Construction of eukaryotic vector pcDNA3.1-flag-pygo2 and expression in C6 cell

目的构建pcDNA3.1-flag-pygo2真核表达载体并在C6细胞中进行表达。方法从小鼠脑胶质细胞中提取总RNA,RT-PCR法反转录合成c DNA,设计引物,调取目的片段,与pcDNA3.1-flag载体连接后转化大肠杆菌DH5α,LB平板筛选菌落,提取质粒。重组质粒pcDNA 3.1-flag-pygo2经过酶切鉴定及测序后,阳离子脂质体法转染C6细胞并经免疫细胞化学染色及蛋白印迹检测重组体的表达。结果重构质粒pcDNA 3.1-flag-pygo2经限制性核酸内切酶EcoRⅠ,HindⅢ酶切分析及测序检查,表明真核表达载体构建正确;瞬时转染C6细胞后,免疫细胞荧光染色及蛋白印迹检测表明转染细胞能够表达外源Pygo2基因。结论成功构建了pcDNA3.1-flag-pygo2真核表达载体并能够在真核细胞中进行表达,这为今后研究pygo2基因在胶质瘤中的作用机制奠定了基础。Objective To construct the eukaryotic expression vector pcDNA3.1-flag-pygo2 and express the combined protein in C6 cell line.Methods To extract total RNA from primary glial cell of mouse and to synthesize c DNA by RT-PCR,then design primer and clone whole segment of pygo2 gene.After the targeted gene was inserted into vector pcDNA3.1-flag,the recombined plamid was transformed into E coli DH5α for LB agar plate screening and the recombined plasmid were extracted and purified.After verification by double enzyme digestion and sequencing.,the constructed eukaryotic expression plamid was transfected to C6 cell line by lipofectamin method,finally the protein expression was detected by immunocytochemical staining as well as western blot.Results The new constructed vector was confirmed by restricted enzyme Eco R I,HindIII digestion assay and correct Pygo2 was verified by sequenceing.finally,pcDNA3.1-flag-hpygo2 can express exogenous pygo2 gene in glioma C6 cell line after transient transfection by the determination of immunocytofluorescent staining and western blot.Conclusion The new plamid pcDNA3.1-flag-pygo2 was constructed successfully,and can express fused protein in eukaryotic cell,which establish the foundation for future research on pygo2 gene function in human glioma

Over-expression of Pygo2 Promotes C6 Cells Proliferation of Glioma

目的通过构建过表达PygO2的重组体上调PygO2表达,探讨其在大鼠胶质瘤C6细胞增殖中的作用及机制。方法重组体经ECOr I和HIndⅢ双酶切鉴定和dnA测序后,用脂质体2000将其转染大鼠胶质瘤C6细胞,采用WESTErn blOT检测外源PygO2蛋白表达,应用克隆形成实验和MTT法检测细胞增殖,流式细胞术检测细胞周期,采用WESTErn blOT检测过表达PygO2对C6细胞中CyClInd1、β-CATEnIn水平的影响,并采用细胞免疫荧光法检测其对C6细胞中CyClInd1、β-CATEnIn亚细胞定位的影响。结果双酶切和测序鉴定结果证实插入序列正确,重组体能有效上调PygO2表达。将重组体转染C6细胞上调PygO2表达后,细胞的生长增殖被显著促进,克隆形成显著增多,细胞周期进程从g1期至S期转变显著增强;且CyClInd1水平随之增高,亚定位无改变,β-CATEnIn水平和亚细胞定位无明显改变。结论成功构建了过表达PygO2的重组体,过表达PygO2通过增高CyClInd1水平,促进细胞从g1期进入S期,从而促进大鼠胶质瘤C6细胞增殖。Objective To up-regulate expression of Pygopus2(Pygo2) by construction of the recombinant vectors of over-expression of Pygo2 protein,and to explore the role and mechanism of over-expression of Pygo2 in C6 cells proliferation of glioma.Methods The recombinant plasmids were digested with EcoRⅠ and Hind Ⅲ to execute the restriction endonuclease identification,then the sequence analysis was assayed by DNA sequencing.The recombinant plasmids were transfected into cultured glioblastoma C6 cells using lipofectamineTM 2000.The exogenous Pygo2 protein level of C6 cells was detected by Western blot analysis.Colony forming assay and MTT assay were used to detect the cell proliferation,and cell cycle analysis was performed by flow cytometry analysis.The effect of Pygo2 over-expression on the level of cyclinD1 and β-catenin of C6 cells was detected by Western blot analysis,and the expression and subcellular location of cyclinD1 and β-catenin of C6 cells were further quantified by immunofluorescent staining.Results The recombinant plasmids were completely coincided with the designs by the restriction map and the sequence analysis,which up-regulated Pygo2 expression of C6 cells efficiently.After Pygo2 expression were up-regulated by transfected C6 cells with the recombinant plasmids,cells proliferation was promoted and colony forming was increased significantly,cell cycle progression from G1 to S transition was enhanced notably.Furthermore,the expression level of cyclinD1 was significantly increased without change of subcellular location,and the expression level and subcellular location of β-catenin were not changed obviously.Conclusion The recombinant vectors of Pygo2 over-expression were constructed successfully.Over-expression of Pygo2 promotes the growth of glioma cells by an increased expression of cyclinD1 to improve G1/S transition.重庆市自然科学基金资助项目(cstc2011jjA10110);重庆市教委科技基金资助项目(KJ100504);福建省自然科学基金资助项目(2009D002

