绿色荧光蛋白GFP的研究进展及应用

源于多管水母属等海洋无脊椎动物的绿色荧光蛋白(GFP),是一种极具应用潜力的标记物,有着极其广泛的应用前景.我们就GFP的理化性质、荧光特性、改进和应用研究进行了综述.     

病毒与非病毒介导的绿色荧光蛋白基因转导的比较研究

自 1992年绿色荧光蛋白 (ＧＦＰ)基因的ｃＤＮＡ被成功克隆以来 ,由于ＧＦＰ结构稳定、在受到蓝光或紫外光照射时可自发发出绿色荧光 ,而这一过程不需要其他共刺激因子、底物或其他基因产物的协助 ,故在生物医学领域的应用日益广泛[1,2 ] 。因此 ,深入研究各种基因转导方法在ＧＦＰ基因转导方面的效率及优缺点、分析ＧＦＰ对细胞生长速度和生物学行为的影响等 ,对于合理应用ＧＦＰ十分重要。一、材料与方法1 重组质粒 :ＧＦＰ真核表达质粒ｐＥＧＦＰ Ｎ1购自Ｃｌｏｎｔｅｃｈ公司。ＧＦＰ重组逆转录病毒质粒ｐＬＮＣＸ ＥＧＦＰ是我们自己构…     

细胞穿透肽PEP-1介导增强型绿色荧光蛋白在小鼠体内跨膜转导

目的 研究细胞穿透肽PEP-1介导的大分子物质在小鼠体内的跨膜转导能力.方法 用基因工程的方法制备并纯化增强型绿色荧光蛋白EGFP和PEP-1-EGFP融合蛋白,分别将500 μg的EGFP蛋白和PEP-1-EGFP融合蛋白通过尾静脉注射入昆明小鼠体内,2 h后麻醉小鼠,PBS充分灌流并取心、脑、肝、脾、肾快速冷冻切片后立即置荧光显微镜下观察.结果 2 h后PEP-1-EGFP融合蛋白处理的小鼠大脑、心肌、肝、脾和肾组织里出现均一的明亮绿色荧光,而EGFP蛋白处理的小鼠各脏器内均未见到绿色荧光.结论 细胞穿透肽PEP-1能携带增强型绿色荧光蛋白穿透小鼠细胞膜并分布于心、脑、肝、脾、肾组织内,为将来用PEP-1介导各种大分子药物跨膜转导进行各种疾病的蛋白治疗提供实验依据.     

5-HT_(1A)受体蛋白在PC12细胞膜转运的动态变化

目的在神经元样PC12活细胞上进行实时、可视和定量研究5-羟色胺1A受体的时空分布、膜转运和信号传导机制。方法运用RT-PCR方法获得小鼠5-HT1A基因,并插入到pEGFP-N1真核表达载体中。采用阳离子脂质体方法将质粒转染至PC12细胞和HEK293细胞中,通过G418筛选出稳定表达5-HT1A-EGFP的PC12细胞系。运用激光共聚焦成像系统观察活细胞中5-HT1A-EGFP的表达情况,利用光漂白荧光恢复(FRAP)技术在PC12细胞膜局部漂白后观察5-HT1A-EGFP荧光蛋白在细胞膜上转运的情况。结果克隆所获得的小鼠5-HT1A基因是准确的。5-HT1A-EGFP蛋白清晰的分布于PC12和HKE293细胞膜上。通过FRAP技术观察到漂白区域的细胞膜在100s内有部分恢复,说明受体在细胞膜上发生转运。结论建立了稳定表达5-HT1A-EGFP融合蛋白的PC12细胞系,并利用活细胞成像和FRAP技术观察分析并证实了5-HT1A受体在PC12细胞的膜表面的表达和转运的动态变化。     

