BrdU-Hoechst-Giemsa方法的进一步改进以及青鱼和草鱼复制带核型的研究

对BrdU-Hoechst-Giemsa方法进行了一定的改进和补充;并将它应用于青鱼和草鱼的核型研究中。实验进一步阐明,BrdU-Hoechst-Giemsa方法的关键性步骤之一是要先测算实验鱼的细胞周期;BrdU、AMD、Hoechst 33258,EB和AO虽都有抑制鱼类染色体浓缩、促进染色体分带的作用,但其中以DNA碱基特异性给合物BrdU、AMD和Hoechst 33258较佳,特别用AMD和Hoechst 33258同时处理活细胞的分带效果最好。

Use of lymphocyte cultures for BrdU replication banding patterns in anuran species (Amphibia)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains

Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK+ yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8–9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 ‘early’ origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.

Ovine cortical osteoblasts outperform bone marrow cells in an ectopic bone assay

Reviewing the available literature, one could conclude that marrow-derived mesenchymal stem cells (BMSCs) are the ‘gold standard’ source for bone tissue engineering applications, due to their multilineage differentiation potential and easy accessibility. However, comprehensive studies comparing their osteogenic potential with bone-derived osteoblasts (OBs) to justify the preferred application of BMSCs based on performance are few. To address these shortfalls, in the present study, ovine BMSCs and OBs seeded onto scaffolds were characterized in vitro and transplanted subcutaneously into NOD/SCID mice in combination with and without recombinant human bone morphogenetic protein 7 (rhBMP-7). It was hypothesized that cell origin, ossification type and degree of vascularization and ossification depends on the nature and commitment of transplanted cells and stimulating growth factors, such as rhBMP-7. After retrieval, specimens were analysed by biomechanical testing, µCT analysis, scanning electron microscopy/energy-dispersive X-ray spectroscopy and histo- and immunohistochemistry for osteocalcin, type II collagen and BrdU. The results showed a high degree of cell survival and proliferation ectopically, resulting in active contribution to endochondral osteogenesis. When compared to BMSCs, OBs showed a higher degree of bone deposition while OB-derived bone was of higher maturation. Stimulation with rhBMP-7 increased the rate of bone synthesis for both BMSCs and OBs, additionally promoting neovascularization and osteoclast activity. These results suggest that the origin and commitment of transplanted cells highly influence the type and degree of ossification, that rhBMP-7 represents a powerful adjuvant for bone tissue-engineering applications, and that mature bone is an adequate alternative cell source for bone tissue-engineering applications.

EdU, a new thymidine analogue for labelling proliferating cells in the nervous system

The standard method of labelling proliferating cells uses the thymidine analogue, bromodeoxyuridine (BrdU), which incorporates into the DNA during S-phase of the cell cycle. A disadvantage of this method is that the immunochemical processing requires pre-treatment of the cells and tissue with heat or acid to reveal the antigen. This pre-treatment reduces reliability of the method and degrades the specimen, reducing the ability for multiple immuno-fluorescence labelling at high resolution. We report here the utility of a novel thymidine analogue, ethynyl deoxyuridine (EdU), detected with a fluorescent azide via the “click” chemistry reaction (the Huisgen 1,3-dipolar cycloaddition reaction of an organic azide to a terminal acetylene). The detection of EdU requires no heat or acid treatment and the incorporated EdU is covalently conjugated to fluorescent probe. The reaction is quick and compatible with fluorescence immunochemistry and other fluorescent probes. We show here that EdU is non-toxic in vitro and in vivo and can be used in place of BrdU to label cells during neurogenesis and the progeny identified at least 30 days later. The fluorescent labelling of EdU, markedly improves the detection of proliferating cells and allows concurrent high resolution fluorescence immunochemistry.

