聚乙烯吡咯烷酮预处理培养细胞促进TAT穿膜效率

目的:探讨PVP预处理细胞对TAT穿膜效率的影响及其作为促渗剂的安全性,以优化TAT穿膜条件。方法:人工合成荧光标记短肽TAT-FITC和作为阴性对照的无意义肽NCO-FITC作用于PVP预处理各种细胞株,荧光显微镜观察TAT-FITC的穿膜效率及其定位;荧光酶标仪定量检测不同预处理的各种细胞摄取荧光标记短肽;MTT检测PVP对各种细胞的存活率及溶血实验检测其对细胞膜结构完整性的影响。结果:经PVP预处理细胞后,TAT-FITC可有效穿膜进入细胞内,MTT结果显示适当浓度的PVP对细胞活力几乎没有影响;溶血实验也证实,PVP不影响红细胞的细胞膜完整性。结论:本实验观察显示,PVP可以明显提高TAT对培养细胞的穿膜效率,但对细胞活力影响很小。     

HPV阳性宫颈癌细胞特异性穿膜肽PTP的穿膜特性研究

目的：证实宫颈癌细胞特异性细胞膜穿透肽特异性穿膜并优化穿膜条件。方法：将编码人乳头瘤病毒（HPV）阳性宫颈癌细胞特异性穿膜肽（PTP）的cDNA片断与编码绿色荧光蛋白(GFP)的基因片断融合,克隆至原核表达质粒pET15b克隆位点,构建pET15b-GFP-PTP重组原核表达载体。转化大肠杆菌BL21(DE3)并表达GFP-PTP融合蛋白,纯化后的GFP-PTP融合蛋白作用于体外培养的HPV阳性宫颈癌细胞株HeLa,Siha,肺癌细胞株A549及正常脐静脉内皮细胞ECV304。利用合成的标记短肽探讨优化穿膜条件。结果：荧光显微镜观察发现,与融合蛋白GFP-PTP共孵育后的Hela和Siha细胞胞浆及胞核内有绿色荧光均匀分布,而A549及ECV304胞内无绿色荧光。与GFP共孵育的任何细胞内均无绿色荧光。流式细胞术分析结果显示PTP对HeLa,Siha细胞的穿膜效率分别为33.2％和28.9％。10% DMSO处理细胞后可增强PTP特异性穿透HPV阳性宫颈癌细胞株的胞膜并改善胞浆内分布。结论：本研究结果为进一步探讨PTP作为宫颈癌防治的靶向导入载体奠定了基础。     

新穿膜融合蛋白His-T1-绿色荧光蛋白的跨膜效率及其对细胞存活的影响

目的研究穿膜融合蛋白His-T1-绿色荧光蛋白(GFP)的跨膜效率及其对细胞存活的影响。方法以浓度为500mg·L-1的His-T1-GFP与人鼻咽癌CNE2或大鼠肾小管上皮NRK52E细胞孵育6h,应用荧光显微镜观察His-T1-GFP跨膜进入细胞的情况。应用多功能酶标仪检测细胞荧光强度,研究His-T1-GFP跨膜的动力学因素:以浓度为500mg·L-1的His-T1-GFP与CNE2或NRK52E细胞孵育10min至24h,观察孵育时间对穿膜作用的影响;以浓度为25mg·L-1至1.0g·L-1的His-T1-GFP与两种细胞孵育6h,观察蛋白浓度对跨膜效率的影响;以浓度为500mg·L-1的His-T1-GFP与两种细胞分别在4℃和37℃的条件下孵育6h,观察温度对蛋白跨膜效率的影响。用细胞乳酸脱氢酶(LDH)试剂盒和MTT法来评价5.0g·L-1浓度的His-T1-GFP对细胞存活的影响。结果在一定浓度范围内,His-T1-GFP能够有效穿透CNE2和NRK52E细胞膜,且对NRK52E细胞的跨膜效率明显高于His-TAT-GFP。His-T1-GFP在10min内就能有效跨膜进入细胞,并且在6h内进入细胞的量与时间成正相关。在一定浓度范围(25mg·L-1~1.0g·L-1)内,该蛋白进入细胞的量与自身浓度成正相关,而在4℃时该蛋白仍具有跨膜能力。当其终浓度高达5.0g·L-1时,对CNE2和NRK52E两种细胞几乎无毒性作用。结论His-T1-GFP蛋白是一种跨膜效率高且低毒的穿膜融合蛋白。     