内源性PS1在活体HEK293和HeLa细胞膜上的定位

目的 检测内源性PS1在活体细胞膜上的定位及其表达.方法 为防止细胞固定及通透使胞膜被破坏,在未经固定和通透的活体细胞上利用PS1抗体,通过间接免疫荧光法观察了内源性PS1在活细胞膜外表面的定位和表达;利用分子标签技术,构建GFP-PS1融合蛋白,瞬时转染细胞,检测到外源性PS1在细胞核膜的定位及在胞质中的少量分布;同时利用PS1抗体,将细胞固定及通透,用间接免疫荧光技术,检测到内源性PS1在细胞核膜中的定位及在细胞质中的少量不均匀分布.结果 内源性PS1在定位于亚细胞结构的膜同时,也定位于细胞外膜.结论 成功地在活体细胞外膜上检测到内源性PS1.     

慢病毒介导的红色荧光蛋白基因在大鼠坐骨神经细胞的表达

目的 研究慢病毒载体介导的红色荧光蛋白(RFP)基因在坐骨神经雪旺细胞体内转染情况.方法 将60只Wistar大鼠随机分成A组、B组、C组和D组四个组,每组15只.四组均行右坐骨神经损伤模型,术毕向B组、C组和D组坐骨神经内注入滴度分别为2×10-6 TU、2×10-7 TU、2×10-8 TU的Lenti-RFP悬液8μl,向A组注入8μl平衡盐液(BSS)作为对照组.术后第1、2、4周观察红色荧光蛋白的表达情况,计算转染效率;并进行神经组织学检查.结果 术后第1周B、C、D组均出现RFP表达,术后第4周D组的转染效率最高.同一时间不同滴度组以及同一滴度组不同时间比较差异有统计学意义(P＜0.05).组织学检查病毒载体对神经未见明显炎性反应及组织学损伤.结论 慢病毒载体能安全有效的在体内转染雪旺细胞,并有浓度依赖性和时间依赖性,对组织无明显毒性作用.     

硫氧还蛋白和氧化/还原因子的融合荧光蛋白载体的构建及在293T细胞中的分布

目的构建硫氧还蛋白和氧化/还原因子的融合荧光蛋白真核表达载体,并在293T细胞中得到表达和定位。方法以RT-PCR方法从PC12细胞中克隆氧化/还原因子(APE/ref-1)的cDNA,然后亚克隆构建APE/ref-1-GFP融合真核表达载体。以PCR方法将质粒pQE30-TRX上的硫氧还蛋白cDNA亚克隆到pDsred1-1质粒上,然后将硫氧还蛋白-DsRed融合基因序列亚克隆到pCMV5质粒上,构建硫氧还蛋白-DsRed融合真核表达载体.通过磷酸钙转染293T细胞,以荧光显微镜分析融合蛋白的表达及其亚细胞定位。结果成功构建了硫氧还蛋白-DsRed和APE/ref-1-GFP融合表达载体,并在293T细胞中得到表达,APE/ref-1-GFP融合蛋白定位在293T细胞核内;硫氧还蛋白-DsRed融合蛋白定位在293T细胞质和细胞核内。结论为进一步研究硫氧还蛋白和APE/ref-1之间的动态相互作用奠定基础。     

质粒型HSV载体介导的GDNF和GFP基因在BHK细胞和骨骼肌细胞中的转移和表达

为了进一步探索治疗中枢神经损伤的新途径 ,本实验以质粒型单纯疱疹病毒 ( H SV)为载体 ,分别构建了含有胶质细胞源性神经营养因子 ( GDN F)和绿色荧光蛋白 ( GFP)基因的重组载体 H SV - GDNF和 H SV- GFP,用包装出的混合毒株分别感染体外培养的 BHK细胞及原代培养的大鼠骨骼肌细胞。 2 d后 ,对感染 HSV - G DNF组的细胞进行 G DNF免疫组织化学反应 ;感染HSV- GFP组的细胞在荧光显微镜下进行观察。结果发现 :质粒型 H SV载体可成功地将外源性 GDN F和 GFP基因导入 BH K细胞和原代培养的大鼠骨骼肌细胞。提示 :质粒型 H SV载体可以作为转基因 GDN F治疗中枢神经系统损伤的转移载体 ,为中枢神经系统损伤的治疗展示了广阔的前景。 GFP作为报告基因 ,也可因其适用广谱、观察简便等特性而得到更加广泛的应用     

