光动力疗法治疗金黄地鼠创伤性口腔溃疡的实验研究

目的:探讨以亚甲基蓝为光敏剂的光动力疗法对金黄地鼠创伤性口腔溃疡的治疗效果。方法:金黄地鼠随机分成4组:空白对照组、光敏剂组、激光组、光动力组。取直径5 mm的金属棒,加热后置于金黄地鼠颊粘膜上5 s,肉眼可见形成直径约为5 mm的溃疡模型;按分组给予不同处理后,在6小时、24小时、5天、7天,处死制备病理切片,经HE、MASSON染色定性观察中性粒细胞数量、再生上皮、成纤维细胞、新生血管和胶原纤维情况。结果:模型建立24小时后,肉眼及组织病理学观察可见金黄地鼠口腔溃疡模型建立成功。在治疗24小时后,光动力治疗组的中性粒细胞数量与其他组相比显著增高(P0.01)。治疗后第五天和第七天,新生的血管和胶原纤维情况光动力组明显优于空白对照组和光敏剂组(P0.01),也多于激光组(P0.05),上皮再生情况激光组优于空白对照组和光敏剂组(P0.01),光动力组上皮再生也优于空白对照组和光敏剂组(P0.05)。成纤维细胞数量各组间无明显差异。结论:光动力疗法可加快口腔溃疡的愈合。     

Protoporphyrin-induced photodynamic effects on transport processes across the membrane of human erythrocytes

Previous studies have shown that illumination of erythrocytes with visible light in the presence of protoporphyrin results in cross-linking of membrane proteins and deterioration of several membrane functions, e.g. active transport of K⁺ and Na⁺.In the present study it is shown that carrier-mediated transport of glucose, l-leucine, sulphate and glycerol is also inhibited by the photodynamic process, whereas non-specific permeability of glycerol and thiourea is increased.It is shown that these effects are not caused by lipid peroxidation, but by photooxidation of membrane proteins. The inhibition of carrier-mediated transport is caused either by photodynamic oxidation of susceptible essential amino acid residues of the carrier molecules, or by an aspecific perturbation of the membrane structure, leading to inhibition of carrier functions.     

Hematoporphyrin-induced photo-oxidation and photodynamic cross-linking of nucleic acids and their constituents

The pH-dependency of photo-oxidation of the physiological purine and pyrimidine bases and some of their derivatives was studied, with hematoporphyrin as sensitizer. At high pH these bases (adenine, guanine, uracil, thymine and cytosine) were photo-oxidizable. In the physiological pH range only guanine, and to a much less extent thymine, were sensitive to photo-oxidation. At physiological pH values a slow photo-oxidation of RNA and DNA took place. The photo-oxidation of nuclei acids was strongly augmented by perturbation of their structure in 8 M urea. In model experiments photodynamic cross-linking of tryptophan and cysteine to DNA was demonstrated. No covalent binding of purine or pyrimidine bases to DNA was observed. In similar model experiments covalent photodynamic coupling of guanosine and guanosine-monophosphate to proteins could be shown, whereas no coupling of the other bases occured. These studies confirm the preferential photo-oxidation of guanine in nucleic acids and demonstrate the possible photodynamic cross-linking of proteins to the guanine moiety in other molecules.     

Protoporphyrin-induced photodynamic effects on band 3 protein of human erythrocyte membranes

In previous studies it has been shown that protoporphyrin-induced photodynamic effects on red blood cells are caused by photooxidation of amino acid residues in membrane proteins and by the subsequent covalent cross-linking of these proteins. Band 3, the anion transport protein of the red blood cell membrane, has a relatively low sensitivity to photodynamic cross-linking. This cannot be attributed to sterical factors inherent in the specific localization of band 3 in the membrane structure. Solubilized band 3, for instance, showed a similar low sensitivity to cross-linking. By extracellular chymotrypsin cleavage of band 3 into fragments of 60 000 and 35 000 daltons it could be shown that both fragments were about equally sensitive to photodynamic cross-linking. The 17 000 dalton transmembrane segment, on the other hand, was completely insensitive. Inhibition of band 3-mediated sulfate transport proceeded much faster than band 3 interpeptide cross-linking, presumably indicating that the inhibition of transport is caused by photooxidation of essential amino acid residues or intrapeptide cross-linking. A close parallel was observed between photodynamic inhibition of anion transport and decreased binding of 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate (H₂DIDS), suggesting that a photooxidation in the immediate vicinity of the H₂DIDS binding site may be responsible for transport inhibition.     

