蛋白转导在基因治疗中的应用

蛋白转导是近几年生命科学领域发现的一种独特现象,具有蛋白转导功能的蛋白通过几个短的碱性小肽组成的蛋白转导域(proteintransductiondomain,PTD)的介导,能将与其共价连接的DNA、多肽或蛋白质以及其他大分子物质通过非经典途径穿过细胞膜甚至血脑屏障,并在细胞间自由传递。PTD这种能携带融合蛋白自由进入细胞的独特功能为人类一些疾病的基因治疗提供了一种新的运载工具,在基因治疗中有着美妙的应用前景。     

Intracellular delivery of Neuroglobin using HIV-1 TAT protein transduction domain fails to protect against oxygen and glucose deprivation

Neuroglobin (Ngb) is a heme protein that is primarily localised in the retina and the brain. Its physiological role is largely unknown. It has been reported that its overexpression protects neurons from hypoxia in vitro and in vivo, suggesting that the rapid modulation of the Ngb level in the nerve cells may be a promising stroke treatment strategy.     

基于支持向量机预测蛋白质转导域的研究

目的 蛋白转导域（PTD）是一类能携带分子穿越细胞膜的短肽,利用支持向量机对多肽片段PTD进行预测.方法 对来源于SwissProt数据库的多肽序列用68个特征值描述其整体和局部的理化特性以及空间结构特征,利用支持向量机（SVM）和直推式支持向量机（TSVM）并结合聚类的方法进行PTD的预测.结果 5次交叉验证的结果显示,TSVM的预测准确率达到（94±4）%,SVM预测准确率达到（94±5）%.2种预测方法共同预测了1210个可能的PTD片段.结论 TSVM和SVM均显示了很好的预测性能,预测的PTD为实验方法有目的 地发现、确认PT提供了基础.     

Protein kinase C in IL- 2 signal transduction

Interleukin-2 (IL-2), secreted principally by activated helper T-cells, plays a pivotal role in the generation and regulation of the immune response. The various biologic functions of IL-2 have been the focus of intensive study over the years and have been well worked out. By contrast, an understanding of the intracellular signals coupled to the IL-2 receptor and responsible for mediating IL-2 effects in T-cells is far less developed, and the role that protein kinase C (PKC) may play in the various cellular responses to IL-2 receptor activation is unclear. In this article we will discuss IL-2, its receptors, and IL-2 signal transduction in relation to the physiological roles PKC activation may play in IL-2-mediated activation of T-cells and other hematopoietic cells.     

Protein kinase mediators of integrin signal transduction

 Protein kinases are important mediators of signal transduction initiated by soluble growth factors and cytokines. Cellular interactions with the extracellular matrix are mediated largely by members of the integrin class of cell adhesion molecules, which also subsume signal transduction functions required for cell growth, differentiation, and survival. Here we review the involvement of protein kinases in mediating integrin intracellular signal transduction and the possible role for these molecules in regulating integrin adhesion. Although in most cases mechanistic details are incomplete, the emerging theme of protein kinases mediating cross-talk between growth factor receptor and integrin signalling systems provides a timely backdrop against which to present new developments in this area. The contribution of the actin cytoskeleton to integrin signal transduction is discussed, with respect to the concept of ’solid-state’ signalling providing a mechanism for imposing order on the protein-protein interactions which underlie signal discrimination. Moreover, we review evidence that dysregulated integrin signalling contributes to pathological processes including arthritis, thrombasthenia, leucocyte adhesion deficiencies, and tumour angiogenesis and invasion. Received: 14 May 1996 / Accepted: 2 July 1996     

