白藜芦醇对CdCl_2暴露所致斑马鱼胚胎发育毒性干预作用及机制的初步研究

为考察天然抗氧化剂白藜芦醇(Res)对Cd Cl2暴露所致斑马鱼胚胎发育毒性的干预作用,并初步探讨其作用机制。采用单独应用Res联合Cd Cl2共同处理受精后2 h(2 hpf)的斑马鱼胚胎,观察至96 hpf胚胎死亡率和孵化率;采用荧光探针DCFH-DA、Mito SOX、JC-1分别检测胚胎总体活性氧、线粒体源ROS水平和胚胎线粒体的膜电势;采用吖啶橙(AO)染色法检测细胞凋亡情况;采用Real-time PCR方法检测胚胎线粒体单链DNA结合蛋白(mt SSBP)、OPA1的表达水平。结果显示,100μmol/L Res持续暴露96 h可明显增加斑马鱼胚胎死亡率(P0.05)。20~100μmol/L Res与20 mg/L Cd Cl2联合暴露较单独Cd Cl2暴露组相比,胚胎死亡率和未孵化率明显增加(P0.01)。100μmol/L Res与20 mg/L Cd Cl2联合处理明显增加了胚胎总体ROS(P0.01)和线粒体内ROS(P0.01)的生成,降低了线粒体的膜电势(P0.05),胚胎细胞凋亡细胞数量明显增加。与Cd Cl2单独暴露组相比,Res联合处理明显下调了mt SSBP和OPA1的m RNA表达水平(P0.01)。由此可知,高浓度Res明显增强Cd Cl2所致的斑马鱼发育毒性,胚胎总体ROS和线粒体源ROS水平升高,线粒体膜电势下降,mt SSBP和OPA1表达下降,表明线粒体损害可能介导了Res对Cd Cl2育毒性的增敏作用。     

hESC衍生和组织源MSC标记GFP基因的特性分析

干细胞标记示踪在研究细胞治疗机理中具有重要作用,而标记物对干细胞的安全可靠性是细胞标记示踪的重要前提。文章对人胚胎干细胞衍生的间充质干细胞（hESC-MSC）、人脂肪间充质干细胞（hADSC）和人脐带间充质干细胞（hUC-MSC）进行了绿色荧光蛋白（GFP）基因慢病毒转导并比较它们在转导前后的增殖、多项分化潜能和细胞表型等生物学特性以及它们的转导效率。结果表明,在等量pLVX-IRES-Puro-GFP慢病毒载体转导下,hESC-MSC细胞慢病毒转导效率显著高于h ADSC(P<0.01)和hUC-MSC(P<0.01);但是hESC-MSC、hADSC和h UC-MSC标记GFP基因前后细胞增长形态、增殖力、细胞表面抗原的表达和分化潜能均没有明显差异。文章提示不同来源的间充质干细胞均可进行安全可靠的GFP基因标记,而人胚胎干细胞衍生的间充质干细胞可能更适合用以慢病毒感染的示踪标记或者基因修饰。     