Strategic targeting of the PI3K–NFκB axis in cisplatin-resistant NSCLC

Chemoresistance is a major therapeutic challenge to overcome in NSCLC, in order to improve the current survival rates of <15% at 5 years. We and others have shown increased PI3K signaling in NSCLC to be associated with a more aggressive disease, and a poorer prognosis. In this study, targeted inhibition of three strategic points of the PI3K–NFκB axis was performed with the aim of exploiting vulnerabilities in cisplatin-resistant NSCLC cells. Cisplatin-resistant cell lines were previously generated through prolonged exposure to the drug. Expression of PI3K and NFκB pathway-related genes were compared between cisplatin-resistant cells and their matched parent cells using a gene expression array, qRT-PCR, DNA sequencing, western blot, and immunofluorescence. Targeted inhibition was performed using GDC-0980, a dual PI3K–mTOR inhibitor currently in Phase II clinical trials in NSCLC, and DHMEQ, an inhibitor of NFκB translocation which has been used extensively both in vitro and in vivo. Effects of the two inhibitors were assessed by BrdU proliferation assay and multiparameter viability assay. NFKBIA was shown to be 12-fold overexpressed in cisplatin-resistant cells, with no mutations present in exons 3, 4, or 5 of the gene. Corresponding overexpression of IκBα was also observed. Treatment with DHMEQ (but not GDC-0980) led to significantly enhanced effects on viability and proliferation in cisplatin-resistant cells compared with parent cells. We conclude that NFκB inhibition represents a more promising strategy than PI3K–mTOR inhibition for treatment in the chemoresistance setting in NSCLC.

The effects of organic environmental toxicants on hard tissue formation in developing tooth : An in vitro study in mice

Dioxins are organic toxicants that are known to impair tooth development, especially dental hard tissue formation. The most toxic dioxin congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Further, clinical studies suggest that maternal smoking during pregnancy can affect child s tooth development. One of the main components of tobacco smoke is the group of non-halogenated polycyclic aromatic hydrocarbons (PAHs), a representative of which is 7,12-dimethylbenz[a]anthracene (DMBA). Tributyltin (TBT), an organic tin compound, has been shown to impair bone mineralization in experimental animals. In addition to exposure to organic toxicants, a well-established cause for enamel hypomineralization is excess fluoride intake. The principal aim of this thesis project was to examine in vitro if, in addition to dioxins, other organic environmental toxicants, like PAHs and organic tin compounds, have adverse effects on tooth development, specifically on formation and mineralization of the major dental hard tissues, the dentin and the enamel. The second aim was to investigate in vitro if fluoride could intensify the manifestation of the detrimental developmental dental effects elicited by TCDD. The study was conducted by culturing mandibular first and second molar tooth germs of E18 NMRI mouse embryos in a Trowell-type organ culture and exposing them to DMBA, TBT, and sodium fluoride (NaF) and/or TCDD at various concentrations during the secretory and mineralization stages of development. Specific methods used were HE-staining for studying cell and tissue morphology, BrdU-staining for cell proliferation, TUNEL-staining for apoptosis, and QPCR, in situ hybridization and immunohistochemistry for the expressions of selected genes associated with mineralization. This thesis work showed that DMBA, TBT, TCDD and NaF interfere with dentin and enamel formation of embryonic mouse tooth in vitro, and that fluoride can potentiate the harmful effect of TCDD. The results suggested that adverse effects of TBT involve altered expression of genes associated with mineralization, and that DMBA and TBT as well as NaF and TCDD together primarily affect dentin mineralization. Since amelogenesis does not start until mineralization of dentin begins, impaired enamel matrix secretion could be a secondary effect. Dioxins, PAHs and organotins are all liposoluble and can be transferred to the infant by breast-feeding. Since doses are usually very low, developmental toxicity on most of the organs is difficult to indentify clinically. However, tooth may act as an indicator of exposure, since the major dental hard tissues, the dentin and the enamel, are not replaced once they have been formed. Thus, disturbed dental hard tissue formation raises the question of more extensive developmental toxicity.

Combination of single walled carbon nanotubes/graphene oxide with paclitaxel: a reactive oxygen species mediated synergism for treatment of lung cancer

Heterogeneity in tumors has led to the development of combination therapies that enable enhanced cell death. Previously explored combination therapies mostly involved the use of bioactive molecules. In this work, we explored a non-conventional strategy of using carbon nanostructures (CNs) single walled carbon nanotube (SWNT) and graphene oxide (GO)] for potentiating the efficacy of a bioactive molecule paclitaxel (Tx)] for the treatment of lung cancer. The results demonstrated enhanced cell death following combination treatment of SWNT/GO and Tx indicating a synergistic effect. In addition, synergism was abrogated in the presence of an anti-oxidant, N-acetyl cysteine (NAC), and was therefore shown to be reactive oxygen species (ROS) dependent. It was further demonstrated using bromodeoxyuridine (BrdU) incorporation assay that treatment with CNs was associated with enhanced mitogen associated protein kinase (MAPK) activation that was ROS mediated. Hence, these results for the first time demonstrated the potential of SWNT/GO as co-therapeutic agents with Tx for the treatment of lung cancer.