Tat-p53融合蛋白的表达、纯化及其转导活性

目的:原核表达野生型p53与TatPTD(proteintransductiondomain)的融合蛋白,检测TatPTD介导p53进入肝细胞的效率。方法:利用RT-PCR方法从A549细胞系中分离野生型p53基因,将该基因分别克隆入pTAT-HA和pET-32a原核表达载体,在大肠杆菌BL21(DE3)LysS内诱导表达并进行纯化。以纯化的p53蛋白经腹腔免疫BALB/c小鼠,制备高效价的抗血清。将Tat-p53融合蛋白加入HepG2细胞培养上清,利用间接免疫荧光法检测Tat-p53融合蛋白导入HepG2细胞的效率。结果:成功地构建了含有野生型p53基因的原核表达载体,表达纯化了Tat-p53融合蛋白及p53蛋白,制备p53特异的抗血清,证实Tat-p53可以高效的转入HepG2细胞内。结论:Tat-p53融合蛋白的表达纯化及活性分析,为应用Tat-p53融合蛋白治疗肝癌的实验研究奠定了基础。     

重组蛋白PTD-HSP27的制备及其穿细胞膜和角膜组织的功能研究

目的:制备PTD-HSP27蛋白并研究其能否穿过人晶状体上皮细胞SRA01/04细胞膜及新西兰大白兔的角膜组织进入眼前房。方法:利用重叠延伸PCR法获得重组目的基因片段PTD-HSPB1-6His,构建重组质粒p KYB-PTD-HSP27-6His,原核诱导PTD-HSP27蛋白表达及纯化重组蛋白后对其进行Western blotting鉴定;用异硫氰酸荧光素(FITC)标记重组蛋白PTD-HSP27并检测其穿透SRA01/04细胞膜的能力及兔眼角膜组织的能力。结果:成功构建并纯化制备目的蛋白PTD-HSP27,在PTD-HSP27孵育后的SRA01/04细胞及结膜囊滴注PTDHSP27兔眼前房内均可检测出重组蛋白PTD-HSP27。结论:通过重叠延伸PCR法及镍柱层析纯化法可以获得较纯的重组蛋白PTD-HSP27;重组蛋白PTD-HSP27能够穿透SRA01/04细胞膜及穿越兔眼角膜进入房水。     

T细胞受体基因转导的研究和应用

T细胞受体（TCR）作为T细胞识别抗原的分子,在免疫应答和免疫调节中发挥重要的作用。利用转基因技术,将识别特定抗原的TCR基因转导到正常T细胞中,使其强制性表达了识别特异抗原TCR,从而达到发挥特异性免疫效应的目的,为产生有效的特异性免疫治疗开辟了一条新途径。     

Cytokines and signal transduction

Cytokines are signaling molecules that coordinate cellular interactions in immune and hematopoietic systems. During the past 5 years many cytokines and their receptors have been identified and cloned. With a few exceptions, cytokine receptors do not contain any known signaling domains and therefore, in order to trigger a specific cellular response, new and unusual features of signaling pathways must be assumed. A major advance in the field has come with the discovery of new family of cytoplasmic protein tyrosine kinases that associate with occupied cytokine receptors and make them competent for intracellular signal generation. This review describes some general characteristics of cytokine signaling.Abbreviations CSF colony-stimulating factor - TGF transforming growth factor Correspondence to: J. M. Pfeilschifter     

Enhanced transduction of CAR-negative cells by protein IX-gene deleted adenovirus 5 vectors

In human adenoviruses (HAdV), 240 copies of the 14.3-kDa minor capsid protein IX stabilize the capsid. Three N-terminal domains of protein IX form triskelions between hexon capsomers. The C-terminal domains of four protein IX monomers associate near the facet periphery. The precise biological role of protein IX remains enigmatic. Here we show that deletion of the protein IX gene from a HAdV-5 vector enhanced the reporter gene delivery 5 to 25-fold, specifically to Coxsackie and Adenovirus Receptor (CAR)-negative cell lines. Deletion of the protein IX gene also resulted in enhanced activation of peripheral blood mononuclear cells. The mechanism for the enhanced transduction is obscure. No differences in fiber loading, integrin-dependency of transduction, or factor-X binding could be established between protein IX-containing and protein IX-deficient particles. Our data suggest that protein IX can affect the cell tropism of HAdV-5, and may function to dampen the innate immune responses against HAdV particles.     

