巨细胞病毒感染致MRC-5细胞周期及Cyclin E表达的变化

本文研究了巨细胞病毒感染人二倍体细胞MRC-5后诱导产生高水平Cyclin E,Cyclin E/cdk2激酶活性增加和导致细胞周期阻滞.应用流式细胞仪分析表明10PFU/cell的病毒量感染MRC-5细胞72h后,29%细胞位于S期,69%细胞位于G2/M期,只有2%细胞位于G1/G0期.应用双抗夹心ELISA法检测,感染病毒20h后,MRC-5细胞中Cyclin E含量比对照细胞高出8倍.感染细胞中CyclinE/cdk2激酶活性基本上与CyclinE含量相关联.     

苜蓿银纹夜蛾核型多角体病毒的感染对Sf9细胞周期的影响

病毒的感染导致细胞内部发生一系列变化。应用流式细胞仪FACS的荧光检测 ,测出Sf9细胞完成整个周期循环大约需要 18h ,G1、S、G2 /M各时相的时间间隔约为 6h ;AcNPV感染Sf9细胞 12 18h ,细胞被抑制于G2 /M期 ;Sf9细胞同步于G1/S期后释放细胞并用AcNPV感染 ,12h后 ,2 / 3的细胞处于G2 /M期 ,1/ 3的细胞处于S期     

苜蓿银纹夜蛾核型多角体病毒的感染对Sf9细胞周期的影响

病毒的感染导致细胞内部发生一系列变化.应用流式细胞仪FACS的荧光检测,测出Sf9细胞完成整个周期循环大约需要18h,G\-1、S、G\-2/M各时相的时间间隔约为6h;AcNPV感染Sf9细胞12-18h,细胞被抑制于G\-2/M期;Sf9细胞同步于G\-1/S期后释放细胞并用AcNPV感染,12h后,2/3的细胞处于G\-2/M期,1/3的细胞处于S期.     

DNA损伤反应中细胞周期检查点蛋白p27~(Kip1)表达及其调控机制

为了研究DNA损伤反应中p2 7Kip1的表达及其调控机制 ,应用免疫印迹的实验结果表明 :10Gy 60 Coγ射线照射后 3h ,HeLa细胞中p2 7Kip1蛋白水平开始下降并持续到 2 4h ,进而失去它对CDKs的抑制功能 .Northern印迹结果显示 ,电离辐射 (IR)对p2 7Kip1mRNA表达水平无明显影响 ,说明电离辐射诱导p2 7Kip1表达水平的降低主要与蛋白质降解相关 ,但其具体的调控机制还不清楚 .已知在G1—S期p2 7Kip1蛋白的降低主要依赖细胞周期蛋白E Cdk2激酶将其磷酸化后的泛素化蛋白酶体途径 (ubiquitin proteasomepathway) .酶动力学研究结果揭示 :电离辐射后细胞周期蛋白E Cdk2激酶活性增高 ,12h细胞周期蛋白E Cdk2激酶活性达到最大 .当在照前用细胞周期蛋白E Cdk2抑制剂olomoucine (10 μmol L)抑制细胞周期蛋白E Cdk2激酶活性时 ,p2 7Kip1蛋白表达水平增加 .此外 ,还观察到电离辐射可诱导p2 7Kip1泛素化水平的增高 ,而在使用蛋白酶体抑制剂MG 132 (5 μmol L)处理HeLa细胞后 ,可抑制辐射诱导p2 7Kip1蛋白水平的下调 .研究结果提示 :泛素化蛋白酶体途径参与了辐射诱导P2 7Kip1蛋白表达下调的降解机制 .     