Encapsulation of methylene blue in polyacrylamide nanoparticle platforms protects its photodynamic effectiveness

The ability to prevent methylene blue (MB), a photosensitizer, from being reduced by plasma reductases will greatly improve its efficacy in photodynamic therapy (PDT) applications. We have developed a delivery approach for PDT by encapsulating MB using nanoparticle platforms (NPs). The 30-nm polyacrylamide-based NPs provide protection for the embedded MB against reduction by diaphorase enzymes. Furthermore, our data shows the matrix-protected MB efficiently induces photodynamic damage to tumor cells. The unprecedented results demonstrate the significant in vitro photodynamic effectiveness of MB when encapsulated within NPs, which promises to open new opportunities for MB in its in vivo and clinical studies.     

胆管癌的光动力疗法

胆管癌(Cholangiocarcinoma CC)是一种罕见的胆道原发性恶性肿瘤,预后较差。根治性手术只适用于一小部分早期诊断的患者。姑息性的经皮或内镜下支架置入胆管引流术能通过减轻瘙痒,疼痛,和胆管炎的程度从而提高生活质量,但对于延长生存时间作用甚微。光动力疗法(PDT)是对于不能手术切除的CC的一种较新颖的局部微创性的姑息疗法。其原理是PDT中使用的光敏性分子能特异性的聚集在增生组织如肿瘤,在特定波长的光照下光敏剂激活生成活性氧类物质,从而选择性的导致肿瘤细胞的死亡。经过初步的可行性性研究和充满前景的前瞻性II期临床研究后,关于研究比较胆管支架置入联合光动力治疗与单纯胆管内支架置入的两组前瞻性随机对照试验已经发表。其中一组试验结果显示PDT组(493天)与非PDT组(98天)相比在中位生存期方面的显著优势(P0.0001),且PDT组患者的体力状态也明显改善。另一组试验也进一步明确了PDT治疗对于提高生存率方面的优势(PDT组630天而单纯支架置入组210天,P0.01)。整个治疗过程耐受性良好。有报道称PDT作为CC的辅助或新辅助疗法已取得了令人满意的结果,因此PDT可以被看作是治疗不能手术切除的胆管癌的一种标准性姑息治疗方法。     

Histone cross-linking by transglutaminase

Transglutaminases irreversibly catalyze covalent cross-linking of proteins by forming isopeptide bonds between peptide-bound glutamine and lysine residues. Among several transglutaminases, tissue-type transglutaminase (tTGase) is most ubiquitously found in every type of cells and tissues in animals, but its natural substrate has yet to be identified. In an attempt to identify the natural substrate for tTGase, we examined in vitro if core histones were subject to cross-linking by tTGase. We found core histone subunits, H2A and H2B, were specifically cross-linked by tTGase. The cross-linking was between either one or both glutamines at C-terminal end of H2A (-VTIAQ104 GGVLPNTQ112 SVLLPKKTESSKSK-C' end) and the first and/or third lysine from C-terminal end of H2B (-AVESEGK116 AVTKYTSSK125-C' end). The cross-linking occurred only when these subunits were released from nucleosome but not when these were organized in nucleosome. Most interestingly, in chicken erythrocyte the cross-linked H2A-H2B was present in a significant amount. From these results, it can be proposed that tTGase-mediated cross-linking is an another form of core histone modification and it may play a role of chromatin condensation during erythrocyte differentiation.     

