制之间的联系。方法:通过Western blot分析小鼠神经母细胞瘤N2a细胞中Y435X突变体的蛋白表达水平;利用免疫荧光检测N2a细胞及原代小鼠大脑皮质神经元中Y435X突变体的亚细胞定位;采用小鼠子宫内胚胎基因转染技术,分析Y435X突变体对大脑皮质神经元迁移、最终定位及顶树突发育的影响。结果:Western blot结果显示,Y435X突变体在N2a细胞中产生大约56 kD的截短蛋白,与野生型Foxp1相比,表达量明显增加;免疫荧光结果说明,Y435X突变体在胞质中形成聚集体,并导致核膜皱缩;Y435X突变体改变了小鼠出生后大脑皮质神经元的正常定位及顶树突的生长方向。结论:Foxp1 Y435X突变体在皮质神经元中产生聚集体,并干扰出生后皮质神经元的正常定位及顶树突的生长。     

TRPC1缺失对小鼠脑神经细胞生存的影响

 目的: 研究瞬时受体电位通道1(TRPC1)缺失对小鼠海马神经元生存的影响。方法: 选用6月龄TRPC1基因敲除小鼠及其对照小鼠,通过神经元特异性标志物NeuN免疫染色、尼氏染色及TUNEL染色检测TRPC1敲除小鼠海马CA1、CA3及齿状回(DG)神经细胞的变化情况。通过Western blot检测TRPC1基因敲除小鼠促凋亡因子C/EBP同源蛋白(C/EBP homologous protein,CHOP)及凋亡相关蛋白cleaved caspase-3的表达水平。结果: NeuN免疫荧光和尼氏染色发现,TRPC1基因敲除小鼠海马CA1、CA3及DG区神经细胞显著减少;TUNEL染色检测发现TRPC1基因敲除小鼠以上区域神经细胞凋亡显著增加;Western blot结果显示,与对照小鼠相比,TRPC1基因敲除小鼠海马组织CHOP及cleaved caspase-3的水平均显著升高。结论: TRPC1基因缺失可导致小鼠海马神经元数量显著减少,其机制可能与TRPC1缺失促进神经细胞凋亡有关。     

利用经典的Zbed3敲除鼠探索ZBED3在小鼠大脑皮质发育过程中的作用

目的利用敲除鼠探索锌指BED结构域超家族成员ZBED3在小鼠皮质发育过程中的作用。方法利用原位杂交和免疫荧光技术检测Zbed3敲除鼠的敲除效率。利用免疫荧光技术检测Zbed3敲除对小鼠皮质神经前体细胞增殖的影响。利用EdU标记技术和免疫荧光技术检测Zbed3敲除对皮质层次形成的影响。利用RT-qPCR和免疫印迹技术探索Zbed3敲除鼠中其他分子的表达变化。结果 Zbed3敲除鼠中检测不到Zbed3 mRNA和蛋白的表达。Zbed3敲除不影响神经祖细胞增殖和皮质层次形成。在Zbed3敲除鼠中,β-catenin的表达水平上调。结论 Zbed3敲除对小鼠皮质发育无明显影响。     

Smad4对小鼠眼睑发育的影响

目的: 利用眼特异性 Smad4 基因敲除小鼠,观察其眼睑表型并探讨其眼睑发育异常的可能机制。方法: 选择 PAX6 第一启动子P0驱动的晶体外胚层特异性表达 Cre重组酶的转基因小鼠(Le-Cre)作为介导敲除的工具鼠,将其与 Smad4 条件基因小鼠( Smad4 ^fl/fl)交配获得 Le-Cre特异性Smad4 基因敲除小鼠,通过HE染色揭示其眼睑组织形态学的改变,采用免疫染色技术对某些关键蛋白的表达进行检测并与野生型小鼠进行比较,并检测其眼睑上皮细胞凋亡和增殖的改变。结果: Smad4 在眼睑的失活导致眼睑在发育过程中不能融合,生后眼睑保持开放;Smad4在眼睑的表达缺失不影响眼睑睑缘上皮细胞的增殖和凋亡,但导致上皮细胞内c-Jun磷酸化过程受损,表皮生长因子受体(EGFR)核转位受影响,引起细胞内肌动蛋白束装配异常而导致上皮细胞移行受损,出现眼睑发育时融合不能。结论: Smad4在眼睑发育中对于眼睑的闭合是必需的。     