穿膜融合多肽TAT N24对Jurkat细胞增殖的影响

摘要目的观察穿膜融合多肽TAT N24对急性T细胞白血病Jurkat细胞增殖的影响。方法以穿膜融合多肽TAT N24处理Jurkat细胞,噻唑蓝（MTT）法观察细胞的生长情况,应用流式细胞仪检测Jurkat细胞周期进程。结果穿膜融合多肽TAT N24处理急性T细胞白血病Jurkat细胞48 h,细胞生长受到抑制,随着多肽浓度的增加,抑制作用增强,表现出明显的浓度依赖性（P＜0.05）;48 h后细胞生长明显受抑,并随时间延长生长受抑更加明显,表现出时间依赖性（P＜0.05）。空白对照组Jurkat细胞中G0/G1期细胞数为（41.91±1.96）%,S期和G2/M期细胞数分别为（52.16±1.76）%和（5.93±0.30）%;对照多肽组Jurkat细胞中G0/G1期细胞数为（46.38±2.31）%,S期和G2/M期细胞数分别为（44.22±1.69）%和（9.40±0.98）%;TAT N24组Jurkat细胞中G0/G1期细胞数为（54.83±1.92）%,S期和G2/M期细胞数分别为（30.75±2.27）%和（14.42±0.68）%。结论融合多肽TAT N24可以有效抑制急性T细胞白血病Jurkat细胞的增殖,阻滞其周期进程。     

穿膜肽TAT修饰的自噬诱导肽Beclin1抗肿瘤的初步研究

目的 研究自噬诱导肽Beclin 1及其经穿膜肽TAT修饰后所得TAT-Beclin 1对乳腺癌的体外自噬诱导作用及体内外疗效.方法 采用透射电子显微镜、eGFP-LC3细胞转染技术和蛋白免疫印迹法综合对比评价Beclin 1和TAT-Beclin 1对4T1细胞自噬流的影响,通过体外细胞毒性和体内抑瘤实验评价其抗肿瘤疗效.结果 TAT-Beclin 1能显著提高Beclin 1在4T1细胞上的自噬诱导能力;TAT-Beclin 1在体内具有一定的抗4T1肿瘤能力.结论 TAT-Beclin 1较Beclin 1能更有效地诱导自噬并发挥治疗乳腺癌的效果.     

亲水性稀释剂对芦丁降解动力学的影响

目的:研究3种常用亲水性稀释剂在固态和溶液状态下对芦丁降解动力学的影响。方法:将芦丁分别与亲水性稀释剂配伍,制成溶液态或固态的混合物,在40、60℃下定时取样并测定芦丁含量,用降解动力学方程对不同芦丁溶液的浓度-时间数据进行拟合。结果:在固态下,使用乳糖或混合稀释剂可显著提高芦丁稳定性,但甘露醇和蔗糖与芦丁配伍加速了药物的降解;在溶液状态下,3种辅料的加入均提高了芦丁的稳定性,大部分溶液的降解过程符合一级动力学方程。结论:在固态和溶液状态下,亲水性辅料对芦丁稳定性有不同的影响。     

穿膜肽TAT和脑肿瘤靶向肽T7双修饰脂质体的制备和体外靶向性评价

本文旨在制备T7肽和穿膜肽TAT双修饰的脂质体(T7 and TAT dual modified liposomes,T7-TAT-LIP)用于血脑屏障和脑肿瘤细胞双级靶向药物递送。研究以CFPE为荧光探针,T7修饰的PEG-DSPE、TAT修饰的PEG-DSPE、卵磷脂、PEG-DSPE和胆固醇为材料,采用成膜水化法制备脂质体,对T7浓度、TAT浓度、连接T7和TAT的PEG长度进行优化,表征其粒径、zeta电位、形态和稳定性。以b End.3细胞和C6细胞为模型,考察T7-TAT-LIP的细胞摄取能力,表征其穿过血脑屏障和脑肿瘤细胞靶向能力。结果表明,T7用量为脂质的6%、修饰T7所用PEG链长为2000、TAT用量为脂质的0.5%、修饰TAT所用PEG链长为1000时所得到的双修饰脂质体被C6细胞摄取能力最强。优化后T7-TAT-LIP粒径为118 nm,zeta电位为-6.32 m V,透射电镜下形态圆整。脂质体在PBS中较为稳定,37℃放置24 h,浊度和粒径无明显变化;4~8℃放置1个月,粒径和PDI无明显变化。在不同时间点,b End.3和C6细胞摄取T7-TAT-LIP的强度均高于单配体修饰脂质体,且随着孵育时间提高,摄取浓度逐渐提高。这些结果说明,双修饰脂质体具有血脑屏障和脑肿瘤细胞双级靶向能力,且效果优于单配体修饰脂质体。     