5-氨基乙酰丙酸的光动力应用研究进展

对光合细菌、藻类及其它细菌的5-氨基乙酰丙酸(5-ALA)产量进行了对比,类球红细菌(Rhodobacter sphaeoides)在黑暗、厌氧条件下培养,可产生大量的胞外5-ALA。5-ALA在农业生产中作为光动力除草剂、杀虫剂取得了很好效果。并且,ALA对提高植物的抗盐、抗冷冻能力也有一定作用。近年来,ALA在癌症治疗、肿瘤诊断方面也得到了广泛应用。     

光动力疗法治疗细菌和病毒性疾病

目前控制细菌和病毒疾病的方法很多,但尚无良方,本文介绍了细菌和病毒的基本特性和常用的防治方法,着重对一种有希望的新疗法－－光动力疗法的研究现状,尚存问题和发展方向作一概述。     

Effect of protein structural integrity on cross-linking by tyrosinase evidenced by multidimensional heteronuclear magnetic resonance spectroscopy

Enzymatic cross-linking of proteins can be catalyzed either by transferase-type enzymes, e.g., transglutaminases, or by oxidoreductases, e.g., tyrosinases or laccases. Three-dimensional structure of protein substrate plays a key role in these reactions, that is, the reactivity and end product are strongly modulated by the accessibility of target amino acid residues to the cross-linking enzyme. Typically structural integrity of protein can be distorted by heat, pH, or mechanical action, as well as by varying ionic concentration of the solution. In this study we used partially unfolded protein (wild-type DrkN SH3) and its structurally stabilized mutant (T22G) to investigate the impact of folded/unfolded conformations on cross-linking by Trichoderma reesei tyrosinase. Our results clearly showed formation of intermolecular cross-links solely between unfolded conformations, making them superior substrates to folded proteins when using tyrosinase as a cross-linking enzyme. Multidimensional heteronuclear magnetic resonance experiments in solution state were employed to investigate cross-linked end-products. The results presented in this study form basis for application development in food, medical, cosmetic, textile, packing and other sectors. In addition, the outcome of this study has a high value for the basic understanding of reaction mechanism of tyrosinases on proteins.     

Photodynamic therapy for American cutaneous leishmaniasis: the efficacy of methylene blue in hamsters experimentally infected with Leishmania (Leishmania) amazonensis

The aim of this study was to investigate the effectiveness of Photodynamic Therapy (PDT) using Methylene Blue (MB) as the photosensitizing compound and a Light-Emitting Diode (LED) in American cutaneous leishmaniasis (ACL). Hamsters were experimentally infected with Leishmania (Leishmania) amazonensis. After the development of the lesions in the footpad, the animals were treated with MB three times a week for 3 months. Ten minutes after each application of MB, the lesions were irradiated with LED for 1 h. The lesions were evaluated weekly by the measurement of the hamster footpad thickness. At the end of the treatment the parasitic load was quantified in the regional lymph node of the hamsters. The treatment promoted a decrease in the thickness of infected footpad (P = 0.0001) and reduction in the parasitic load in the regional lymph node (P = 0.0007) of the animals from group treated with MB + LED. PDT using MB + LED in ACL caused by L. amazonensis shows a strong photodynamic effect. This therapy is very promising, once it is an inexpensive system and the own patient can apply it in their wound and in their house without the need of technical assistance.     

Effects of glyoxal cross-linking on baked starch foam

Starch trays and plates were prepared from native corn starch and corn starch cross-linked with glyoxal at different concentrations (0.126, 0.269, 0.271, 0.468 g/kg) using a foam baking process. The effect of cross-linking on baking parameters, including density, colour, water absorption, and tensile and flexural properties, was determined. Also, the morphologies of the trays were examined using a scanning electron microscope. Cross-linking considerably reduced the baking time, density and water absorption of the trays. Trays made from starch cross-linked with 0.126 and 0.269 g/kg glyoxal presented the best properties, with a homogenous microstructure and smooth surface quality. These trays had the lowest density (0.27 g/cm³) and had approximately 53% reduction in water absorption. Moreover, both the tensile and flexural strain of these foams was significantly higher than other foams.     

藻红蛋白介导光动力治疗的光化学机制研究

为探讨藻红蛋白介导的光动力治疗的光化学机制,观察藻红蛋白浓度和活性氧抑制剂对藻红蛋白光漂白作用的影响,研究藻红蛋白介导的光敏反应产生活性氧分子的途径和影响因素。表明：藻红蛋白浓度太高会降低光动力的效率,其活性氧产生的机制为Ⅰ和Ⅱ型,用Ⅰ型反应抑制使反应向Ⅱ型方向转变,产生更多的单线态氧,可显著提高光动力效率。     