中国干旱区的湖泊

湖泊作为地表水的重要载体,参与自然系统的水分循环,这在世界干旱地区显得格外突出和重要。应用浏览软件(Google Earth),对2001-2005年中国干旱区湖泊进行判别,并结合有关实地调查资料得知,我国干旱区有大小湖泊近400个,其中,10 km2以上的有29个,10 km2以下的有334个,在我国3大自然地理区域中位居第二。开展干旱区湖泊的研究,对破解干旱区的气候变化和湖泊演变过程、形成特征,以及湖泊水生生物资源的科学开发利用和保护具有重要的理论和现实意义

离心场强化晶硅切割废料sisic分离过程油水分相

对切割料中Si和Si C的高效分离进行了研究,利用晶硅切割废料中Si和Si C表面性质的差异,向浆料中加入柴油并充分乳化,使SiC吸附在油滴上实现Si/SiC分离,对乳化后的浆料施加离心力强化油水分相,调节浆料pH值改变颗粒表面Zeta电位,调控乳化后的油滴大小,研究了Si/Si C分离效果、分相时间与浆料pH的关系及附有Si C的油滴表观密度与油滴直径的关系,对乳化后的浆料分别施加超重力系数为10, 50, 100, 150和200的离心力,考察了离心时间2 min时的分相效果和Si/SiC分离效果。结果表明,常重力场中,油滴尺寸越小,分相时间越长,但Si C去除效果变好,pH=7时,水相Si C含量为4.23wt%。油滴直径小于64?m时,油滴在浆料中不可上浮。离心场中,超重力系数为100, p H=7时,水相中Si C含量为5.47wt%,分相时间由460 min缩短为2 min。通过对离心场中Si C的受力分析解析了离心场中Si C在油滴表面的赋存状态,证实离心场作用下,Si C沿油滴表面向离心力方向移动使油滴对Si C的吸附力减小

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轻载型电动载物爬楼机

为了解决老户型无电梯小区搬运大件物品不便的问题,设计了基于滑块机构电助力爬楼机,采用曲柄滑块机构作为助力爬楼机的主要运动机构,以24 V锂电池及减速电机作为爬楼机的动力提供和动力输出机构。通过UG软件的motion进行滑块导路倾角的优化分析,模拟出滑块在运动过程中最低点与最高点之间的距离差为17.674 cm,表明爬楼车的运动满足我国建筑楼梯模数协调标准,物理样机的实验也验证了这一点;通过ANSYS分析,表明小车爬楼运动过程中强度满足受力要求,爬楼机可在楼梯上平稳运行,爬楼过程中的安全性和可靠性将通过机械结构做保障

1;3;5-三甲基苯的热解研究

替代燃料主要包括直链烷烃、支链烷烃、环烷烃及芳烃等。本文利用分子束质谱结合同步辐射单光子电离技术研究了芳烃类替代燃料1,3,5-三甲基苯在流动管反应器中的热解过程。通过扫描光电离效率谱探测到了质量数为2-202的60多种热解产物,包括大量同分异构体和自由基。通过测量不同温度下的光电离质谱获得了燃料的初始分解温度、热解产物的初始生成温度等一系列重要信息,对于1,3,5-三甲基苯分解路径的基元反应研究具有重要意义。根据实验结果和理论分析得到了1,3,5-三甲基苯主要的初始分解路径