基于斑马鱼模型的海洋中药厚藤安全性评价

采用模式生物斑马鱼模型评价海洋中药厚藤水提物（Ipomoea pes-caprae（Linn.） Sweet water extract,IWE）及醇提物（Ipomoea pes-caprae（Linn.） Sweet alcohol extract,IAE）的安全性。采用受精后3 dpf （Day post fertilization） AB系斑马鱼进行急性毒性评价,将受精后10 hpf （Hours post fertilization）斑马鱼用于胚胎发育毒性及心脏毒性评价;将受精后3 dpf的gz15Tg/+（AB）肝脏荧光蛋白转基因斑马鱼用于肝脏毒性评价。结果表明,急性毒性实验中厚藤水提物半数致死浓度（LC₅₀）为2.84 mg/mL,10%致死浓度（LC₁₀）为1.46 mg/mL;厚藤醇提物LC₅₀为1.32 mg/mL,LC₁₀为0.76 mg/mL。胚胎发育毒性实验中厚藤水提物最大非致死浓度（MNLC）组及LC₁₀组干预24 h对斑马鱼胚胎未见尾部自主运动现象且发育迟缓,斑马鱼发育至72 hpf出现心脏和软黄囊肿大现象;厚藤醇提物LC₁₀组与空白对照组比较出现心包水肿,血瘀,观察中各组未见胚胎发育畸形现象。心脏毒性实验中空白对照组斑马鱼胚胎心跳节律规则、搏动强有力;厚藤水提物及醇提物在24 hpf,48 hpf,72 hpf时间点1/3MNLC组、MNLC组及LC₁₀组分别与空白对照组比较,均能明显减慢斑马鱼胚胎的心率且呈现剂量依赖性（P<0.01）;厚藤水提物及醇提物1/3MNLC组、MNLC组及LC₁₀组分别与空白对照组比较,均能明显增加斑马鱼胚胎的SV-BA间距（P<0.01）。肝脏毒性实验中厚藤水提物中各给药组与空白对照组比较,肝脏荧光面积均有减少趋势,组间比较无差异性;厚藤醇提物中1/3MNLC组、MNLC组及LC₁₀组与空白对照组比较,肝脏荧光面积均有增加趋势,但组间比较无差异性,各给药组斑马鱼的丙氨酸氨基转移酶（ALT）、天冬氨酸转氨酶（AST）水平与空白对照组比较均无显著差异。本实验中厚藤水提物的LC₅₀显著高于醇提物。高剂量LC₁₀组均对斑马鱼胚胎发育产生一定毒性作用。厚藤水提物及醇提物随着给药剂量增加,明显减慢斑马鱼胚胎的心率,产生一定心脏毒性作用。厚藤水提物及醇提物均未引起明显的肝脏毒性。     

顺铂类配合物肝毒性的自由基机理研究

运用自旋捕捉电子自旋共振技术直接捕捉到兔肝、肾微粒体脂质过氧化形成的脂自由基Ｌ，上亚细胞水平的实验研究表明各种顺铂类配合物促进Ｌ形成的作用和ＭＤＡ的增长，血浆抗坏血酸（ＶＣ）库水平及血液ＧＳＨ－Ｐｘ活性的降低呈相似的变化趋势。作用的强弱与配合物的化学结构有关。顺铂的肝毒性与肾毒性一样，可能与自由基介导的氧化性损伤有关。     

一种新颖的阳离子非病毒基因载体 Chitosan-g-PEI-g-PEG-OH 的性能研究

研究了新型阳离子聚合物Chitosan-g-PEI-g-PEG-OH的性能，重点考察其粒度，基因转染效率与细胞毒性，探讨了其作为基因载体的可能性.通过动态光散射仪（DSL）、透射电镜（TEM）观察了Chitosan-g-PEI-g-PEG-OH与DNA自组装形成的颗粒形态及粒径，Chitosan-g-PEI-g-PEG-OH可复合DNA形成粒径160～210 nm的纳米复合物，适合进入细胞.使用MTT比色法分析Chitosan-g-PEI-g-PEG-OH的毒性并与PEI，PEI-g-PEG-OH比较.选用增强型绿色荧光蛋白(EGFP) 转染Hela细胞，应用流式细胞术检测转染效率.新型阳离子多聚物Chitosan-g-PEI-g-PEG-OH在提高基因转染效率的同时降低了其细胞毒性，有望成为基因转移的有效载体.     

鸡与鹌鹑杂交种早期胚胎发育特异表达基因的初步筛选

利用mRNA差异显示技术，对入孵67～91h的鸡（♀）与鹌鹑（♂）杂交种胚胎中mRNA差异表达进行比较，筛选出与杂交种胚胎早期发育相关的特异表达基因，并克隆测序这些序列，分析这些基因在杂交受精蛋早期胚胎发育中的作用，及其对杂交种早期胚胎死亡是否有影响。分析结果表明，已经获得6个杂交种孵化不同时期的基因片段与鸡的mRNA序列具有高度同源性。其中，孵化第72h时胚胎mRNA表达的序列与鸡雌激素受体结合位点相关抗原mRNA具有高度同源性，孵化第79h时产生的差异片段经过比对，发现：与鸡（♀）肽酰-脯氨酰异构酶类似的mRNA序列具有高度同源性。而杂交种胚胎发育到第72h时，可检测到鸡雌激素受体结合位点相关抗原mRNA的表达，比前人所得到的表达时间（胚胎发育在第84h时检测到）提前了12h，这很可能是造成杂交种早期胚胎大量死亡的原因之一。     

