基于AFM的活体状态外泌体纳米结构及机械特性研究

外泌体在细胞生理病理活动过程中起着重要的调控作用,研究外泌体的行为特性对于揭示生命活动及疾病发生发展的内在机理具有重要的基础意义.然而由于缺乏合适的观测手段及方法,目前对于活体状态下外泌体结构及特性的认知仍然很不足.原子力显微镜(AFM)的发明为研究溶液环境下天然状态生物样本提供了强大的技术工具,已成为生物学重要研究手段.本文利用AFM对单个活体状态外泌体的纳米结构及机械特性进行了研究.通过多聚赖氨酸静电吸附作用将从淋巴瘤患者骨髓中分离的外泌体吸附至基底,在溶液环境下实现了对单个活体状态外泌体的高质量AFM形貌成像并通过与空气中成像结果进行对比揭示了空气干燥处理对外泌体形貌的影响.在此基础上,分别利用AFM压痕试验和多参数成像技术实现了对单个活体状态外泌体机械特性的定量测量和可视化表征.最后基于所建立的方法技术揭示了化学处理后外泌体结构和机械特性的动态变化.研究结果为研究纳米尺度下活体状态外泌体的结构及特性,以更好理解天然状态外泌体的生理行为提供了新的方法和思路,对于外泌体研究具有潜在积极的意义.     

云南松苗木生长与生物量的相关性及通径分析

为了解云南松苗木生长对生物量的影响程度和相对重要性,以采自云南省昆明市宜良县禄丰村林场种子培育的2年生云南松苗木为材料,对苗高、地径、主根长、侧根长、针叶长和侧根数6个生长性状及各组分生物量进行测定,运用相关性分析和逐步回归分析剔除了对生物量影响不大的性状,在此基础上采用通径分析方法研究了云南松苗木生长性状指标与生物量指标间的关系。结果表明：云南松苗木生长性状和生物量之间均呈现极显著的正相关关系。各生长性状对生物量均有直接或间接的正向效应,直接影响云南松苗木生物量的优势因素为地径和苗高,针叶长、侧根长和主根长对生物量的影响也起正向效应。各生长性状对生物量的直接效应均较大,而通过其他性状的间接效应均较小,其中侧根长通过其他性状对生物量产生的间接影响最大。依据相关性分析和通径分析,建立了云南松各组分生物量与生长性状之间的数学模型,可用于云南松苗期生物量的估测。     

The formation of Ca<Superscript>2+</Superscript> gradients at the cleavage furrows during cytokinesis of Zebrafish embryos

In dividing embryos, a localized elevation in intracellular Ca²⁺ ([Ca²⁺]_i) at the cleavage furrow has been shown to be essential for cytokinesis. However, the underlying mechanisms for generating and maintaining these [Ca²⁺]_i gradients throughout cytokinesis are not fully understood. In the present study, we analyzed the role of inositol 1,4,5-trisphosphate receptors (IP₃Rs) and endoplasmic reticulum (ER) distribution in determining the intracellular Ca²⁺ gradients in early zebrafish blastomeres. Application of the injected Ca²⁺ indicator, Indo-1, showed that during the first cell division a standing Ca²⁺ gradient was formed ∼35 min after fertilization, with the [Ca²⁺]_i spatially decaying from 500–600 nmol/L at the cleavage furrow to 100–200 nmol/L around the nucleus. While the IP3R immunohistochemical fluorescence was relatively concentrated in the peri-furrow region, ER labeling was relatively enriched in both peri-furrow and peri-nuclear regions. Numeric simulation suggested that a divergence in the spatial distribution of IP3R and the locations of Ca²⁺ uptake within the ER was essential for the formation of a standing Ca²⁺ gradient, and the Ca²⁺ gradient could only be well-established under an optimal stoichiometry of Ca²⁺ uptake and release. Indeed, while inhibition of IP3R Ca²⁺ release blocked the generation of the Ca²⁺ gradient at a lower [Ca²⁺]_i level, both Ca²⁺ release stimulation by inositol 1,4,5-trisphosphate (IP3) injection and ER Ca²⁺ pump inhibition by cyclopiazonic acid also eliminated the Ca²⁺ gradients at higher [Ca²⁺]_i levels. Our results suggest a dynamic relationship between ER-mediated Ca²⁺ release and uptake that underlies the maintenance of the perifurrow Ca²⁺ gradient and is essential for cytokinesis of zebrafish embryos.     