长链非编码RNA AK089560在间质干细胞成骨与成脂分化中的表达

背景：最近研究发现众多长链非编码RNA调控干细胞多潜能性和分化。长链非编码RNA AK089560在干细胞多向分化中的表达变化及作用,目前尚不清楚。目的：观察间质干细胞C3H10T1/2成骨分化与成脂分化过程中AK089560的表达。方法：重组人骨形态发生蛋白2诱导间质干细胞C3H10T1/2成骨分化,碱性磷酸酶染色鉴定早期成骨分化。地塞米松、吲哚美辛和胰岛素三因子联合诱导C3H10T1/2成脂分化,油红O染色鉴定成脂分化结果。采用qRT-PCR检测诱导前后不同时间点AK089560表达的变化。采用RNAfold软件预测AK089560二级结构,UCSC基因组浏览器分析AK089560邻近编码蛋白基因,fancyGENE在线软件构件长链非编码RNA与编码基因关系图。结果与结论：C3H10T1/2经成骨诱导后,70%以上细胞碱性磷酸酶染色阳性;成脂分化诱导后,80%以上细胞油红O染色阳性。qRT-PCR结果显示,长链非编码RNA AK089560在成骨分化与成脂分化第2,4,6天表达均明显下降,成骨分化与成脂分化第2,4,6天与相应时间未分化组相比均差异有显著性意义(P < 0.05)。生物信息学分析表明,LncRNA AK089560具有复杂茎环结构,与编码基因Sema3a形成sense overlap关系。结果表明AK089560在间质干细胞成骨与成脂分化中下调表达,提示其可能参与调控间质干细胞多向分化。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

CX3CR1通过调节Ca2+内流介导神经元凋亡影响缺血性卒中小鼠预后

目的观察脑缺血后趋化因子受体1(CX3CR1)在神经元上的表达变化,并探讨其作用及机制。方法将野生型C57/BL6小鼠和CX3CR1基因敲除小鼠,分别设置对照组(假手术组)和小鼠中动脉永久闭塞(pMCAO)模型,用磁共振成像(MRI)检测30 min后脑缺血范围和24 h后梗死面积;免疫荧光三重染色法检测凋亡;Western blot检测CX3CR1蛋白的表达。在体外,培养原代神经元,建立氧糖剥离(OGD)细胞缺血模型;用MTT法检测细胞存活率;免疫荧光检测神经元CX3CR1的表达;激光共聚焦显微镜观察神经元内Ca^2+浓度变化。结果在野生型小鼠中,与对照组和健侧相比,pMACO后,患侧CX3CR1表达显著升高(P<0.05),且CX3CR1与凋亡蛋白caspase-3在神经元上共表达;与野生型小鼠相比,CX3CR1基因敲除小鼠pMACO 30 min后缺血损伤面积相似,但24 h后CX3CR1基因敲除小鼠梗死面积小于对照组(P<0.05);在体外,原代神经元OGD后CX3CR1表达显著升高(P<0.05),敲除神经元上CX3CR1,可减轻谷氨酸介导的兴奋性损伤,细胞存活率显著上升,同时观察到,敲除CX3CR1可降低谷氨酸介导的Ca^2+内流到神经元的速度和总量。结论缺血可诱导神经元CX3CR1的表达,且神经元CX3CR1可以通过调节Ca^2+内流来介导神经元的凋亡。     

小鼠H2B-GFP真核表达载体的构建与鉴定

目的 构建小鼠H2B-绿色荧光蛋白（GFP）真核表达载体，为动态观察小鼠细胞中染色体的形态变化，进一步研究小鼠耐药细胞中双微体（DMs）的形成机制提供有效的分子工具。方法 利用RT-PCR的方法获得小鼠H2B cDNA，以羧基端插入pcDNA3.1/CT-GFP-TOPO载体上，构建H2B-GFP真核表达载体，经菌液PCR、酶切及DNA测序鉴定插入片段大小、方向及序列的正确性，提取质粒转染小鼠胚胎成纤维细胞系NIH3T3进行鉴定。结果 经菌液PCR、酶切和测序，证明小鼠H2B-GFP真核表达载体含有大小、方向及序列正确的H2B cDNA片段，转染NIH3T3后在细胞核中表达。结论 作者成功构建了同时携带有G418筛选位点及多酶切位点的小鼠H2B-GFP真核表达载体，为其在小鼠体外培养细胞中的表达奠定了基础。     

