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

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以特种玉米为指示作物的有机施肥,连续两年田间试验,结果表明:施肥后的土壤耕层氮、磷、钾养分盈亏幅度为1.9%15.7%、2.6%12.0%、-0.29%0.13%,比单施化肥增加3.016.8、2.812.2、0.250.67个百分点;氮、磷、钾盈亏量与施肥投入量呈极显著正相关。参试的3种有机肥中,猪粪肥表现为土壤生态培肥效果比较好,施肥两年耕层土壤氮、磷、钾含量均有盈余,存量比例为0.17∶1.00∶0.04

焦炭中硫的空间分布规律研究

采用炼焦混合煤模拟工业焦化过程，研究了焦炭中硫的空间分布规律。结果表明，焦炭柱同一高度的有机硫、无机硫的质量分数从中心到边缘逐渐升高；相同取样位置处裂纹表面的有机硫、无机硫比对应内部位置硫的质量分数高；对于炭化室直径为230mm的模拟实验，有机硫增加约0．035％，无机硫增加约0．08％。XPS分析显示，有机硫与无机硫的质量分数的差异是由噻吩硫及金属硫化物的质量分数不同造成的。二维相似模拟实验进一步证实焦炭柱中硫的质量分数从中心沿径向到边缘逐渐升高

梯度温度分布下半焦／焦炭收缩规律的研究

利用炉侧安装了移动测量标尺的加热炉（φ150mm×300mm），分别以三种加热速率（1．0℃／min、1．5℃／min和3．0℃／min）及两种堆密度（880kg／m^3和1080kg／m^3）模拟工业炼焦，研究了1500g的炼焦用煤在焦化过程中径向收缩与焦化时间、中心温度、焦化升温速率及梯度温度的关系。结果表明，中心温度为280℃～360℃时煤柱开始收缩，900℃左右收缩结束，径向收缩值5mm～8．5mm，收缩率7％～12％；加热速率和堆密度增大，煤柱径向温度梯度增大，径向收缩值减小；加热速率增大，开始收缩时的中心温度降低，第二收缩高峰逐渐减弱，各梯度温度降低，煤柱收缩系数减小；堆密度大，开始收缩时的中心温度和各梯度温度均较高，对收缩系数及收缩高峰无明显影响。在焦化的不同时期，煤柱不同位置的升温速率不同

梯度温度分布下半焦／焦炭收缩规律的研究

利用炉侧安装了移动测量标尺的加热炉（φ150mm×300mm），分别以三种加热速率（1．0℃／min、1．5℃／min和3．0℃／min）及两种堆密度（880kg／m^3和1080kg／m^3）模拟工业炼焦，研究了1500g的炼焦用煤在焦化过程中径向收缩与焦化时间、中心温度、焦化升温速率及梯度温度的关系。结果表明，中心温度为280℃～360℃时煤柱开始收缩，900℃左右收缩结束，径向收缩值5mm～8．5mm，收缩率7％～12％；加热速率和堆密度增大，煤柱径向温度梯度增大，径向收缩值减小；加热速率增大，开始收缩时的中心温度降低，第二收缩高峰逐渐减弱，各梯度温度降低，煤柱收缩系数减小；堆密度大，开始收缩时的中心温度和各梯度温度均较高，对收缩系数及收缩高峰无明显影响。在焦化的不同时期，煤柱不同位置的升温速率不同