Anti-apoptotic activity of gentiopicroside in d-galactosamine/lipopolysaccharide-induced murine fulminant hepatic failure

This study investigated the hepatoprotective effects of gentiopicroside on d-galactosamine (d-GalN) and lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were administrated orally with gentiopicroside (40 or 80 mg/kg body weight) at 12 h and 1 h before d-GalN (700 mg/kg)/LPS (10 μg/kg) injection. Gentiopicroside markedly reduced the increases in serum aminotransferase activities and lipid peroxidation. The glutathione content decreased in d-GalN/LPS alone group, and this decrease was attenuated by gentiopicroside. Increases in serum tumor necrosis factor-α (TNF-α), which were observed in d-GalN/LPS alone group, were significantly reduced by gentiopicroside. Importantly, gentiopicroside attenuated d-GalN/LPS-induced apoptosis of hepatocytes, as estimated by the caspase-3 cleavage, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation. d-GalN/LPS-induced caspase-8 and -9 activation was significantly suppressed by gentiopicroside. Moreover, increased cytosolic cytochrome c protein was reduced by gentiopicroside. Also, the increased ratio of Bax and Bcl-2 protein was significantly attenuated by gentiopicroside. After 6 h of d-GalN/LPS injection, phosphorylated c-jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) was significantly increased, whereas phosphorylation JNK and ERK were attenuated by gentiopicroside. Our results suggest that gentiopicroside offers remarkable hepatoprotection against damage induced by d-GalN/LPS related with its anti-apoptotic activities.     

The anti-fibrotic effect of betulinic acid is mediated through the inhibition of NF-κB nuclear protein translocation

The purpose of the study was to investigate the anti-fibrotic effect and the potential mechanisms of action of betulinic acid (BA) against hepatic fibrosis in vivo and in vitro. BA is an active compound isolated from the bark of the birch tree Betula spp. (Betulaceae). Liver fibrosis was induced by intraperitoneal injections of thioacetamide (TAA, 200mg/kg) twice weekly for 6weeks in Wistar rats. The administration of BA (20 or 50mg/kg) was started following TAA injections and was continued for 6 or 8weeks to evaluate both the preventive and the protective effects. BA demonstrated great efficacy in preventing and curing hepatic fibrosis via attenuating the TAA-mediated increases in liver tissue hydroxyproline and α-smooth muscle actin (α-SMA). In vitro, BA effectively decreased the HSC-T6 cell viability induced by TNF-α and showed low toxicity in normal human chang liver cells. Moreover, BA significantly attenuated the expression of α-SMA and tissue inhibitor of metalloproteinase-1 (TIMP-1) and increased the levels of matrix metalloprotease (MMP)-13. BA also inhibited the expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and the activation of nuclear factor-κB (NF-κB) in a time-dependent manner. This study provides evidence that BA exerts a significant anti-fibrosis effect by modulating the TLR4/MyD88/NF-κB signaling pathway.     

水稻白叶枯病抗性基因Xa23鉴别菌株突变体库的构建及无毒基因突变体的筛选

利用in vivo转座技术构建了白叶枯病抗性基因Xa23鉴别菌株的突变体库,特异性引物PCR扩增和转座子插入位点旁侧序列分析结果表明转座子插入到白叶枯病菌的基因组中。经人工接种鉴定,筛选到4个毒力发生变化的突变体。为进一步克隆Xa23无毒基因提供了条件。     

热休克蛋白70在妊娠早期小鼠子宫中的表达及雌激素的诱导作用

采用Western印迹、免疫组织化学、免疫电镜和图像分析技术研究了妊娠早期小鼠子宫热休克蛋白70(Heat shock protein,HSP70)的表达变化以及雌二醇对子宫HSP70表达的影响。结果表明：(1)与正常小鼠相比,孕鼠HSP70含量显著增多,且随妊娠日龄的增加而增加(P＜0．01);(2)小剂量(0．28μg／g体重)和大剂量(1．10μg／g体重)雌二醇均可诱导小鼠子宫HSP70免疫反应阳性细胞数显著增加(P＜0．01),但不表现剂量依赖关系;(3)Western印迹显示雌二醇使子宫HSP70蛋白谱带发生改变,正常小鼠仅有73kDl条蛋白带,小剂量组检出68kD、72kD、73kD3条蛋白带,大剂量组检出72kD、73kD2条蛋白带;(4)电镜下,HSP70免疫阳性反应定位于子宫内膜基质细胞胞浆与细胞核。这些结果提示,HSP70可能与蜕膜反应中基质细胞增殖密切相关,雌二醇对子宫HSP70的表达具有明显的诱导作用[动物学报49(3)：345—352,2003]。     

转基因水稻T—DNA侧翼序列的扩增与分析

利用现有的转抗白叶枯病基因Xa21的水稻材料,通过TAIL－PCR技术扩增出携带Xa21基因的T－DNA的侧翼序列,对24个有效扩增片段的序列分析结果表明,其中14个侧翼序列是水稻DNA,9个含载体主干序列,1个是外源基因Xa21片段,14个T－DNA侧翼的水稻DNA序列与直接转化法外源基因整合位点的基因组序列具有不同的特点,这些T－DNA在水稻染色体上整合后其两端序列的特点类似于在转基因双子叶植物中观察到的现象,在含主干序列的侧翼序列（37．5％,9／24）,中,载体主干序列是以不同的类型出现的。