甘露醇对脑出血后脑水肿的影响

［摘要］目的探讨甘露醇对脑出血后脑水肿的影响。方法对51例脑出血患者进行回顾性分析，将其分为发病24 h内入院应用甘露醇组35例（A组）和24 h后入院应用甘露醇组16例（B组）；入院48 h和2周后复查脑CT，通过对比脑水肿的变化，分析甘露醇对脑水肿的影响。结果A组48 h出现脑水肿加重者5例（14.3%），2周后出现脑水肿加重者21例（60.0%）；B组48 h出现脑水肿加重者1例（6.3%），2周后出现脑水肿加重者3例（18.8%）。两组48 h CT比较差异无显著性（P＞0.05），2周后CT比较差异有极显著性（P＜0.01）；2周后疗效差异有显著性（P＜0.05）。结论脑出血患者发病24 h内应用甘露醇可使脑水肿和病情加重。因此，脑出血患者早期使用甘露醇应慎重。     

卡介苗对大鼠肺巨噬细胞膜的影响

本文观察了卡介苗(GCG)对大鼠肺巨噬细胞(AM)膜的影响。实验结果表明:(1)BCG使AM膜的流动性增高。(2)AM经BCG处理后,胞浆的标志酶LDH和溶酶体标志酶β-Glu漏出量增多,说明BCG明显地损伤了AM的胞浆膜和溶酶体膜。(3)AM经BCG刺激后,释放出大量OH·。     

穿膜肽介导紫杉醇脂质体对肿瘤的治疗作用

目的制备和表征F3修饰紫杉醇脂质体,并进行细胞学和体内抗肿瘤评价。方法采用薄膜分散法制备脂质体,人肺腺癌细胞A549进行摄取评价;小鼠肝癌细胞H22荷瘤小鼠进行体内抗肿瘤活性评价。结果 F3修饰脂质体粒径为90 nm;该修饰后脂质体被细胞摄取能力明显增强,进而提高了药物的抗肿瘤活性,给药16天后,F3-Lipo组瘤体增长仅为11倍,相比于生理盐水组26倍,Taxol 18倍,未修饰脂质体14倍,有更好的抗肿瘤效果。结论 F3穿膜肽可以明显提高载体入胞能力,进而改善药物治疗作用。     

Cell Penetrating Peptides in Drug Delivery

Protein transduction domains (PTDs) are small cationic peptides that can facilitate the uptake of large, biologically active molecules into mammalian cells. Recent reports have suggested that PTDs may be able to mediate the delivery of cargo to tissues throughout a living organism. Such technology could eliminate the size restrictions on usable drugs, enabling previously unavailable large molecules to modulate in vivo biology and alleviate disease. In this article, we review the evidence that PTDs can be used both to deliver active molecules to pathological tissue in vivo and to treat models of disease such as ischemia, inflammation, and cancer.     

穿膜肽提高纳米银穿膜活性的研究

目的合成纳米银,在其表面修饰穿膜肽(TAT),并检测修饰后纳米银粒对人乳腺癌耐阿霉素细胞(MCF-7/ADR)的穿膜活性。方法通过化学还原法制备纳米银(AgNP),并通过Ag-S共价键与TAT连接修饰AgNP。通过粒度仪、透射电镜、激光共聚焦显微镜、流式细胞仪以及二喹林甲酸法等仪器及方法对其表征、共价连接及TAT的介导活性进行测定。结果成功制备了10 nm以下的AgNP,且修饰TAT后的AgNP(AgNP-TAT)表现出了比AgNP更强的穿膜活性。结论 TAT修饰AgNP后能显著提高其穿膜活性。     

Cell Penetrating Peptides: Intracellular Pathways and Pharmaceutical Perspectives

Cell penetrating peptides, generally categorized as amphipathic or cationic depending on their sequence, are increasingly drawing attention as a non-invasive delivery technology for macromolecules. Delivery of a diverse set of cargo in terms of size and nature ranging from small molecules to particulate cargo has been attempted using different types of cell penetrating peptides (CPPs) in vitro and in vivo. However, the internalization mechanism of CPPs is an unresolved issue to date, with dramatic changes in view regarding the involvement of endocytosis as a pathway of internalization. A key reason for the lack of consensus on the mechanism can be attributed to the methodology in deciphering the internalization mechanism. In this review, we highlight some of the methodology concerns, focus more on the internalization pathway and also provide a novel perspective about the intracellular processing of CPPs, which is a crucial aspect to consider when selecting a cell penetrating peptide as a drug delivery system. In addition, recent applications of cell penetrating peptides for the delivery of small molecules, peptides, proteins, oligonucleotides, nanoparticles and liposomes have been reviewed.     