The noncoding RNA AK127244 in 2p16.3 locus: A new susceptibility region for neuropsychiatric disorders

The presence of redundant copy number variants (CNVs) in groups of patients with neurological diseases suggests that these variants could have pathogenic effect. We have collected array comparative genomic hybridization (CGH) data of about 2,500 patients affected by neurocognitive disorders and we observed that CNVs in 2p16.3 locus were as frequent as those in 15q11.2, being both the most frequent unbalances in our cohort of patients. Focusing to 2p16.3 region, unbalances involving NRXN1 coding region have been already associated with neuropsychiatric disorders, although with incomplete penetrance, but little is known about CNVs located proximal to the gene, in the long noncoding RNA AK127244. We found that, in our cohort of patients with neuropsychiatric disorders, the frequency of CNVs involving AK127244 was comparable to that of NRXN1 gene. Patients carrying 2p16.3 unbalances shared some common clinical characteristics regardless NRXN1 and AK127244 CNVs localization, suggesting that the AK127244 long noncoding RNA could be involved in neurocognitive disease with the same effect of NRXN1 unbalances. AK127244 as well as NRXN1 unbalances seem to have a particular influence on language development, behavior or mood, according with the topographic correlation between NRXN1 expression and prefrontal cortex functions.     

Gerbil Angiotensin II AT1 receptors are highly expressed in the hippocampus and cerebral cortex during postnatal development

Increasing evidence suggests that Angiotensin II, classically known from its many effects regulating salt and water homeostasis, is also involved in brain development and cognitive functions through activation of AT₁ Angiotensin II receptors. The recently cloned gerbil AT₁ receptor is expressed in brain areas controlling hydro-mineral homeostasis, and particularly highly expressed in limbic areas such as the hippocampal formation. We quantified the gerbil AT₁ receptor messenger RNA expression and receptor binding by quantitative in situ hybridization and receptor autoradiography, respectively, in the hippocampal formation and cerebral cortex of gerbils during postnatal development. The receptor messenger RNA and binding were present from birth and showed a gradual and sustained increase through postnatal maturation in the CA1 and CA2 regions of the hippocampus and in the dentate gyrus. Conversely, in the CA3 region, no binding was detected while receptor messenger RNA peaked at 15 days after birth and disappeared in the adult. The highest receptor messenger RNA expression and binding were found in the septomedial portions of the CA1 region and at septal levels of the CA2 region.We detected the highest receptor messenger RNA expression at postnatal day one in the frontolateral pole of the cerebral hemispheres. In these areas, and in the frontoparietal and insular cortex, receptor messenger RNA dramatically decreased during postnatal life. Similarly, we found receptor messenger RNA expression in the cingulate, retrosplenial, perirhinal and infralimbic cortex with higher values during the first two weeks of development and decreased expression in the adult. However, receptor binding in the cerebral cortex, did not decrease during postnatal life.The differential profile of receptor messenger RNA expression and binding in the gerbil cortex and hippocampus during postnatal maturation suggest a role for AT₁ receptors in the development and function of the corticohippocampal system.     

Gerbil angiotensin II AT1 receptors are highly expressed in the hippocampus and cerebral cortex during postnatal development