内毒素诱导的细胞内信息转导

Depending on LBP/CD14 systems, LPS activates a series of signal - transducing systems in cells. Protein tyrosin kinase(PTK)system, ceramide activated kinase(CAK)system might play an important role in cells signal - transducing. This article give a summary about signal transduction in cells induced by en-dotoxin.     

IL-2 gene therapy of solid tumors: an approach for the prevention of signal transduction defects in T cells

The identification of tumor-associated antigens has focused attention on the mechanisms that underlie the failure of T cells to destroy tumor cells. A deeper understanding of the process of signal transduction following the binding of ligand by the T cell receptor can help to identify underlying defects that may be involved. Gene therapy using tumor cells genetically modified to express cytokines or surface determinants is a promising technique for stimulating antitumor responses. A potential pitfall in its application to cancer, however, is that some patients' T cells are immune suppressed and may resist stimulation by such genetically engineered vaccines. Recent studies have demonstrated that T cells from tumor-bearing patients exhibit abnormalities in signal transduction events, possibly rendering them unable to respond to activation signals. Gene therapy with interleukin 2 secreting tumor cells in an animal model has been shown effective in preventing the onset of signaling defects. A more precise definition of the molecular mechanisms that enable cytokine-secreting tumor cells to stimulate specific antitumor responses may make it feasible to optimize immunotherapeutic approaches resulting in better clinical results.     

IL—6信号转导与转录激活子活性研究

目的　探讨 Ｉ Ｌ６ 生物效应与胞内磷酸化蛋白和转录激活子活性变化之间的关系。方法　应用原位杂交和 Ａ Ｐ Ａ Ａ Ｐ 方法检测细胞内 Ｉ Ｌ６ Ｒ ｍ Ｒ Ｎ Ａ 和蛋白的表达;采用 Ｄ Ｎ Ａ 结合蛋白凝胶阻滞电泳分析 Ｉ Ｌ６ 信号转导与 Ａ Ｐ Ｒ Ｆ 活性的相关性;用免疫沉淀和 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 观察 Ｉ Ｌ６ 信号转导中磷酸化蛋白的变化情况。结果　（１） Ｉ Ｌ６ 受体（ Ｉ Ｌ６ Ｒ） ｍ Ｒ Ｎ Ａ 和蛋白表达阳性的人骨髓瘤细胞系（ Ｓ Ｋ Ｏ００７） 体外对 Ｉ Ｌ６ 有增殖反应;（２） 胞内转录激活子活性与 Ｉ Ｌ６ 诱导时间和剂量有相关性,抗ｇｐ１３０ 单克隆抗体和酪氨酸蛋白激酶抑制剂均可特异性抑制转录激活子活性;（３） Ｉ Ｌ６ 诱导 Ｓ Ｋ Ｏ００７细胞后,胞浆内出现一组相对分子质量为（１３０ 、９０ 、５４ 、３６） ×１０３ 磷酸化蛋白,它们的磷酸化活性与转录激活子活性一样,也具有时间和剂量相关性。结论　结果提示,转录激活子和这组相对分子质量不同的酪氨酸磷酸化蛋白参与 Ｉ Ｌ６ 信号转导调节。     

腺病毒载体介导gfp基因在树突细胞的转导及其影响因素的分析

目的：研究腺病毒载体介导外源基因在人树突细胞转染的有效方法。方法：绿色荧光蛋白（gfp）报告基因重组腺病毒的构建采用直接连接法。人树突细胞的制备通过分离人外周血单核细胞，然后在体外经过诱导过程再生。结果：经腺病毒介导实现了gfp基因在树突细胞的转导和表达。病毒滴度对转导效率影响较大，只有使用高滴度（MOI〉100）的重组腺病毒才能获得较高的转导效率（40％以上）；脂质体和多聚赖氨酸可以明显提高转导效率（提高50％左右）。转导效率最高可达65％左右。结论：由腺病毒介导进行树突细胞的转基因需要较高的病毒滴度；脂质体和多聚赖氨酸可以提高基因的转导效率。     