Differentiation Restricted Endocytosis of Cell Penetrating Peptides in MDCK Cells Corresponds with Activities of Rho-GTPases

Purpose Cellular entry of biomacromolecules is restricted by the barrier function of cell membranes. Tethering such molecules to cell penetrating peptides (CPPs) that can translocate cell membranes has opened new horizons in biomedical research. Here, we investigate the cellular internalization of hCT(9-32)-br, a human calcitonin derived branched CPP, and SAP, a γ-zein related sequence. Methods Internalization of fluorescence labelled CPPs was performed with both proliferating and confluent MDCK cells by means of confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) using appropriate controls. Internalization was further elaborated in an inflammatory, IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies. Activities of active form Rho-GTPases (Rho-A and Rac-1) in proliferating and confluent MDCK cells were monitored by pull-down assay and Western blot analysis. Results We observed marked endocytic uptake of the peptides into proliferating MDCK by a process suggesting both lipid rafts and clathrin-coated pits. In confluent MDCK, however, we noted a massive but compound-unspecific slow-down of endocytosis. This corresponded with a down-regulation of endocytosis by Rho-GTPases, previously identified to be intimately involved in endocytic traffic. In fact, we found endocytic internalization to relate with active Rho-A; vice versa, MDCK cell density, degree of cellular differentiation and endocytic slow-down were found to relate with active Rac-1. To our knowledge, this is the first study to cast light on the previously observed differentiation restricted internalization of CPPs into epithelial cell models. In the inflammatory IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies, CPP internalization was enhanced in a cytokine concentration-dependent way resulting in maximum enhancement rates of up to 90%. We suggest a cytokine induced redistribution of lipid rafts in confluent MDCK to cause this enhancement. Conclusion Our findings emphasize the significance of differentiated cell models in the study of CPP internalization and point towards inflammatory epithelial pathologies as potential niche for the application of CPPs for cellular delivery. This work was supported by the Commission of the European Union (EU project on Quality of Life and Management of Living Resources, Project No. QLK2-CT-2001-01451.     

Cellular Uptake But Low Permeation of Human Calcitonin-Derived Cell Penetrating Peptides and Tat(47-57) Through Well-Differentiated Epithelial Models

PURPOSE: To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers. METHODS: Cellular uptake of hCT(9-32) and permeation of six hCT-derived peptides, namely, hCT(9-32), hCT(12-32), hCT(15-32), hCT(18-32), hCT(21-32), and a random sequence of hCT(9-32) were evaluated in fully organized confluent Madin-Darby canine kidney (MDCK), Calu-3, and TR146 cell culture models. For comparison, Tat(47-57) and penetratin(43-58) were investigated. The peptides were N-terminally labeled with carboxyfluorescein (CF). Uptake in the well-differentiated epithelial models was observed by confocal laser scanning microscopy (CLSM), whereas permeation through the models was analyzed by reversed-phase (RP)-HPLC. RESULTS: In MDCK epithelium hCT(9-32), Tat(47-57) and penetratin(43-58) demonstrated punctuated cytoplasmic distribution. In Calu-3, Tat(47-57) and penetratin(43-58) were simultaneously localized in a punctuated cytoplasmic and paracellular distribution, whereas hCT(9-32) showed strict paracellular distribution. By contrast, in TR146 cells, Tat(47-57) was located strictly paracellularily, whereas penetratin(43-58) showed a punctuated cytoplasmic pattern and hCT(9-32) both. The transepithelial permeability of all tested peptides and their cargo was lower than that of paracellular markers. CONCLUSIONS: The CPP uptake pattern depends on both the type of peptide and the cell culture model. In general, the investigated CPP have no apparent potential for systemic drug delivery across epithelia. Nevertheless, distinct patterns of cellular distribution may offer a potential for localized epithelial delivery.     

细胞穿膜肽在癌症治疗中的应用

细胞膜有选择渗透性,能阻止大多数外来因子进入细胞。然而许多治疗因子必须穿过细胞膜才能进入细胞内起作用。细胞穿膜肽是一组简短的阳离子序列,它具有很强的穿膜能力。自从1988年发现细胞穿膜肽后,细胞穿膜肽被用于携带各种物质,如小分子、核酸、抗体、纳米粒等进入细胞膜。本文重点介绍了近几年来关于细胞穿膜肽穿膜机制的研究进展,以及细胞穿膜肽作为运载体携带抗癌药物靶向进入肿瘤细胞,从而实现对肿瘤细胞的靶向性,同时可克服肿瘤的多药抗药性等研究进展。     

多肽类药物在新生儿缺氧缺血性脑损伤中的研究进展

新生儿缺氧缺血性脑损伤是新生儿死亡和婴幼儿神经系统功能障碍的主要原因,目前唯一公认有效的治疗方法是亚低温治疗.近年来,随着新技术的发展,多肽类药物由于具有高选择性、高效性、耐受性好、制备简单、成本低等优点,得到了广泛关注.利用新生儿缺氧缺血性脑损伤模型的研究发现,多种多肽类药物可直接作用或通过与细胞穿膜肽结合进入神经细...