Increasing evidence suggests that Angiotensin II, classically known from its many effects regulating salt and water homeostasis, is also involved in brain development and cognitive functions through activation of AT1 Angiotensin II receptors. The recently cloned gerbil AT1 receptor is expressed in brain areas controlling hydro-mineral homeostasis, and particularly highly expressed in limbic areas such as the hippocampal formation. We quantified the gerbil AT1 receptor messenger RNA expression and receptor binding by quantitative in situ hybridization and receptor autoradiography, respectively, in the hippocampal formation and cerebral cortex of gerbils during postnatal development. The receptor messenger RNA and binding were present from birth and showed a gradual and sustained increase through postnatal maturation in the CA1 and CA2 regions of the hippocampus and in the dentate gyrus. Conversely, in the CA3 region, no binding was detected while receptor messenger RNA peaked at 15 days after birth and disappeared in the adult. The highest receptor messenger RNA expression and binding were found in the septomedial portions of the CA1 region and at septal levels of the CA2 region. We detected the highest receptor messenger RNA expression at postnatal day one in the frontolateral pole of the cerebral hemispheres. In these areas, and in the frontoparietal and insular cortex, receptor messenger RNA dramatically decreased during postnatal life. Similarly, we found receptor messenger RNA expression in the cingulate, retrosplenial, perirhinal and infralimbic cortex with higher values during the first two weeks of development and decreased expression in the adult. However, receptor binding in the cerebral cortex, did not decrease during postnatal life. The differential profile of receptor messenger RNA expression and binding in the gerbil cortex and hippocampus during postnatal maturation suggest a role for AT1 receptors in the development and function of the corticohippocampal system.     

乙型肝炎病毒E抗原肝细胞作用蛋白AK026018表达谱芯片的研究

目的 应用基因芯片技术,筛选能被乙型肝炎病毒E抗原（HBeAg）肝细胞作用蛋白AK026018反式调节的靶基因,初步研究该蛋白的生物学功能.方法 应用反转录聚合酶链反应（RT-PCR）技术,从HepG2细胞中扩增编码AK026018蛋白的全基因,构建真核表达载体,转染肝母细胞瘤系HepG2,提取总mRNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1的HepG2细胞进行DNA芯片分析并比较.结果 经限制性内切酶分析和DNA序列测定鉴定构建的重组表达载体正确.在8464个基因表达谱的筛选中,发现有122个基因有差异表达,其中78种基因表达水平显著下调,45种基因表达水平显著上调.结论 成功地应用DNA芯片技术筛选出HBeAg结合蛋白新基因AK026018的反式调节蛋白,证明该基因对于肝细胞基因表达谱有显著影响.     

Expression of C5a receptor in mouse brain: role in signal transduction and neurodegeneration.

In this study we explored the potential role of the complement derived anaphylatoxin C5a and the expression of its receptor in mouse brain. Using in situ hybridization, we found that C5a receptor messenger RNA is expressed in mouse brain. In response to intraventricular kainic acid injection, there was marked increase in the C5a receptor messenger RNA expression, particularly in hippocampal formation and cerebral cortex. C5a ligand-binding autoradiography confirmed the functional expression and elevation of the C5a receptor post-lesioning. The expression of C5a receptor messenger RNA in brain was confirmed by northern blot hybridization of total RNA from neuronal and glial cells in vitro. Based on these findings we explored the role of C5a in mechanisms of signal transduction in brain cells. Treatment of primary cultures of mouse astrocytes with human recombinant C5a resulted in the activation of mitogen-activated extracellular signal-regulated protein kinase. This response appeared to be mediated by the C5a receptor since astrocyte cultures derived from C5a receptor knockout mice were not responsive to the treatment. Understanding the regulation of C5a receptor in brain and mechanisms by which pro-inflammatory C5a modulates specific signal transduction pathways in brain cells is crucial to studies of inflammatory mechanisms in neurodegeneration.     

Expression of the peplomer glycoprotein of murine coronavirus JHM using a baculovirus vector

The gene encoding the E2 peplomer glycoprotein of coronavirus mouse hepatitis virus JHM strain (JHMV) has been inserted into the genome of Autographa californica nuclear polyhedrosis baculovirus (AcNPV) in lieu of the coding region of the AcNPV polyhedrin gene. This recombinant virus produced E2 protein in insect cells under the control of the baculovirus polyhedrin promotor. The expressed E2 protein was shown in size and antigenic properties to be similar to the E2 protein produced in mouse cells infected by JHMV. The expressed E2 protein was glycosylated and transported to the cell surface; however, no proteolytic cleavage was detected in insect cells. The sera from rats immunized with partially purified E2 protein derived from insect cells reacted in immunoprecipitation and immunofluorescence experiments with the E2 protein produced in JHMV-infected mouse cells. The antiserum failed to neutralize the infectivity of JHMV. These results suggest that the E2 protein expressed by the recombinant baculovirus in insect cells is similar but not identical to the E2 protein produced in JHMV-infected mouse cells. The inability of the E2 protein expressed in insect cells to produce neutralizing antibody is discussed.