GFP基因重组病毒在神经解剖研究中的应用

绿色荧光蛋白 (GFP)基因重组病毒标记技术是神经解剖研究的新方法。此方法可以用于观察神经元的形态学特点、神经活性物质及其受体的化学构筑、神经元形态与投射终止部位和功能的关系以及有关的局部神经环路。该法弥补了以往形态学研究方法的一些缺陷 ,能为机能学研究提供直接的形态学证据。     

携带绿色荧光蛋白的外源性胶质纤维酸性蛋白基因稳定转染对胶质瘤细胞分化及增殖的影响

目的观察增强胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)基因表达对恶性胶质瘤细胞生物学特性的影响,为胶质瘤诱导分化及基因治疗研究提供理论基础。方法构建携带1．1kb的GFAP cDNA和绿色荧光蛋白(GFP)基因的真核表达载体,采用脂质体转染法将其导入人恶性胶质瘤细胞系SHG-44,G418结合荧光动态监测筛选阳性克隆;采用原位杂交、免疫细胞化学及Western蛋白印迹等方法检测GFAP基因及其蛋白表达;并通过形态学、细胞生长曲线、软琼脂克隆形成及流式细胞分析等观察GFAP基因对胶质瘤细胞形态、增殖和细胞周期的影响。结果转染阳性的SHG-44细胞GFAP mRNA及其蛋白表达增强,瘤细胞形态趋向成熟,突起增多变细,细胞增殖速度减缓,G0／G1期、G2／M期细胞比例降低。结论增强GFAP基因表达可显著抑制胶质瘤细胞增殖并诱导其分化成熟,提示通过基因治疗策略或诱导分化方法上调GFAP基因表达是恶性胶质瘤治疗的新途径。     

可视化鼻咽癌细胞模型CNE2-EGFP的建立

目的利用pEGFP-N1质粒转染鼻咽癌细胞株,建立稳定表达绿色荧光蛋白的鼻咽癌细胞株CNE2-EGFP．为整体鼻咽癌可视化研究提供良好的试验材料。方法利用脂质体转染法,将pEGFP—N1质粒转染鼻咽癌细胞株．通过G418抗性筛选、亚克隆扩增获得GFP稳定表达的人鼻咽癌细胞株CNE2-EGFP。结果成功获得多个稳定表达GFP的人鼻咽癌细胞CNE2-EGFP细胞株,比较CNE2和CNE2-EGFP发现,两者生长曲线、细胞周期分布、裸鼠成瘤能力等均无显著性差异。结论建立了非G418依赖、稳定表达GFP且保持母株细胞特性的人鼻咽癌细胞株CNE2-EGFP,为鼻咽癌整体可视化研究打下了坚实的基础。     

包裹E1A基因的纳米粒子制备及转染人肺腺癌细胞A549实验研究

目的制备包裹E1A基因（腺病毒早期表达基因）的纳米粒子,并观察其介导E1A基因转染人肺腺癌细胞A549的可行性和效率。方法应用聚乳酸聚乙醇酸共聚物和聚乙烯醇包载E1A基因,制备纳米级粒子混合物,检测其包埋率、体外释放情况及粒径大小。用制备的包裹DNA纳米粒子转染人肺腺癌细胞A549,并以阳离子脂质体为对照,用PCR、RT-PCR方法分别检测转染细胞中E1A基因DNA整合和mRNA表达。结果制备的纳米粒子粒径为150～280nm,包埋率为0.78%,体外释放约为22d;在转染相等质量的DNA情况下,纳米组所得克隆数较脂质体组多（P〈0.05）;PCR、RT-PCR结果表明纳米粒子和脂质体转染细胞均有E1A基因整合和mRNA表达。结论成功制备了纳米粒子,纳米粒子可携带外源基因进行基因转染。     