流式细胞术BrdU／DNA双染法测定云芝糖肽诱导的Molt-4细胞周期阻滞

目的：探究云芝糖）Ik（PSP）对人急性淋巴母细胞白血病Molt-4细胞周期的影响。方法：采用流式细胞术BrdU／DNA双染法获得各时相细胞分布状况和细胞周期的动力学参数。结果：0．1mg／mlPSP处理12h后,G2／M期细胞百分比由对照组的11．09％减少至3．69％。DNA合成时间由12．10h延长至108．40h。24h处理组中,S期细胞百分比由对照组的43.29％增加至67．26％,而G0／G1期和G2／M期细胞百分比均减少,G0／G1期细胞百分比由对照组的37．47％减少至27．43％,G2／M期细胞百分比由对照组的19．24％降低至5.31％。DNA合成时间更是由11．95h延长至114．52h。结论：PSP对人急性淋巴母细胞白血病Molt-4细胞周期的阻滞作用在于S期．该作用与DNA合成抑制有关。     

人肿瘤坏死因子相关凋亡诱导配体稳定过表达基因工程修饰人脐带间充质干细胞亚细胞系的建立

目的通过基因工程修饰法建立肿瘤坏死因子相关凋亡诱导配体(TRAIL)稳定过表达的人脐带间充质干细胞亚系(h UC-MSC_TRAIL)。方法人TRAIL全长蛋白编码序列(CDS)由PCR法扩增而得,PCR产物经NotⅠ和MluⅠ双酶切并纯化后,亚克隆至经同样双酶切的慢病毒表达载体p LEX-MCS。重组载体经PCR法和限制性内切酶酶切法鉴定,再行DNA直接测序验证后命名为人TRAIL表达慢病毒载体pLEX-h TRAIL。p LEXh TRAIL与相应包装质粒ps PAX2和p MD2.G经聚乙烯亚胺介导共转染HEK293T细胞以包装慢病毒。P4代h UC-MSC经慢病毒感染24 h,再行嘌呤霉素筛选2周后,抽提细胞基因组DNA,行PCR法鉴定h TRAIL c DNA在h UC-MSC基因组中的整合情况;同时抽提细胞总RNA,并行RT-PCR法检测外源h TRAIL在h UC-MSC中的m RNA表达水平,以及实时定量RT-PCR法检测周期调控相关蛋白Cyclin D1、Cyclin E1、p21~(WAF1/CIP1和p27的表达,采用方差分析和t检验进行统计学分析。结果 PCR法和限制性内切酶酶切法鉴定结果表明,本研究已成功构建人TRAIL慢病毒表达载体p LEX-h TRAIL,直接DNA测序结果证实克隆得到的人TRAIL蛋白编码序列准确无误;病毒包装及细胞感染的鉴定结果说明,慢病毒感染法可成功介导外源人TRAIL在h UC-MSC的稳定整合和高表达;实时定量RT-PCR法检测结果则显示,与对照慢病毒感染后的细胞相比,h TRAIL表达慢病毒感染后其细胞周期调控相关蛋白Cyclin D1、Cyclin E1、p21~(WAF1/CIP1)和p27的m RNA表达水平分别是对照组的1.19倍(P=0.141)、0.94倍(P=0.745)、0.95倍(P=0.047)和1.01倍(P=0.567),表明外源TRAIL高表达对体外培养的h UC-MSC生长增殖等表型无显著影响。结论本研究经基因工程修饰法成功构建了具外源TRAIL稳定高表达的h UC-MSC亚细胞系,该亚细胞系的建立为后续靶向攻击TRAIL敏感肿瘤细胞的细胞治疗的探索奠定了基础。     