质子滴线附近的新核素~(129)Pm

通过重离子引起的融合蒸发反应 92 Mo (40 Ca ,p2n)合成了稀土区未知核素 12 9Pm ,并且配合氦喷嘴快速带传输系统利用X γ符合方法对它进行了首次鉴别 .实验观测到了经 12 9Pm的 (EC +β+ )衰变产生的对应于子核 12 9Nd中 5 2 -→ 1 2 -跃迁的一条 99keVγ线 .根据这条 99keVγ线的时间衰变曲线 ,提取出 12 9Pm的半衰期为 (2 .4± 0 .6 )s

Effect of Pygo2 expression inhibition on proliferation of glioblastoma U251 cells

目的构建rnA干扰(rnAI)重组体抑制PygO2的表达,并探讨其对脑胶质瘤u251细胞增殖的影响。方法针对PygO2 CdnA序列设计并合成一对特异性的含有“短发卡“的寡核苷酸序列及其随机对照序列,经退火后插入PSuPEr中构建重组体。经ECOrⅠ和HIndⅢ双酶切鉴定和dnA测序后,用脂质体2000将其转染脑胶质瘤u251细胞,采用rT-PCr和WESTErn blOT检测PygO2 SHrnA对u251细胞PygO2 MrnA和蛋白的干扰效果,应用克隆形成实验和MTT法检测细胞的增殖情况,流式细胞术检测细胞周期,brdu掺入法检测dnA合成。结果双酶切和测序鉴定证实插入序列完全正确;PygO2 SHrnA显著抑制了其MrnA和蛋白的表达,MTT分析其细胞增殖也被显著抑制(P<0.01)。与SCr SHrnA组的克隆形成数(78.9±6.8)%相比,PygO2 SHrnA组克隆数(55.4±5.2)%显著减少(P<0.05)。与对照组的brdu掺入率(20.96±2.19)%相比,PygO2 SHrnA组的brdu掺入率(8.81±0.56)%显著减少(P<0.05),而SCr SHrnA组的brdu掺入率是(20.35±1.73)%无显著变化。而且,PygO2 SHrnA使u251细胞处于S期百分比显著减少、g1期显著增加(P<0.01),致细胞周期阻滞于g1期。结论成功构建了抑制PygO2表达的重组载体,下调PygO2表达能有效地抑制脑胶质瘤u251细胞dnA合成,使细胞阻滞于g1期,抑制细胞增殖。Objective To construct RNA interference recombinant to inhibit Pygo2 expression and explore its effect on proliferation of glioblastoma U251 cells.Methods A pair of specific short hairpin RNA(shRNA) and scrambled(scr) control shRNA were designed according to Pygo2 cDNA sequence,and then cloned into eukaryotic expression vector pSuper to construct recombinants.After EcoRⅠ and HindⅢ digestion identification and DNA sequencing,the recombinants were used to transfect glioblastoma U251 cells with lipofectamineTM 2000.RT-PCR and Western blotting were applied to detect the RNA interference effects of Pyro2 shRNA.Colony-forming assay and MTT assay,flow cytometry,and BrdU incorporation assay were employed to detect cell proliferation,cell cycle,and DNA synthesis,respectively.Results Double digestion and sequencing results verified that the inserted sequences were correct.Pygo2 shRNA remarkably inhibited the mRNA and protein expression of Pygo2 as well as the proliferation of U251 cells.Comparing with the colony formation rate in the scr shRNA group [(78.9±6.8)%],that in the Pygo2 shRNA group [(55.4±5.2)%] was significantly reduced(P<0.05).The BrdU incorporation rate in the control group [(20.96±2.19)%] was significantly higher than that in the Pygo2 shRNA group [(8.81±0.56)%](P<0.05),but it was similar to that in the scr shRNA group [(20.35±1.73)%].Pygo2 shRNA decreased the U251 cell percentage in S phase and increased that in G1 phase obviously(P<0.01),inducing cell cycle arrest in G1 phase.Conclusion Down-regulation of Pygo2 expression can effectively inhibit DNA synthesis of U251 cells,induce cell cycle arrest in G1 phase,and inhibit cell proliferation.厦门市科技局基金(3502z20089001);福建省自然科学基金(2009D002);中国博士后基金(20080440728);重庆市教委科技基金(KJ100504)---