竹红菌素光敏剂与微血管病变光动力治疗

光动力疗法已被用于临床治疗除实体肿瘤以外的一些微血管类疾病,包括鲜红斑痣(portwine stains,PWS)和老年视网膜黄斑变性(age-related macular degeneration,AMD)等。竹红菌素是一种苝醌类光敏剂,因为在光疗窗口(600～900 nm)的吸收较少,它不适用于实体肿瘤的光动力治疗,但对于治疗微血管类疾病却有其独特的优势。本文根据竹红菌素光物理特性提出其主要适应症范围,并根据其临床实用化问题提出应对策略。通过构造竹红菌素水溶性纳米制剂或具有优化脂水双亲性的衍生物,实现脂溶性光敏剂既可以安全给药又最大程度保持生物利用度和光动力活性。生物学实验证明竹红菌素类光敏剂对生物靶体具有超强的光动力活性。由此推测:竹红菌素类光敏剂在光动力治疗微血管疾病(如鲜红斑痣和老年黄斑变性)及其它浅表型疾病方面具有广阔的应用前景。     

钌化合物对铜绿假单胞菌的光动力杀伤作用

【摘 要】 目的 探讨光动力疗法（PDT）对体外培养的铜绿假单胞杆菌的杀伤效应。方法 以多重耐药铜绿假单胞杆菌（P. aeruginosa）为对象,用457 nm和532 nm的激光作为光源,对不同浓度钌化合物孵育的细菌悬液进行光照,用菌落计数法观测PDT对铜绿假单胞杆菌的杀伤作用;同时观察了铜绿假单胞菌临床耐药菌与标准菌株对相同PDT作用的敏感性差异。结果 PDT处理组具有剂量－效应关系,在光照剂量相同的情况下,457 nm激光作为光源有更好的PDT杀伤效果;对多重耐药的铜绿假单胞菌,光动力同样有效,其作用甚至强于标准株。结论 钌化合物介导的光动力作用对体外培养的铜绿假单胞杆菌具有很强的杀伤作用,其效果和剂量关系密切。     

Association of a lower molecular weight protein to the mu-opioid receptor demonstrated by (125)I-beta-endorphin cross-linking studies

Cross-linking experiments using the (125)I-beta-endorphin revealed the presence of several receptor-related species in cell lines expressing endogenous opioid receptors, including a small molecular mass protein (approximately 22 kDa). Previous reports have suggested that this 22-kDa (125)I-beta-endorphin cross-linked protein could be the degradative product from a higher molecular mass species, i.e., a fragment of the receptor. To determine if this protein is indeed a degraded receptor fragment, (125)I-beta-endorphin was cross-linked to the (His)(6) epitope-tagged mu-opioid receptor (His-mu) stably expressed in the murine neuroblastoma Neuro(2A) cells. Similar to earlier reports with cell lines expressing endogenous receptors, two major bands of 72- and 25-kDa proteins were specifically cross-linked. Initial cross-linking experiments indicated the absolute requirement of the high-affinity (125)I-beta-endorphin binding to the mu-opioid receptor prior to the appearance of the low molecular weight species, suggesting that the 22-kDa protein could be a degraded fragment of the receptor. However, variations in the ratios of these protein bands being cross-linked by several homo- or heterobifunctional cross-linking agents were observed. Although neither the carboxyl terminus mu-opioid receptor-specific antibodies nor the antibodies against the epitope at the amino terminus of the receptor could recognize the 22-kDa protein, this (125)I-beta-endorphin cross-linked species could be coimmunoprecipitated with the receptor antibodies or could be isolated with a nickel resin affinity chromatography. The direct physical association of the 22-kDa protein with the receptor was demonstrated also by the observation that the 22-kDa protein could not bind to the nickel resin alone, but that its binding to the nickel resin was restored in the presence of the His-mu. Taken together, these results suggest that the 22-kDa protein cross-linked by (125)I-beta-endorphin is not a degradative product, but a protein located within the proximity of the mu-opioid receptor, and that it is tightly associated with the receptor.     