Intranasal Delivery of Plasma and Platelet Growth Factors Using PRGF-Endoret System Enhances Neurogenesis in a Mouse Model of Alzheimer’s Disease

[EN] Neurodegeneration together with a reduction in neurogenesis are cardinal features of Alzheimer’s disease (AD) induced by a combination of toxic amyloid-β peptide (Aβ) and a loss of trophic factor support. Amelioration of these was assessed with diverse neurotrophins in experimental therapeutic approaches. The aim of this study was to investigate whether intranasal delivery of plasma rich in growth factors (PRGF-Endoret), an autologous pool of morphogens and proteins, could enhance hippocampal neurogenesis and reduce neurodegeneration in an amyloid precursor protein/presenilin-1 (APP/PS1) mouse model. Neurotrophic and neuroprotective actions were firstly evident in primary neuronal cultures, where cell proliferation and survival were augmented by Endoret treatment. Translation of these effects in vivo was assessed in wild type and APP/PS1 mice, where neurogenesis was evaluated using 5-bromodeoxyuridine (BdrU), doublecortin (DCX), and NeuN immunostaining 5 weeks after Endoret administration. The number of BrdU, DCX, and NeuN positive cell was increased after chronic treatment. The number of degenerating neurons, detected with fluoro Jade-B staining was reduced in Endoret-treated APP/PS1 mice at 5 week after intranasal administration. In conclusion, Endoret was able to activate neuronal progenitor cells, enhancing hippocampal neurogenesis, and to reduce Aβ-induced neurodegeneration in a mouse model of AD.