超氧化物歧化酶在斑马鱼早期胚胎中的表达特点

以斑马鱼早期胚胎为研究对象，通过免疫组织化学的方法研究了超氧化物歧化酶（SOD）在斑马鱼体节分化时期的表达特点。结果发现：在体节分化的不同时期，SOD在脊索细胞的胞质部位有不同强度的表达；SOD在脑组织中表达较强，到孵化期，在大脑区域的表达略强于间脑区域。提示SOD不仅存在于成体的抗氧化防线中，在胚胎早期发育过程中以SOD为代表的酶性抗氧化系统就开始逐步建立，维持胚胎组织器官细胞中的正常活性氧浓度，确保胚胎的正常发育。     

胰岛素样生长因子及其受体在绵羊早期胚胎的表达

旨在研究IGFs及其受体（IGFR）在绵羊卵母细胞和早期胚胎中的表达．对绵羊进行超数排卵处理,手术法采卵得到卵母细胞和着床前早期胚胎,结合荧光免疫染色方法和激光共聚焦成像系统,研究了绵羊未成熟卵母细胞、成熟卵母细胞和着床前各期胚胎中IGF—Ⅰ、IGF-Ⅱ、IGF—ⅠR、IGF—ⅡR的表达情况．结果表明,IGF-Ⅰ、IGF-Ⅱ在未成熟卵母细胞和成熟卵母细胞中均有表达;受精后,直到囊胚的整个早期发育阶段也检测到了这两个因子的表达,且胚胎免疫染色在各卵裂球之间呈现不均匀的分布．IGF—ⅠR和IGF—ⅡR在未成熟卵母细胞、成熟卵母细胞和早期胚胎均有表达,在胚胎期主要分布于卵裂球细胞的顶端（apical surface）,而在囊胚阶段主要在滋胚层细胞中表达,内细胞团没有表达．     

茶多酚对肿瘤细胞多药耐药性逆转作用的研究

用免疫组化法和流式细胞仪对肿瘤细胞系MCF－7和MCF－7／Adr的P－糖蛋白表达水平进行定性定量研究。用噻唑蓝比色法（MTT）研究茶多酚的细胞毒性及其对耐药性的逆转作用,并与Pgp抑制剂－奎尼定进行了比较。免疫组化法检测P－糖蛋白表达水平,MCF－7／Adr呈强阳性,而CF－7呈阴性;流式细胞仪定量检测结果MCF－7／Adr细胞系细胞阳性率为15％,MCF－7细胞系细胞阳性率为1.8%。MCF－7／Adr细胞系存在Pgp的过度表达。噻唑蓝比色法（MTT）检测结果茶多酚、奎尼定的加入对阿霉素对MCF－7的细胞毒性几乎没有影响。而茶多酚、奎尼定的加入明显增加了MCF－U／Adr对阿霉素的敏感性。免疫组化与流式细胞技术结合,可用于肿瘤细胞系P－糖蛋白表达水平的定性定量检测。茶多酚不仅具有一定的抗癌活性,还与奎尼定一样具有多药耐药性逆转作用。     

组蛋白修饰与ES细胞的多能性

胚胎干细胞（ESCs）来源于早期胚胎内细胞群，具有分化和发育多能性和无限增殖与更新能力。组蛋白修饰对ES细胞的自我更新和无限增殖能力及多能性保持具有重要作用。组蛋白修饰是表观遗传调控的关键因素，细胞通过表观遗传状态改变控制基因的选择性表达，实现对细胞分化的调控。并且可以建立调控网络调节ES细胞多能性维持。     