葡萄糖6-磷酸脱氢酶调控细胞周期蛋白E1和细胞周期蛋白依赖性激酶2促进细胞增殖及其在肾透明细胞癌中的预后价值

肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)是一种转移率高、预后差的细胞代谢性疾病,对其有效诊疗及预后分子标志物的研究十分重要。葡萄糖6-磷酸脱氢酶(glucose 6-phosphatedehydrogenase, G6PD)在ccRCC中高表达,并提示患者不良预后,其促进ccRCC细胞增殖的分子机制有待进一步揭示。本研究发现,降低G6PD可抑制细胞周期G₁/S期转化并显著抑制ccRCC细胞增殖。G6PD可在细胞水平调控G₁/S期转化及增殖相关因子Cyclin D1,CDK4,CDK6,Cyclin E1和CDK2基因表达。TCGA数据库分析结果表明,ccRCC 中Cyclin D1,Cyclin E1 和 CDK2的mRNA 水平显著升高,而CDK4表达无明显差异,CDK6表达却显著降低。相关性分析结果显示,G6PD与Cyclin D1呈显著负相关(P<0.0001),G6PD与CDK4,CDK6之间无显著相关性(P>0.05),G6PD与Cyclin E1(P<0.0001)以及CDK2(P<0.05)显著正相关。进一步免疫组化检测结果表明,Cyclin E1和 CDK2在ccRCC肿瘤组织中表达显著升高。生存预后分析结果显示,Cyclin D1高表达提示ccRCC患者整体预后更为良好,CDK4和CDK6表达水平在ccRCC患者总生存率预测中无意义;而Cyclin E1和CDK2高表达均可提示ccRCC患者预后不良。进一步细胞水平检测发现,Cyclin E1、CDK2表达降低可显著逆转G6PD促进ccRCC细胞增殖的能力。综上,与增殖相关因子Cyclin D1,CDK4和CDK6相比,G6PD有可能通过促进Cyclin E1和CDK2表达升高而发挥促进 ccRCC肿瘤细胞增殖的作用,并且这3者的异常高表达有望成为ccRCC患者不良预后的独立生存预测因素。     

慢病毒载体系统介导hUC—MSC绿色荧光蛋白和荧光素酶共表达技术体系的建立

目的建立慢病毒载体系统介导的人脐带间充质干细胞（hUC-MSC）绿色荧光蛋白（GFP）和荧光素酶共表达技术体系。方法 GFP和荧光素酶共表达慢病毒载体与相应包装质粒psPAX2和pMD2.G经聚乙烯亚胺介导共转染HEK293T细胞以包装病毒;病毒感染P4代hUC-MSC 12 h后,再行嘌呤霉素筛选24 h,普通光学显微镜观察细胞形态,荧光显微镜下观察GFP的表达情况,IVIS Kinetic成像系统拍照以观察和记录慢病毒感染后hUC-MSC荧光素酶的表达情况;MTS法行细胞生长曲线作图,同时,普通和实时定量RTPCR法检测细胞周期调控相关蛋白Cyclin D1、Cyclin E1和p21WAF1/CIP1的表达。采用方差分析和t检验进行统计学分析。结果慢病毒感染并不会造成体外培养hUC-MSC形态的明显改变,而荧光显微镜和IVIS Kinetic成像系统的观察结果则分别证实,GFP和荧光素酶经慢病毒载体系统的介导可在hUC-MSC中成功地共表达。此外,细胞生长曲线作图结果表明,对照和GFP及荧光素酶共表达慢病毒感染后hUC-MSC的生长增殖速率相仿（P〉0.01）;实时定量RT-PCR法检测结果则显示,与对照慢病毒感染相比,GFP和荧光素酶共表达慢病毒感染后其细胞周期调控相关蛋白Cyclin D1、Cyclin E1和p21WAF1/CIP1mRNA表达水平分别是对照组的1.11倍（P=0.130）、0.54倍（P=0.000）和0.78倍（P=0.005）,表明外源GFP和荧光素酶共表达对体外培养的hUC-MSC生长增殖等表型无显著影响。结论慢病毒载体系统可有效介导外源基因在hUC-MSC中的表达;同时,GFP和荧光素酶在hUC-MSC中的共表达也将极大地方便其体内转归的示踪。     

