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

维持性血透患者的心理状态研究

研究我国血透患者的心理状况并进行针对性的心理治疗。方法 采用症状自评量表(SCL90-R)、多维度健康状况心理控制源量表(MHLC)、艾森克个性问卷(EPQ)及终末期肾脏病(ESRD)患者专用的生活质量表对北京六个医院透析中心的92名维持性血透患者进行了心理状态的研究,并与美国及加拿大相同的研究进行了比较分析。结果 本组的血透存在着抑郁、焦虑、恐怖等心理障碍,出现心理障碍的比例显著高于美国同类患者(P&lt;0.01),且焦虑的发生率高于美国同类患者(P&lt;0.01)。这些障碍与MHLC中机遇项分(CED)显著相关(P&lt;0.01)。EPQ中神经质项分高者倾向于发生抑郁、焦虑等心理障碍。本组的血透患者客观生活质量较加拿大同类患者低(P&lt;0.01),但在总的生活满意度上没有显著性差异。生活质量与心理及躯体因素均呈显著相关,心理障碍与躯体症状也显著相关(P&lt;0.01)。结论 本组的血透患者心理状态与美国加拿大同类患者相比既有相同之处,又有特殊之处。我们应该兼顾病人的躯体和精神两方面的健康,努力提高他们的生活质量。</p

miR-126 Inhibits Invasion of Human Brain Glioma Cells by Decreasing MMP-2

目的初步探讨MIr-126抑制人胶质瘤细胞侵袭的可能机制。方法化学合成MIr-126,脂质体转染人脑胶质瘤u87细胞,应用rT-PCr、WESTErn blOT检测MMP-2基因和蛋白的表达情况,并应用TrAnSWEll小室检测转染前后细胞侵袭力的变化。结果 MIr-126上调后u87细胞的MMP-2基因和蛋白表达降低,并且细胞侵袭力明显降低。结论化学合成的MIr-126在抑制人胶质瘤细胞侵袭过程中发挥重要作用,可能成为胶质瘤基因治疗的新靶点。Objective To explore the mechanism underlying the inhibiting effects of miR-126 on the invasion of human brain glioma cells.Methods miR-126 was synthesized and transfected into the U87 malignant glioma cells by liposome.The relative expression of MMP-2 and MMP-9 gene was quantified by RT-PCR,the expression of MMP-2 and MMP-9 protein was detected by Western blot,and cell invasion was assessed by Transwell chamber assay.Results miR-126 inhibited the activity of MMP-2 and the invasion of human U87 glioma cells.Conclusion The chemically synthesized miR-126 plays an important role in cell invasion of human brain glioma cells,which may be a promising target for glioma gene therapy.福建省自然科学基金面上项目资助课题(2009D002);厦门市科技局基金资助项目(3502220077055

Down-regulated Pygo2 expression suppresses proliferation,invasion,and cyclin D1 expression 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、侵袭的影响及其机制。方法针对PygO2CdnA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阴性对照序列,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,采用实时定量PCr和蛋白质印迹方法检测PygO2SHrnA对u251细胞PygO2MrnA和蛋白表达的干扰效果,MTT法检测细胞增殖,流式细胞术检测细胞周期分布,brdu掺入法检测dnA合成,TrAnSWEll检测细胞侵袭。采用蛋白质印迹和免疫荧光法检测PygO2SHrnA对u251细胞CyClIn d1、β-CATEnIn蛋白水平和亚细胞定位的影响。结果双酶切和测序鉴定证实插入序列完全正确;PygO2SHrnA显著抑制了u251细胞PygO2MrnA和蛋白的表达;PygO2SHrnA抑制u251细胞PygO2表达后,细胞增殖显著降低,细胞更多地阻滞在g1期,且brdu掺入显著减少,侵袭细胞数显著减少。此外,抑制PygO2表达可显著下调u251细胞CyClIn d1的表达但不改变其亚细胞定位。u251细胞β-CATEnIn表达及其亚细胞定位无明显改变。结论成功构建了抑制PygO2表达的重组载体。抑制PygO2表达能有效地抑制脑胶质瘤u251细胞dnA合成,可能通过下调WnT信号靶基因CyClIn d1的表达,使细胞阻滞于g1期而抑制细胞增殖和侵袭。Objective To construct recombinant vectors for RNA interference(RNAi)targeting Pygo2,and to assess its influence on the proliferation,invasion of glioblastoma U251 cells and the related mechanism.Methods A pair of oligonucleotides containing short hairpin structure targeting Pygo2 cDNA sequences were designed and synthesized,and their negative control sequences were also synthesized.After annealed,they were inserted into pSuper vector to generate the recombinant plasmids.Then the recombinant plasmids were digested with EcoRⅠand HindⅢ for identification,and the sequence was assayed by DNA sequencing.The recombinant plasmids were transfected into cultured glioblastoma U251 cells using Lipofectamine--TM 2000.The effect of Pygo2 shRNA on Pygo2 mRNA and protein in U251 cells was detected by real-time PCR and Western blotting analysis,respectively.MTT assay was used to detect the cell proliferation;cell cycle was analyzed by flow cytometry;Bromodeoxyuridine(BrdU) incorporation analysis was used to examine DNA synthesis;and cell invasion assay was performed using Transwell chambers.The effect of Pygo2 shRNA on the protein level and subcellular location of cyclin D1 and β-catenin was detected by Western blotting analysis and immunofluorescent staining.Results The recombinant plasmids were completely coincided with the design by the restriction map and the sequence analysis.Pygo2 mRNA and protein expression was significantly suppressed by Pygo2 shRNA.Furthermore,the proliferation of cells in Pygo2 shRNA group was notably inhibited,cell cycle was arrested at the G_1 phase,and BrdU incorporation and migrating cells were significantly inhibited.In addition,Pygo2 knockdown significantly down-regulated cyclin D1 expression without altering the subcellular location,and the expression level and subcellular location of β-catenin had no noticeable changes.Conclusion The recombinant vectors for specific suppression of Pygo2 expression have been constructed successfully.Inhibition of Pygo2 expression can suppress cell proliferation and invasion of glioma U251 cells,decrease DNA synthesis,arrest cell cycle at the G_1 phase,and decrease expression of the Wnt target gene cyclin D1.厦门市科技局资助项目(3502z20089001);福建省自然科学基金面上项目(2009D002);中国博士后基金(20080440728);重庆市教委科技项目(KJ100504)---

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)---

JUNO Sensitivity on Proton Decay p→νˉK+p\to \bar\nu K^+ Searches

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this paper, the potential on searching for proton decay in $p\to \bar\nu K^+$ mode with JUNO is investigated.The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits to suppress the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar\nu K^+$ is 36.9% with a background level of 0.2 events after 10 years of data taking. The estimated sensitivity based on 200 kton-years exposure is $9.6 \times 10^{33}$ years, competitive with the current best limits on the proton lifetime in this channel

JUNO sensitivity on proton decay p → ν K + searches*

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this study, the potential of searching for proton decay in the $p\to \bar{\nu} K^+$ mode with JUNO is investigated. The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits suppression of the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar{\nu} K^+$ is 36.9% ± 4.9% with a background level of $0.2\pm 0.05({\rm syst})\pm$$0.2({\rm stat})$ events after 10 years of data collection. The estimated sensitivity based on 200 kton-years of exposure is $9.6 \times 10^{33}$ years, which is competitive with the current best limits on the proton lifetime in this channel and complements the use of different detection technologies