长日光周期对水稻幼穗发育的影响及其对光敏核不育水稻育性转换的作用研究

光敏核不育水稻晚粳农垦58S具有长日照下不育、短日照下可育的特点，是目前二系法杂交水稻应用的基础。对于其长日光周期引起雄性败育的特性已得到很多实验的支持，但这种光周期反应特性是光敏不育材料所特有，还是在水稻穗发育中普遍存在，目前尚不清楚。对这一问题的认识涉及到对光敏不育性本质的了解及对这一性状的有效利用，本文对此进行了系统的研究分析。 本研究以24种水稻品种包括光敏核不育系及常规水稻品种为材料，在控制光周期下进行。即利用16h长日照处理(LD)和l0h短日照处理(SD)及其不同组合，以抽穗期、叶龄、抽穗叶片总数、花粉育性、结实率、穗长、穗粒密度为指标，结合光敏不育系幼穗发育的形态解剖学特征，探讨了在整个水稻发育中包括叶片生长、幼穗分化以及穗发育等过程中，不同材料的光周期反应特征，尤其是二次枝梗期后的穗发育过程中的光周期反应特征。此外还分析了温度与光周期反应的关系及温度在光敏不育现象中的作用，并研究了代谢抑制剂对光敏不育特征的影响。 研究表明，光周期对水稻的出叶速度基本没有影响，但对水稻的抽穗叶龄有影响，长日照使抽穗叶龄增加而延迟其穗分化及抽穗。光周期还对幼穗分化后的穗发育过程有抑制延迟，作用，影响大小因品种而异，以对早稻、籼稻的影响最弱，对晚稻、粳稻的影响最强，与其穗分化中的感光性有明显的相关性。 除对抽穗期有影响外，穗发育阶段的长日光周期还影响着穗发育的其它性状，如使穗长增加，芒较长、稳粒密度降低，花粉育性降低，结实率下降。此外植株发育的其它性状也可受到影响，如剑叶发育不良表现为叶片缺少仅有叶鞘、倒二叶生长旺盛、植株较高等。同时几组不同组合的光周期处理结果均表明，长日光周期对水稻穗发育的影响主要发生在穗发育的前5-10天即颖花原基分化期、雌雄蕊原基分化期至花粉母细胞形成期。这些结果表明水稻的光周期反应不仅表现在茎端从营养生长向生殖生长的转换上（幼穗分化），而且还表现在幼穗分化完成后的穗发育过程中。长日光周期对晚稻穗发育均有抑制效应，且日长对稳发育的影响时期与光敏核不育水稻的‘育性转换敏感期’完全一致。因此在农垦58S中引起‘光敏不育’的原因很可能不是一种特殊的光周期反应，而是该材料雄性器官发生过程不能对长日光周期做出适当的反应。 对24种不同品种水稻的光周期反应表明，不同材料光周期反应特性不同。光敏不育系农垦58S与农垦58在对长日照的反应上也有较大不同，表现为前者在短日照下穗分化较快，在自然日照下抽穗较早。这表明除了育性不同外，农垦58S与农垦58在光周期反应特征上也有所不同，然而我们认为这种不同不是农垦58S表现光敏不育的主要原因。因为本研究中还发现，光敏不育系农垦58S与其可育回复突变体农垦58S(r)在抽穗期等光周期反应特征上相当一致，但在育性反应上却有较大不同，长日照下农垦58S(r)表现为雄性可育，而农垦58S表现为雄性败育。根据上述几方面的比较，我们认为光敏不育的机制很可能在于农垦58S突变体其雄性器官发育对环境不利信号的反应能力的变弱所致。 在本研究中发现，温度对水稻穗发育的影响表现在两个方面：一方面是通过影响光周期反应强弱而起作用，如高温可加强短日照下的穗分化和发育过程，高温亦可加强长日照对穗分化发育的抑制作用;另一方面是直接对器官发生过程产生影响，如在对短日照下光敏不育系和常规稻不同温度条件下处理时的结实率比较分析发现，常规稻的结实率与其抽穗扬花期的平均温度显著负相关，而光敏核不育水稻的结实率虽与抽穗扬花期的温度有一定相关性，但更与穗发育期的平均温度呈显著负相关，二者在受温度影响的作用时期上有显著差异，因此温度也可直接对雄性器官发育起作用。区分温度对光敏不育的两方面影响，同时考虑到光敏不育机制更有可能在于光敏不育系农垦58S雄性器官发育对环境信号反应能力的变弱的假设。我们就可以较好地理解农垦58S‘光敏不育’性状经杂交转育到对光周期弱感的籼稻中所出现的‘温敏不育性’。 核酸代谢抑制剂5-FU，2-TU对SD下的幼穗分化有较强抑制作用，使幼穗分化被迟滞，而2-BrDU和蛋白质合成抑制剂CHX、CL对其影响较小。抑制剂处理也不能诱导LD下的穗分化。 短日照下，5-FU可对穗发育有强烈抑制作用，可使常规品种农垦58及光敏不育系农垦58S穗畸形，颖花减少并发育不良，穗长缩短，枝梗减少，花粉败育甚至无花粉，结实率显著降低，其有效作用时期为穗发育的二次枝梗分化期至雌雄蕊原基分化期，与长日照诱导农垦58S败育的作用时期也完全吻合，5-FU对SD下穗发育的影响还可被核酸抑制剂的恢复剂乳清酸所部分恢复。其它代谢抑制剂如2-TU、CHX、CL等也可使农垦58S育性明显降饭，而所有这些抑制剂对常规可育的农垦58及农垦58S(r)的育性影响较小，表明它们与光敏不育系对抑制信号的反应能力有显著不同。 长日照下5-FU对LD下的农垦58S的幼穗发育也有很强的抑制作用，使稳长缩短，颖数减少，但它还可使部分LD下处理植株抽穗期较LD对照明显提前，并可使农垦58S育性部分恢复而有结实，说明5-FU还可对LD的抑制作用有抑制，通过对LD抑制作用的抑制使LD下的育性转换有部分恢复。其它代谢抑制剂在穗发育前期处理LD下农垦58S叶片均可看到植株在抽穗期较LD下提前5—8天的同时，其花粉育性有不同程度的提高，在高温长日下甚至有一定程度的结实率，表明各种抑制剂均可对穗发育中的光周期作用产生影响。 总之，本研究结果表明，短日植物水稻的光周期反应不仅存在于幼穗分化上，还存在于幼穗发育和花器官发生等发育过程中。幼穗发育的光周期效应表现为抽穗期、穗长、穗粒密度、结实率等多方面的变化，作用时期以穗发育早期的花器官发生阶段影响最大。作用强弱因品种不同而异，以粳稻和晚稻中作用较强。光敏不育突变的更主要变化可能在于农垦58S的雄性器官分化发育时对环境不利信号的反应能力变弱，导致其正常发育受阻，育性不能正常表达。温度在水稻穗发育上既可通过影响光周期反应而起作用，还可直接对穗器官发育产生影响而对育性表达起作用。此外我们还发现农垦58S与农垦58不仅在雄性育性上有显著不同，而且其光周期反应特性也有较大的差异。抑制剂处理结果也支持光敏不育系农垦58S的雄性器官发生过程较农垦58更易受抑制剂影响而育性降低，而抑制剂对长日光周期抑制作用的部分解除，可以使其育性有一定程度的恢复，也表明光周期对雄性育性的影响最为显著。这些结果可以帮助我们更加全面地认识光敏不育水稻的基本特性，从而为进一步开展光敏不育的转育及应用研究提供可靠的科学依据。