犬2型腺病毒E1转化细胞系的特性研究

对筛选到的一株CAV—2 E1转化细胞(DK／E1)进行了初步的特性研究。以DK细胞为对照,观察到转化细胞的形态与DK细胞有明显区别,细胞变长,易聚集生长。通过测定DK及DK／E1细胞分别在2％、5％和10％牛血清培养基中的生长曲线,结果发现DK／E1细胞的生长速度比DK细胞快。用蚀斑和TCID50测定了病毒在DK和DK／E1细胞上的病毒滴度,结果表明DK／E1细胞产生的病毒蚀斑比DK细胞的大,TCID50测得的病毒滴度比DK细胞产生的病毒滴度高。CAV—2全基因组DNA对DK／E1细胞和DK细胞的转染试验结果表明,5μg和10μg的CAV—2 DNA转染的DK／E1细胞,分别在转染后第14天和第11天均出现了典型的腺病毒细胞病变,而相同量CAV—2 DNA转染的DK细胞未出现病变,说明DK／E1细胞有助于CAV—2 DNA的复制和病毒粒子的包装。在DK／E1细胞内的重组试验表明,DK／E1细胞也有利于重组病毒的产生。以上转化特性在DK／E1细胞传至80代时仍保持不变,说明本试验获得了一株CALV—2 E1基因的转化细胞系。     

HearNPV在生长对数期与平台期宿主细胞中的复制差异分析

使用实时荧光定量PCR技术对HearNPV在生长对数期和平台期HzAM1细胞的复制差异进行分析。结果表明,HzAM1细胞生长对数期的倍增时间为22 h,生长对数期的细胞以S期细胞为主(48.6%),而平台期细胞中以G2/M期细胞为主(72.6%)。在这两种不同状态的细胞中,病毒的复制主要在感染后60 h内完成,在感染后14~20 h,病毒复制倍增时间分别为1.8 h和1.9 h,几乎没有差别。但是感染生长对数期细胞时,吸附侵入细胞内的BV数量、BV释放的数量、最终的病毒产量以及病毒表达的蛋白产量明显高于被病毒感染的生长平台期细胞。如生长对数期细胞内复制合成的病毒DNA总量的25%装配形成BV病毒粒子出芽释放到细胞外,而对于平台期细胞,病毒DNA仅有13%装配形成BV病毒粒子出芽释放到细胞外。病毒感染两种生长状态的细胞,病毒DNA均从感染后7~8 h开始复制,没有明显差别;而生长对数期细胞从被感染后18~20 h释放子代病毒BV,生长平台期细胞则在感染后22~25 h开始释放病毒BV。在感染后30~60 h,在生长对数期被感染的细胞释放BV的速度约为483 copies/cell/h,而平台期细胞约为100 copies/cell/h。最初吸附侵入到生长对数期细胞内的BV粒子数量明显多于侵入到生长平台期细胞内的BV数量。实验证实,生长对数期与平台期的细胞膜的流动性有很大差别,推测健康细胞表面有活性的病毒受体数量可能决定了侵入细胞内的BV的数量。     

microRNA-16-5p enhances radiosensitivity through modulating Cyclin D1/E1–pRb–E2F1 pathway in prostate cancer cells

Prostate cancer (CaP) is the second most common cancer in men worldwide in 2012, and radiation therapy is one of the most common definitive treatment options for localized CaP. However, radioresistance is a major challenge for the current radiotherapy, accumulating evidences suggest microRNAs (miRNAs), as an important regulator in cellular ionizing radiation (IR) responses, are closely correlated with radiosensitivity in many cancers. Here, we identified microRNA-16-5p(miR-16-5p) is significantly upregulated in CaP LNCaP cells following IR and can enhance radiosensitivity through modulating Cyclin D1/E1–pRb–E2F1 pathway. To identify the expression profile of miRNAs in CaP cells exposed to IR, we performed human miRNA probe hybridization chip analysis and miR-16-5p was found to be significantly overexpressed in all treatment groups that irradiated with different doses of X-rays and heavy ions (¹²C⁶⁺). Furthermore, overexpression of miR-16-5p suppressed cell proliferation, reduced cell viability, and induced cell cycle arrest at G0/G1 phase, resulting in enhanced radiosensitivity in LNCaP cells. Additionally, miR-16-5p specifically targeted the Cyclin D1/E1–3′-UTR in LNCaP cells and affected the expression of Cyclin D1/E1 in both mRNA and protein levels. Taken together, miR-16-5p enhanced radiosensitivity of CaP cells, the mechanism may be through modulating Cyclin D1/Cyclin E1/pRb/E2F1 pathway to cause cell cycle arrest at G0/G1 phase. These findings provided new insight into the correlation between miR-16-5p, cell cycle arrest, and radiosensitivity in CaP, revealed a previously unrecognized function of miR-16-5p–Cyclin D1/E1–pRb–E2F1 regulation in response to IR and may offer an alternative therapy to improve the efficiency of conventional radiotherapy.     