Influence of PrP 106―126 on expression of laminin and fibronectin in astrocyte

Astrogliosis is a hallmark of prion disease, but the metabolic alterations of astrocytes remain poorly documented. A synthetic peptide corresponding to amino acid 106-126 of the human prion protein （PrP） has been shown to be toxic to neurons. In this study, the effects of PrP 106-126 on astrocytes were investigated in vitro. The proliferation of astrocytes was significantly （P 〈 0.05） increased when grown in media conditioned with PrP 106-126 （80 μmol/L） from microglia. The expression of laminin （LN） and fibronectin （FN） was examined at both mRNA and protein levels. The results showed that exposure of astrocytes to PrP 106-126 enhanced the expression of LN and FN. The increase of FN in astrocyte cultures required cytokines previously released by activated microglia. This study reveals the expression of LN and FN affected by PrP106-126.     

Cloning, sequencing and phylogenic analysis of duck prion gene

Duck prion gene was cloned and sequenced. Similar to mammalian prion protein (PrP), duck prion is encoded by a single exon of a single copy in genome, which was confirmed by Southern blot analysis. All of the structural features of mammalian PrP were also identified in the duck PrP. Compared with mammalian PrP, it exhibited a 30 % of general similarity. When compared with chicken PrP, it showed a higher homology of 97%. A phylogenetic tree was constructed to trace evolution of prion gene in animals.     

朊蛋白（PrP）与叠朊（Doppel）蛋白相关性的研究进展

近几年研究发现,与传染性海绵状脑病（TSE）发生密切相关的朊蛋白基因Prn,包括三种基因：Prnp（PrP编码基因）,Prnd（Doppel的编码基因）及Prnt．其中Prnd与Prnp关系更加密切．研究指出,二者的编码蛋白Doppel与PiP。的同源性虽然不高,但其翻译后修饰及空间结构却十分相似．这种相似性造成了二者在功能上的相关．如PrPc的缺失可能引起Doppel过表达;而prpc也似乎能够拮抗Doppel引起的神经毒性．     

Binding of disease-associated prion protein to plasminogen

Transmissible spongiform encephalopathies are associated with accumulation of PrP(Sc), a conformer of a cellular protein called PrP(C). PrP(Sc) is thought to replicate by imparting its conformation onto PrP(C) (ref. 1), yet conformational discrimination between PrP(C) and PrP(Sc) has remained elusive. Because deposition of PrP(Sc) alone is not enough to cause neuropathology, PrP(Sc) probably damages the brain by interacting with other cellular constituents. Here we find activities in human and mouse blood which bind PrP(Sc) and prion infectivity, but not PrP(C). We identify plasminogen, a pro-protease implicated in neuronal excitotoxicity, as a PrP(Sc)-binding protein. Binding is abolished if the conformation of PrP(Sc) is disrupted by 6M urea or guanidine. The isolated lysine binding site 1 of plasminogen (kringles I-III) retains this binding activity, and binding can be competed for with lysine. Therefore, plasminogen represents the first endogenous factor discriminating between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes.     

Fibrils from brains of cows with new cattle disease contain scrapie-associated protein

During the past two years, more than 1,000 cases of a neurological disorder of cattle, bovine spongiform encephalopathy (BSE), have been confirmed from farms throughout Great Britain. The neurological signs and brain pathology of BSE resemble those produced in other species by the pathogens of scrapie and related disorders. The discovery of fibrils similar to scrapie-associated fibrils in detergent extracts o BSE-affected brain supported the clinical and pathological diagnosis of the disease, but has been controversial. Scrapie-associated fibrils are found in brain extracts of all species affected by scrapie and diseases caused by related pathogens. They are pathological aggregates of a neuronal membrane protein termed PrP and a protease-resistant form of PrP is a molecular marker of scrapie-associated fibrils. In this report, we show the major protein of BSE fibrils is the bovine homologue of PrP as judged by its size, protease resistance, immunoreactivity, lectin binding and partial N-terminal protein sequence. This confirms that BSE is a scrapie-like disease.     

Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding.