人胚神经干细胞移植治疗大鼠脊髓损伤

　目的　探讨人胚神经干细胞( hNSC) 移植治疗脊髓损伤(SCI) 的可行性。方法　分 离、培养和鉴定hNSC;用5 溴22 脱氧尿苷嘧啶(BrdU) 标记hNSC ,并将其移植到14 只T10 半横断 的Wistar 大鼠损伤脊髓内(另外14 只T10 半横断损伤的大鼠作为对照组,仅损伤脊髓内注射 DMEM/ F12 培养液) ,用BrdU 的FITC 免疫荧光染色检测移植细胞的存活和迁徙,用NF2200 、 GFAP 免疫组织化学鉴定移植细胞的分化,BBB 评分评定大鼠功能恢复情况。结果　(1) 获得了大 量的hNSC; (2) 用免疫组织化学可以检测到移植的hNSC 能在体内长时间存活(达2 个月) 并向远 处迁徙,并分化为神经元和胶质细胞; (3) 检测到实验组大鼠BBB 得分明显高于对照组大鼠( P < 0. 01) ,在SCI 后第10 周时实验组和对照组BBB 得分最大差距达到2. 1分。结论　hNSC 移植能促 进SCI 大鼠后肢功能恢复,它是SCI 移植治疗较有价值的细胞资源。

人胚胎神经干细胞的分离培养及鉴定研究

目的:研究人胚胎神经干细胞的培养条件及体外分化情况。方法:从12周龄人胚胎脑皮质分离细胞,采用无血清培养技术,协同应用碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)和重组人白血病抑制因子(rhLIF)进行培养;5-溴脱氧尿嘧啶核苷(BrdU)标记检测细胞的增殖能力,间接免疫荧光化学法检测细胞的分化情况。结果:培养得到的大量半悬浮生长的神经干细胞球能够传代培养;BrdU标记阳性,可诱导分化为神经元、星形胶质细胞和少突胶质细胞。结论:人胚胎脑皮质分离细胞培养得到的细胞群具有神经干细胞的基本特征,可进一步用于基础及临床研究。

草鱼体内肾细胞姐妹染色单体分化及交换的初步研究

本文确立了一个以草鱼体内肾细胞姐妹染色单体交换频率为指标的检测环境诱变或致癌物质的短期试验系统。采用硫堇-UV-Giemsa染色法,分析了草鱼体内肾细胞的SCD-2(注射BrdU后第二个细胞周期的中期分裂相的SCD)频率和SCE频率。用500微克/克体重BrdU体内标记5天,草鱼肾细胞SCD-2频率为8.58±0.22%;SCE频率为3.05±2.523 SCE_5/细胞。以丝裂霉素C(Mitomycin C,MMC)作为阳性对照,分析了化合物亚硝基胍(N-methyl-N~1-nitro-N-nitro

猕猴体细胞核移植影响因素的研究：着床前胚胎表观遗传重编程和供体细胞周期同步化

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