反义细胞周期蛋白E真核表达载体的构建及其对体外人乳腺癌细胞的抑制作用

 为了探讨细胞周期蛋白 E(cyclin E)与人乳腺癌细胞恶性特征间的相关性 ,利用反义 RNA抑制基因表达的技术 ,构建了细胞周期蛋白 E反义 RNA的真核表达载体并转入人乳腺癌细胞中 .通过 G41 8筛选出阳性克隆 ,经 PCR和 Western印迹检测 ,确定细胞中含有重组质粒 ,并且细胞周期蛋白 E蛋白的水平明显降低 ,由此获得了反义 RNA表达载体导致的细胞周期蛋白 E表达受抑制的细胞 .细胞模型建立后 ,观察分析了细胞形态 ,细胞生长的血清依赖性以及软琼脂成集落能力 ,与对照细胞相比所发生的变化 .结果显示 ,细胞周期蛋白 E受抑制后 ,乳腺癌细胞体积变大 ,细胞生长对血清依赖性增加 ,低血清培养到第 6d时 ,细胞密度约为对照细胞的五分之一 ,细胞成集落能力也显著下降 ,软琼脂中克隆形成率下降 57% .这些变化都表明乳腺癌细胞恶性程度由于细胞周期蛋白 E表达受抑制而减弱 ,可以推测 cyclin E与乳腺癌细胞的恶性增殖及非锚定依赖性生长有着明显的关系 .     

丙型肝炎病毒NS5A蛋白对Huh7细胞周期的影响

应用PCR技术从含有丙型肝炎病毒（HCV）全长开放阅读框的质粒pBRTM／HCV1～3011中获得NS5A全长基因片段,利用基因重组技术将其克隆至真核表达载体pcDNA3．1（-）中。通过酶切、PCR及测序鉴定证实,NS5A基因已正确插入到pcDNA3．1（-）中。再利用脂质体介导转染Huh7细胞,30h后收获细胞,经Western blot验证,证实HCV的NS5A基因在Huh7细胞中已经获得表达。在培养条件完全一致的条件下,表达NS5A基因的Huh7细胞与pcDNA3．1（-）转染的细胞在转染30h后被收集起来,乙醇固定,PI染色后利用流式细胞仪检测细胞周期变化。G0／G1期由60．6％下降到49．7％,S期由23．9％上升到32．7％,而转染pcDNA3．1（-）细胞的细胞周期与正常的Huh7细胞则差别不大。从而证明HCV NS5A蛋白对Huh7细胞周期具有调节作用。     

A Jekyll and Hyde Role of Cyclin E in the Genotoxic Stress Response: Switching from Cell Cycle Control to Apoptosis Regulation

Cyclin E protein levels and associated kinase activity rise in late G1 phase, reach a peak at the G1/S transition, and quickly decline during S phase. The Cyclin E /Cdk2 complex has a well-established function in regulating two fundamental biological processes: cell cycle progression and DNA replication. However, Cyclin E expression is deregulated in a wide range of tumors. Our recent reports have uncovered a critical role for Cyclin E, independent of Cdk2, in the cell death of hematopoietic tumor cells exposed to genotoxic stress. An 18-kD C-terminal fragment of Cyclin E, p18-Cyclin E, which is generated by caspase-mediated cleavage in hematopoietic cells during genotoxic stress-induced apoptosis has a critical role in the amplification of the intrinsic apoptotic pathway. By interacting with Ku70, p18-Cyclin E liberates Bax, which participates in the amplification of apoptosis by sustaining a positive feedback loop targeting mitochondria. This process is independent of p53 function and new RNA or protein synthesis. Therefore, Cyclin E emerges as an arbiter of the genotoxic stress response by regulating a finite physiological balance between cell proliferation and death in hematopoietic cells.     