Prions are the infectious agents responsible for transmissible spongiform encephalopathies. The principal component of prions is the glycoprotein PrP(Sc), which is a conformationally modified isoform of a normal cell-surface protein called PrP(C) (ref. 1). During the time between infection and the appearance of the clinical symptoms, minute amounts of PrP(Sc) replicate by conversion of host PrP(C), generating large amounts of PrP(Sc) aggregates in the brains of diseased individuals. We aimed to reproduce this event in vitro. Here we report a procedure involving cyclic amplification of protein misfolding that allows a rapid conversion of large excess PrP(C) into a protease-resistant, PrP(Sc)-like form in the presence of minute quantities of PrP(Sc) template. In this procedure, conceptually analogous to polymerase chain reaction cycling, aggregates formed when PrP(Sc) is incubated with PrP(C) are disrupted by sonication to generate multiple smaller units for the continued formation of new PrP(Sc). After cyclic amplification more than 97% of the protease-resistant PrP present in the sample corresponds to newly converted protein. The method could be applied to diagnose the presence of currently undetectable prion infectious agent in tissues and biological fluids, and may provide a unique opportunity to determine whether PrP(Sc) replication results in the generation of infectivity in vitro.     

The most infectious prion protein particles

Neurodegenerative diseases such as Alzheimer's, Parkinson's and the transmissible spongiform encephalopathies (TSEs) are characterized by abnormal protein deposits, often with large amyloid fibrils. However, questions have arisen as to whether such fibrils or smaller subfibrillar oligomers are the prime causes of disease. Abnormal deposits in TSEs are rich in PrP(res), a protease-resistant form of the PrP protein with the ability to convert the normal, protease-sensitive form of the protein (PrP(sen)) into PrP(res) (ref. 3). TSEs can be transmitted between organisms by an enigmatic agent (prion) that contains PrP(res) (refs 4 and 5). To evaluate systematically the relationship between infectivity, converting activity and the size of various PrP(res)-containing aggregates, PrP(res) was partially disaggregated, fractionated by size and analysed by light scattering and non-denaturing gel electrophoresis. Our analyses revealed that with respect to PrP content, infectivity and converting activity peaked markedly in 17-27-nm (300-600 kDa) particles, whereas these activities were substantially lower in large fibrils and virtually absent in oligomers of < or =5 PrP molecules. These results suggest that non-fibrillar particles, with masses equivalent to 14-28 PrP molecules, are the most efficient initiators of TSE disease.     

Identification of scrapie prion protein-specific mRNA in scrapie-infected and uninfected brain

To date no nucleic acid has been found in the purified infectious agent which causes the spongiform encephalopathy known as scrapie. In an attempt to identify a unique scrapie virus-associated messenger RNA in tissues of infected animals, we have synthesized an oligonucleotide probe complementary to the mRNA sequence corresponding to the amino-acid sequence of the prion protein, PrP27-30 (ref. 1). We report here that, with this probe, a complementary DNA clone representing PrP27-30 was obtained from scrapie-infected mouse brain; the DNA sequence of this clone could be translated into a protein that matches exactly the published sequence of PrP27-30. The cDNA clone hybridized to a single 2.4-2.5-kilobase (kb) mRNA from both normal and scrapie-infected brain. Thus, the PrP27-30 mRNA is not uniquely associated with scrapie infectivity, suggesting that PrP27-30 may be a normal component of mouse and hamster brain.     

Amphotericin B treatment dissociates in vivo replication of the scrapie agent from PrP accumulation.

Scrapie and related animal and human disorders are neurodegenerative diseases characterized by the formation of a modified, partly proteinase-resistant protein (PrP) of the host, which tends to aggregate as amyloid fibrils and accumulate in the brain of infected individuals. There is a general consensus that the pathological form of PrP (PrPSc) is essential for the clinical appearance of the disease, but whether it is part of the scrapie agent or a by-product of viral infection is still controversial. Here we report that treatment of scrapie-infected hamsters with amphotericin B delays the accumulation in the brain of the proteinase-resistant portion of PrPSc by about 30 days without affecting scrapie replication. The consequence is that hamsters treated with amphotericin B developed clinical signs of disease later than infected controls. We argue that the proteinase-resistant portion of PrPSc is necessary for the development of the disease but that it is unlikely to be essential for scrapie replication.     

Induction of embryonic stem cells to hematopoietic cells in vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright's staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34+ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34-CD38+. Wright's staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.