可溶性人干细胞因子与EGFP融合基因的构建及真核表达

目的　可溶性人干细胞因子 (SCF)膜外活性区与增强型绿色荧光蛋白 (EGFP)融合基因表达载体的构建及其表达。方法　应用基因工程技术构建重组载体 ,电穿孔转染CHO K1细胞株 ,荧光分光光度计检测上清液中融合蛋白的表达 ,流式细胞仪检测其活性。结果　融合基因在瞬时转染的真核细胞中获得表达 ,并分泌至上清 ,且SCF EGFP融合蛋白中分子标签EGFP未影响可溶性人SCF膜外活性区的结构及其生物学活性。结论　可溶性人干细胞因子与EGFP融合基因载体构建成功 ,表达的融合蛋白具有生物活性 ,为进一步研究SCF对造血干细胞的生物学作用奠定了基础。     

KIF1A is the primary anterograde motor protein required for the axonal transport of dense-core vesicles in cultured hippocampal neurons

Dense-core vesicles (DCVs) are responsible for transporting, processing, and secreting neuropeptide cargos that mediate a wide range of biological processes, including neuronal development, survival, and learning and memory. DCVs are synthesized in the cell body and are transported by kinesin motor proteins along microtubules to pre- and postsynaptic release sites. Due to the dependence on kinesin-based transport, we sought to determine if the kinesin-3 family member, KIF1A, transports DCVs in primary cultured hippocampal neurons, as has been described for invertebrate neurons. Two-color, live-cell imaging showed that the DCV markers, chromogranin A-RFP and BDNF-RFP, move together with KIF1A-GFP in both the anterograde and retrograde directions. To demonstrate a functional role for KIF1A in DCV transport, motor protein expression in neurons was reduced using RNA interference (shRNA). Fluorescently tagged DCV markers showed a significant reduction in organelle flux in cells expressing shRNA against KIF1A. The transport of cargo driven by motors other than KIF1A, including mitochondria and the transferrin receptor, was unaffected in KIF1A shRNA expressing cells. Taken together, these data support a primary role for KIF1A in the anterograde transport of DCVs in mammalian neurons, and also provide evidence that KIF1A remains associated with DCVs during retrograde DCV transport.     

Ebola virus infection of human PBMCs causes massive death of macrophages, CD4 and CD8 T cell sub-populations in vitro

Ebola virus causes an often fatal disease characterized by poor immune response and high inflammatory reaction in the patients. One of the causes for poor immunity is virus-mediated apoptosis of lymphocytes in the host. In this study, we infected human PBMCs with Ebola Zaire virus and study apoptosis of different cell types using flow cytometry. We have shown that Ebola virus causes bystander death of CD4 and CD8 T cells. Cells infected with virus had 30-40% active caspase 3(+), annexin-V(+) and Bcl2(low) phenotype by day 8 PI as compared to inactivated virus-treated cells. 60-70% of the macrophages were also dead by day 8 PI and had similar phenotype. Our data also showed that virus may induce death signals in Fas(+)/FasL(+) T lymphocytes and macrophages but did not upregulate Fas/FasL expression in these cells. Lastly, CD4, CD8 and CD14 cells were purified after infection and were studied for death signals by RNAse protection assay. We found an upregulation of TRAIL mRNA in CD4 and CD8 T cells on day 7 PI. A two-fold increase in CD4 T cells and three-fold increase in CD8 T cells were observed in TRAIL mRNA levels as compared to uninfected controls and inactive virus-treated cells. Surprisingly, we did not find any difference in TRAIL mRNA levels between infected macrophages and uninfected controls. These data suggest that Ebola virus evades the immune response by causing massive lymphocyte death. In addition, they may give an explanation on why the host is unable to produce a good antibody response in the absence of CD4 T cells.     