亚甲兰光化学法对血浆乙肝病毒的灭活

目的:研究灭活病毒对乙肝病毒和血浆成分的影响。方法:在已知HBV-DNA阳性血浆中加入MB,调整MB终浓度分别为1.0μmol/l、5.0μmol/l、10.0μmol/l,光照度为40000LUX,照射时间分别为0min、20min、40min、60min,在各点分别取样抽提基因组DNA用于荧光定量分析,测定HBV-DNA的浓度,推断在不同时间和浓度下的灭活效果。在灭活效果最好的点取样检测血浆正常成分有无显著变化,其中包括生化指标,酶学指标,凝血因子FⅧ:C活性,SDS聚丙烯酰胺凝胶电泳观察蛋白亚基的变化,蛋白免疫印迹观察血浆的抗体蛋白活性变化。结果:当MB为10.0μmol/L,光照60min时,灭活乙肝病毒的效果最好,对于高拷贝数的病毒浓度,在本实验的条件下,尚无法完全灭活病毒,但可以使HBV-DNA浓度明显下降;从病毒灭活的表格看,灭活效果与时间和MB浓度呈正相关,作用60min时血浆中主要成分的生化指标无显著变化及凝血因子FⅧ:C活性无明显改变,部分血浆酶活性有明显下降(P<0.05)。血浆蛋白的亚基数目和迁移速度未见明显变化,血浆的抗体蛋白也具有正常的生物学活性。结论:MB10μmol/L辅助荧光...     

Photodynamic Activity of Plant Extracts from Sarawak,Borneo

Photodynamic therapy (PDT) is a medical treatment that involves the irradiation of an administered photosensitizing drug with light of a particular wavelength to activate the photosensitizer to kill abnormal cells. To date, only a small number of photosensitizers have been clinically approved for PDT, and researchers continue to look for new molecules that have more desirable properties for clinical applications. Natural products have long been important sources of pharmaceuticals, and there is a great potential for discovery of novel chemotypes from under‐explored biodiversities in the world. The objective of this study is to mine the terrestrial plants in Sarawak, Borneo Island, for new photosensitizers for PDT. In a screening program from 2004 to 2008, we prepared and studied 2,400 extracts from 888 plants for their photosensitizing activities. This report details the bioprospecting process, preparation and testing of extracts, analysis of the active samples, fractionation of four samples, and isolation and characterization of photosensitizers.     

Major determinants of photoinduced cell death: Subcellular localization versus photosensitization efficiency

We present a study on whether and to what extent subcellular localization may compete favorably with photosensitization efficiency with respect to the overall efficiency of photoinduced cell death. We have compared the efficiency with which two cationic photosensitizers, namely methylene blue (MB) and crystal violet (CV), induce the photoinduced death of human cervical adenocarcinoma (HeLa) cells. Whereas MB is well known to generate singlet oxygen and related triplet excited species with high quantum yields in a variety of biological and chemical environments (i.e., acting as a typical type II photosensitizer), the highly mitochondria-specific CV produces triplet species and singlet oxygen with low yields, acting mostly via the classical type I mechanism (e.g., via free radicals). The findings described here indicate that the presumably more phototoxic type II photosensitizer (MB) does not lead to higher degrees of cell death compared to the type I (CV) photosensitizer. In fact, CV kills cells with the same efficiency as MB, generating at least 10 times fewer photoinduced reactive species. Therefore, subcellular localization is indeed more important than photochemical reactivity in terms of overall cell killing, with mitochondrial localization representing a highly desirable property for the development of more specific/efficient photosensitizers for photodynamic therapy applications.     

Studies on crystallization and cross-linking of lipase for biocatalysis

The development of robust biocatalysts with increased stability and activity is a major challenge to industry. A major breakthrough in this field was the development of cross-linked enzyme crystals with high specificity and stability. A method is described to produce micro crystals of CLEC lipase, which is thermostable and solvent stable. Lipase from Burkholderia cepacia was crystallized using ammonium sulfate and cross-linked with glutaraldehyde to produce catalytically active enzyme. The maximum yield of CLEC was obtained with 70% ammonium sulfate and cross-linked with 5% (v/v) glutaraldehyde. SEM studies showed small hexagonal-shaped crystals of 2–5 μm size. CLEC lipase had improved thermal and reuse stability. It is versatile, having good activity in both polar and nonpolar organic solvents. CLEC lipase was coated using β cyclodextrin for improving the storage and reuse stability. CLEC was successfully used for esterification of Ibuprofen and synthesis of ethyl butyrate.