蛋白质转导技术及其应用

蛋白质转导结构域(protein transduction domain,PTD)是指一类能将其他生物大分子导入多种哺乳动物细胞的小分子阳离子多肽,它可以用于研究细胞内某些蛋白质的功能,还可以与具有治疗作用的蛋白质、多肽等生物大分子偶联,在缺血、肿瘤等多种疾病的治疗中有潜在的应用价值。     

心肌肥大的信号转导机制

编者按：细胞信号转导在当今生命科学领域占有极其重要的地位,自20世纪90年代以来,与细胞信号转导有关的研究已两度获诺贝尔医学奖,由此可见一斑。以此为契机,探索疾病的信号转导机制已成为当前国际上倍受瞩目的热点课题。心肌肥大和心力衰竭是大多数心血管疾病的严重或终末阶段,研究其细胞信号转导机制改变及其在发病学中的作用具有非常重要的意义。本专题重点介绍目前这一领域的研究进展,内容涉及心肌肥大和心力衰竭时细胞内信号转导通路的改变、心力衰竭时肾上腺素受体及其信号转导机制的改变等方面。尤其值得一提的是,本专题还包…     

Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, could be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68.     

转化生长因子β胞内信号转导与Smads蛋白

转化生长因子 β(ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒ -β,ＴＧＦ -β)是ＴＧＦ -β超家族成员之一 ,分布于多种细胞组织中 ,在调节细胞增殖与分化、机体生长与发育、细胞外基质形成、免疫功能等方面均有重要作用。由于它具有广泛而重要的生物学效应 ,其信号转导通路成为诸多领域研究热点之一。最近发现 ,Ｓｍａｄｓ蛋白是ＴＧＦ -β受体 (ＴβＲ)复合物的下游信号调节蛋白 ,可将信号从胞膜直接转至胞核[1,2 ] ,从而对于ＴＧＦ -β胞内信号转导机制的认识有了进一步发展。1　ＴＧＦ -β的胞外信号转导及跨膜　　ＴＧＦ -β在…     

神经元保护蛋白TAT-Bcl-XL的体外高效表达及其功能的初步研究

目的：在原核表达系统中表达对凋亡神经元具有保护作用的重要蛋白Bcl-XL与蛋白质转导序列（PTD）的融合蛋白，并检测重组蛋白对重金属离子所诱导的细胞凋亡的保护作用。方法：通过RT-PCR的方法，用Bcl-XL特异引物从乳腺癌细胞系MCF-7细胞的总RNA中扩增出Bcl-XL基因，构建相应的原核表达载体，体外表达的TAT-Bcl-XL融合蛋白经镍亲和层析介质纯化后，通过免疫荧光方法检测其转导293T细胞的能力；并用流式细胞仪检测融合蛋白抑制细胞凋亡的能力。结果：经RT-PCR从MCF-7细胞总RNA中得到相应的TAT-Bcl-XL基因片段，并将其克隆入pCRT7/CT-TOPO载体中，重组质粒pTBTOPO转化大肠杆菌后，在SDS-PAGE和Western blot的结果中出现了与预期分子量相同的蛋白条带和阳性信号，纯化的TAT-Bcl-XL重组蛋白经免疫荧光检测，主要分布于细胞的细胞质中。流式细胞仪的检测结果显示融合表达蛋白可以有效地抑制Zn2＋离子所诱导的细胞凋亡，使细胞的存活率提高40%。结论：TAT-Bcl-XL融合蛋白在大肠杆菌中获得高效表达，初步的功能性检测表明融合蛋白具有抑制细胞凋亡的功能。