共转染CDK1、CDK2siRNA对肿瘤细胞周期和细胞凋亡的影响

目的研究共转染CDK1、CDK2siRNA同时抑制CDKI、CDK2蛋白表达对肿瘤细胞周期和细胞凋亡的影响,探讨细胞周期主要调控分子在肿瘤细胞凋亡中的作用。方法以人宫颈癌细胞株HeLa细胞为研究对象,用脂质体lipofectamine2000同时转染CDKl和CDK2siRNA。在转染后48、60h收集细胞,用Western印迹检测CDKl、CDK2蛋白的表达,AnnexinV／PI检测转染细胞的凋亡,流式细胞术DNA含量检测分析细胞周期。转染细胞进行瑞氏一姬姆萨染色（Wright—Giemsa）后在显微镜下观察其形态变化i结果共转染CDKl、CDK2siRNA后48和60h,Western印迹结果显示CDKl和CDK2蛋白的表达都同时降低。共转染CDKl、CDK2siRNA后,细胞周期S期和G1／M期比例与对照相比有明显增加;共转染细胞经瑞氏一姬姆萨染色后在显微镜下可见双核或多核细胞增多;AnnexinV／PI检测结果显示共转染CDK1、CDK2siRNA的细胞在48和60h细胞凋亡率与对照相比有显著的升高。结论siRNA干扰导致的CDKI、CDK2表达同时降低不仅导致细胞周期s期和G1／M期的阻滞,也诱导了肿瘤细胞的凋亡。     

携带siSPK1重组腺病毒的构建及其对N2a细胞凋亡的影响

应用AdMax腺病毒载体系统构建携带siSPK1基因的重组腺病毒,进一步研究SPK1基因对N2a细胞凋亡的影响。设计可形成小发夹结构的SPK1-siRNA模板cDNA序列,克隆至质粒pDC316-siRNA,构建SPK1-siRNA穿梭质粒pDC316-SPK1,经鉴定正确后,将pDC316-SPK1与辅助包装质粒（pBHG loxΔE1,3 Cre）共转染至HEK293细胞,包装纯化并扩增重组腺病毒颗粒,终点稀释法测定病毒滴度;病毒感染N2a细胞,Western blot法检测SPK1蛋白的表达;Hoechst33258染色检测SPK1基因对N2a细胞凋亡的影响。酶切后PCR分析、测序鉴定表明,pDC316-SPK1构建成功,病毒纯化后滴度为2.50E＋10 PFU/mL;重组病毒可在蛋白水平抑制SPK1的表达;SPK1基因抑制后,N2a细胞凋亡增加（P〈0.001）。上述结果表明,含有SPK1-siRNA的重组腺病毒构建成功,且具有抑制SPK1蛋白表达的功能,该基因沉默后,N2a细胞凋亡增加。     

Phospholipase A2 Sensitive Liposomes for Delivery of Small Interfering RNA (siRNA)

Small interfering RNA (siRNA) is potent and highly specific for gene silencing and there is currently a lot of enthusiasm for developing siRNA into a drug. However, for most therapeutic applications of siRNA, delivery systems are needed. These delivery systems have multiple requirements and should on one hand ideally be stable carriers protecting the siRNA from degradation and on the other hand assist the siRNA in overcoming membrane barriers for intracellular delivery to the cytosol. Long-circulating liposomes, which are sensitive to secretory phospholipase A₂ (sPLA₂) are feasible delivery systems for systemic administration of drugs due to their passive targeting to pathological tissue via the enhanced permeability and retention (EPR) effect and their site-specific, enzyme-triggered release of encapsulated drug in response to sPLA₂ which exists locally at elevated levels at, e.g,. sites of inflammation. However, recent data suggest that endosomal membrane destabilizing approaches could be addressed to design sPLA₂-sensitive liposomes as successful delivery systems for siRNA to the RNA interference pathway in the cytoplasm upon systemic administration.     

Biology, herbivory, and host specificity of Antiblemma leucocyma (Lepidoptera: Noctuidae) on Miconia calvescens DC. (Melastomataceae) in Brazil

Miconia calvescens DC. (Melastomataceae) is an invasive tree considered one of the greatest threats to natural ecosystems of Hawaii and other Pacific islands. The potential for using the defoliator Antiblemma leucocyma (Lepidoptera: Noctuidae) as a biological control agent of M. calvescens was evaluated in the native habitat of the insect in Brazil. Impact assessment in the field showed that 37.2% of leaves presented damage by A. leucocyma, and among damaged leaves, 1.5-51.9% of the leaf area (16.3% on average) was affected. Damage was found in young to fully expanded leaves (28-915 cm² in area). Individual A. leucocyma developing as third through sixth instars in the laboratory consumed an average of 1348 cm² of M. calvescens leaf material in about 11 days. Populations of A. leucocyma in the field were heavily impacted (up to 83% parasitism) by a braconid parasitoid. Observations in the field and single- and two-host choice tests in the laboratory indicated that A. leucocyma has a narrow host range restricted to M. calvescens. Usefulness of this herbivore for biological control in Hawaii appears low, however, because of its probable susceptibility to generalist parasitoids.     