Bunyamwera Virus Replication in Cultured Aedes albopictus (Mosquito) Cells: Establishment of a Persistent Viral Infection

Bunyamwera virus replication was examined in Aedes albopictus (mosquito) cell cultures in which a persistent infection is established and in cytopathically infected BHK cells. During primary infection of A. albopictus cells, Bunyamwera virus reached relatively high titers (10⁷ PFU/ml), and autointerference was not observed. Three virus-specific RNAs (L, M, and S) and two virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA synthesis and viral protein synthesis were extremely low, corresponding to <2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h postinfection and was shut down to barely detectable levels at 24 h postinfection. Virus-specific RNA and nucleocapsid syntheses showed similar patterns of change, but later in infection. The proportions of cells able to release a single PFU at 3, 6, and 54 days postinfection were 100, 50, and 1.5%, respectively. Titers fell to 10³ to 10⁵ PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days postinfection and increased gradually until they were 75 to 95% of the total infectious virus at 66 days postinfection. Temperature-sensitive mutants appeared between 23 and 49 days postinfection. No antiviral activity similar to that reported in A. albopictus cell cultures persistently infected with Sindbis virus (R. Riedel and D. T. Brown, J. Virol. 29: 51-60, 1979) was detected in culture fluids by 3 months after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of 10. Virus-specific RNA and protein syntheses were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. We discuss these results in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.     

Golgi-associated RhoBTB3 targets Cyclin E for ubiquitylation and promotes cell cycle progression

Cyclin E regulates the cell cycle transition from G1 to S phase and is degraded before entry into G2 phase. Here we show that RhoBTB3, a Golgi-associated, Rho-related ATPase, regulates the S/G2 transition of the cell cycle by targeting Cyclin E for ubiquitylation. Depletion of RhoBTB3 arrested cells in S phase, triggered Golgi fragmentation, and elevated Cyclin E levels. On the Golgi, RhoBTB3 bound Cyclin E as part of a Cullin3 (CUL3)-dependent RING–E3 ubiquitin ligase complex comprised of RhoBTB3, CUL3, and RBX1. Golgi association of this complex was required for its ability to catalyze Cyclin E ubiquitylation and allow normal cell cycle progression. These experiments reveal a novel role for a Ras superfamily member in catalyzing Cyclin E turnover during S phase, as well as an unexpected, essential role for the Golgi as a ubiquitylation platform for cell cycle control.     

Evidence for post-transcriptional regulation of cyclin B1 mRNA in the cell cycle and following irradiation in HeLa cells.

Cyclin B1 mRNA expression varies markedly through the cell cycle with its peak in G2/M and lowest level in G1. Cyclin B1 mRNA levels are also transiently reduced in HeLa cells after gamma-irradiation, coincident with the radiation-induced G2 block. In order to understand the mechanisms underlying these variations, we have measured cyclin B1 mRNA stability in HeLa cells during different phases of the cell cycle. The half-life of the mRNA measured after actinomycin D administration is 1.1-1.8 h in both early and late G1, 8 h in S and 13 h in G2/M. We therefore conclude that altered RNA stability is important in modulating cyclin B1 mRNA levels through the HeLa cell cycle. Furthermore, 3 h after irradiation of HeLa cells in S phase with 10 Gy, the half-life of cyclin B1 mRNA is reduced to 5 h; it is further reduced to 2-3 h at 14 h after irradiation. Thus, decreased stability contributes to the reduction in cyclin B1 mRNA following irradiation.