Comparison of differing cytopathic effects in human airway epithelium of parainfluenza virus 5 (W3A), parainfluenza virus type 3, and respiratory syncytial virus

Parainfluenza virus 5 (PIV5) infects a wide range of animals including dogs, pigs, cats, and humans; however, its association with disease in humans remains controversial. In contrast to parainfluenza virus 3 (PIV3) or respiratory syncytial virus (RSV), PIV5 is remarkably non-cytopathic in monolayer cultures of immortalized epithelial cells. To compare the cytopathology produced by these viruses in a relevant human tissue, we infected an in vitro model of human ciliated airway epithelium and measured outcomes of cytopathology. PIV5, PIV3 and, RSV all infected ciliated cells, and PIV5 and PIV3 infection was dependent on sialic acid residues. Only PIV5-infected cells formed syncytia. PIV5 infection resulted in a more rapid loss of infected cells by shedding of infected cells into the lumen. These studies revealed striking differences in cytopathology of PIV5 versus PIV3 or RSV and indicate the extent of cytopathology determined in cell-lines does not predict events in differentiated airway cells.     

TULA proteins bind to ABCE-1, a host factor of HIV-1 assembly, and inhibit HIV-1 biogenesis in a UBA-dependent fashion

TULA, a recently identified UBA- and SH3-containing protein, has previously been shown to regulate cell signaling through protein tyrosine kinases. In order to search for novel functions of TULA, we identified, using mass spectrometry, proteins associated with TULA. ABCE-1 also known as RLI and HP68, a host factor of HIV-1 assembly, was found among TULA-associated proteins in these experiments. Considering an important role of ABCE-1 in HIV-1 assembly, we were compelled to analyze the effect of TULA on HIV-1 biogenesis. Our study provides evidence that TULA proteins substantially inhibit production of both sub-genomic and full-length HIV-1 viral particles and that the effect of TULA is dependent on UBA domain-mediated interactions. The primary role of ABCE-1 in the effect of TULA appears to be the recruitment of TULA to the sites of HIV-1 assembly where TULA interferes with the late steps of the HIV-1 life cycle, most likely by disrupting essential ubiquitylation-dependent events that remain to be identified.     

Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS

Analysis of the interplay between cell proliferation and death has been greatly advantaged by the development of CNS slice preparations. In slices, interactions between neurons and neurons and the glial cells are fundamentally preserved in a fashion close to the in vivo situation. In parallel, these preparations offer the possibility of an easy experimental manipulation. Two main types of slices are currently in use: the acute slices, which are short living preparations where the major functions of the intact brain (including neurogenesis) are maintained, and the organotypic cultures, where the maturation and plasticity of neuronal circuitries in relation to naturally occurring neuronal death and/or experimental insults can be followed over several weeks in vitro.     

A review of neurohormone GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis mellifera

G protein-coupled receptor (GPCR) genes are large gene families in every animal, sometimes making up to 1-2% of the animal's genome. Of all insect GPCRs, the neurohormone (neuropeptide, protein hormone, biogenic amine) GPCRs are especially important, because they, together with their ligands, occupy a high hierarchic position in the physiology of insects and steer crucial processes such as development, reproduction, and behavior. In this paper, we give a review of our current knowledge on Drosophila melanogaster GPCRs and use this information to annotate the neurohormone GPCR genes present in the recently sequenced genome from the honey bee Apis mellifera. We found 35 neuropeptide receptor genes in the honey bee (44 in Drosophila) and two genes, coding for leucine-rich repeats-containing protein hormone GPCRs (4 in Drosophila). In addition, the honey bee has 19 biogenic amine receptor genes (21 in Drosophila). The larger numbers of neurohormone receptors in Drosophila are probably due to gene duplications that occurred during recent evolution of the fly. Our analyses also yielded the likely ligands for 40 of the 56 honey bee neurohormone GPCRs identified in this study. In addition, we made some interesting observations on neurohormone GPCR evolution and the evolution and co-evolution of their ligands. For neuropeptide and protein hormone GPCRs, there appears to be a general co-evolution between receptors and their ligands. This is in contrast to biogenic amine GPCRs, where evolutionarily unrelated GPCRs often bind to the same biogenic amine, suggesting frequent ligand exchanges ("ligand hops") during GPCR evolution.