针对SARS冠状病毒重要蛋白的siRNA设计(英文)

NA干涉 (RNAinterference ,RNAi)是一种特异性地导致转录后基因沉默的现象 ,在哺乳动物细胞中小分子干扰RNA双链体 (smallinterferingRNAduplexes ,siRNAduplexes)可以有效地诱导RNAi现象 ,为一些疾病的治疗开辟了新的途径 .针对SARS冠状病毒 (SARScoronavirus ,SARS CoV)中编码 5个主要蛋白质的基因 ,用生物信息学的方法设计了3 48条候选siRNA靶标 .在理论上 ,相应的siRNA双链体能特异地抑制SARS CoV靶基因的表达 ,同时不会影响人体细胞基因的正常表达 ,这为进一步siRNA类药物的实验研究提供了理论基础     

siRNA沉默整合素beta1 基因对子宫内膜癌细胞侵袭、转移的影响

目的：探讨siRNA 沉默整合素茁1 基因对子宫内膜癌细胞侵袭、转移的影响。方法：选取子宫内膜癌ECC-1细胞系(ER阳性) 和KLE细胞系(ER阴性)，分别转染Integrin beta1 siRNA 质粒(Integrin beta1 siRNA 组)、无义序列siRNA质粒(无义序列对照组)和空载 质粒(空载对照组)，利用实时荧光定量PCR 检测各组细胞中Integrin 茁1 mRNA的表达，Western blot 检测各组细胞中Integrin beta1、 beta-catenin 和C-Myc 蛋白的表达，Transwell 小室检测各组细胞迁移和侵袭能力，MTT 法检测各组细胞的增殖情况。结果：Integrin beta1 siRNA 组ECC-1 细胞和KLE 细胞中Integrin beta1 mRNA 和蛋白相对表达量均低于无义序列对照组和空载对照组(P<0.05)； Integrin beta 1 siRNA组ECC-1 细胞和KLE细胞中beta-catenin 蛋白和C-Myc 蛋白相对表达量均低于无义序列对照组和空载对照组， 差异均有统计学意义(P<0.05)；Integrin beta1 siRNA组ECC-1 细胞和KLE 细胞中迁移细胞数和侵袭细胞数均低于无义序列对照组 和空载对照组(P<0.05)；Integrin beta1 siRNA 组ECC-1细胞和KLE 细胞的A 值均低于无义序列对照组和空载对照组(P<0.05)。结 论：特异性抑制Integrin beta1 基因可抑制子宫内膜癌细胞迁移、侵袭和增殖，可能与抑制Wnt信号传导有关。     

不同饥饿程度拟小食螨瓢虫对朱砂叶螨的捕食作用

为明确饥饿程度对拟小食螨瓢虫 Stethorus (Allosstethorus) parapauperculus 控害潜能的影响,本研究通过比较不同饥饿程度下拟小食螨瓢虫对朱砂叶螨 Tetranychus cinnabarinus 的功能反应和自身密度干扰效应,探讨不同饥饿程度拟小食螨瓢虫对朱砂叶螨的捕食作用。结果表明,饥饿程度对拟小食螨瓢虫朱砂叶螨的捕食量有明显的影响,瓢虫的取食量与朱砂叶螨的密度呈正相关,表现为饥饿48 h＞饥饿24 h＞未饥饿;拟小食螨瓢虫对各虫态朱砂叶螨的捕食作用均符合Holling Ⅱ模型。在一定空间范围内,拟小食螨瓢虫个体间存在竞争和相互干扰,Hassell Varley模型能较好地反映拟小食螨瓢虫在捕食各虫态朱砂叶螨时受自身密度的干扰情况。在同样饥饿状态下,随着瓢虫自身密度增加,捕食效率降低,干扰效应增大。     

When does reproductive interference occur? Predictions and data

Reproductive interference is interspecific sexual interactions that are costly to at least one species involved. Although many studies have reported a substantial fitness cost associated with reproductive interference, suggesting its ecological significance, others have not observed reproductive interference in study species. Reproductive interference that incurs a large fitness cost is more likely to occur during secondary contacts than between long-coexisting species. I first explain the rationale underlying this prediction using existing literature. Next, I present a conceptual framework to classify pairs of interacting species into one of four states, defined by the ecological and evolutionary stabilities of the species pairs. I discuss how the stability states of species pairs are likely to change over time, along with changes in the demographic and evolutionary role of reproductive interference. I then perform literature survey to test the prediction that reproductive interference should be more prevalent in secondary contact. Finally, I discuss the implications of the proposed conceptual framework and literature survey result.