【作者】 赵占娟；

【导师】 刘天军；

【作者基本信息】 北京协和医学院 ， 药物化学， 2014， 博士

【摘要】 近年来,由于抗生素的滥用导致了“超级细菌”(superbug)的出现和传播。所谓“超级细菌”是指这些细菌对于目前的抗生素具有多重耐药的特性,这对于临床创伤感染的治疗增添了很大的难度。且耐多重抗生素的超级细菌的爆发性流行尽管只是有潜在的可能,但其可怕之处却引起了世界各国的恐慌。因此发展新的抗感染策略迫在眉睫。光动力抗菌治疗方法(photodynamic antimicrobial chemotherapy,PACT)就是其中最具前景的新疗法之一,对于细菌、真菌和病毒引起的感染,特别是对于耐药菌感染均显示很好的疗效。光动力灭菌是基于光、光敏剂和氧三种因素协同作用的氧化损伤机制,不会因为单一用药、光敏剂的浓度、曝光时间不足等因素产生耐药问题。且超级细菌多发生在伤口、肺部以及血液和尿道中,除了血液感染外,其它部位采用PACT还是易于实现的,因而无论从耐药性、杀菌性以及效果和方便考虑,PACT均有很大的优势。PACT的关键是光敏药物。目前,国外利用不同光敏剂对不同细菌的光动力灭活作用进行了大量的探索,旨在发现理想的光敏药物。理想的抗菌光敏剂,应具有高效低毒和好的选择性,对细胞壁的通透性强,高效灭活微生物,而对于正常组织伤害较小。在国内外以往的文献报道中,作为抗菌的光敏药物选择性不够突出,有些对于革兰氏阳性菌有效,有些对革兰氏阴性菌有效,对于上述两类菌群均有效的高效光敏剂尚不多见,因而设计和发现可以有效治疗上述两类细菌的光敏药物,将会大大拓展PACT的应用。基于上述研究目标,我们开展了体内体外光敏抗菌药物的筛选,具体研究内容涵盖以下两部分：1.高效抗菌光敏剂的设计与筛选1.1水溶性糖酞菁类光敏剂的光动力抗菌作用研究酞菁类光敏剂有其特有的优势,但是水溶性差、易聚集的缺点限制了酞菁的使用。考虑到细菌代谢和繁殖较快,我们利用半乳糖作为修饰基团,不仅可以改善酞菁的水溶性、生物相容性,而且可以提高细菌的选择性摄取可使光敏药物优先聚集于细菌内而非周围组织或细胞中,从而实现选择性灭菌。研究结果表明：细菌对四种酞菁类化合物(T1-T4)的吞噬量在30min时达到最大,随着光照时间的延长,菌落数呈下降趋势,在能量密度达到6J/cm2后,灭活率达到最大,且细菌的灭活率不再随激光能量密度的增加而增加。在四种半乳糖取代的酞菁化合物(T1-T4)中,光照T1对革兰氏阳性耐药菌MRSA有很好的灭活作用且暗毒性较低,但对革兰氏阴性菌(绿脓杆菌和大肠杆菌)的灭活效果却很低。激光共聚焦显微成像显示MRSA对T1的吞噬量最多,T1的荧光最强。1.2卟啉类光敏剂的光动力抗菌作用研究由于半乳糖酞菁类光敏剂对革兰氏阳性菌有较好的杀菌效果,但对革兰氏阴性菌杀菌效率很低,极大的限制了此类光敏剂的使用。卟啉类化合物是源于天然的另外一类光敏剂,但其母核的光敏效率与选择性均不够理想,考虑到细菌的增长分化需要大量的碳源和氮源。我们选择双乙二胺为对原卟啉化合物进行修饰,得到一类新型的抗菌光敏剂。体外研究表明其对革兰氏阴性菌及革兰氏阳性菌在体外均有高效的抗菌灭活作用,以小鼠急性创伤感染为模型的在体实验表明,双乙二胺的PACT作用可以明显的杀死绿脓杆菌,促进创伤面的愈合。双乙二胺光敏剂有其特有的抗菌优势,但是不稳定,见光容易分解的缺点限制了其使用。为了进一步研究氨基数目的取代关系对卟啉光敏剂的稳定性、生物活性的影响,本实验室对双乙二胺进一步设计,得到了五种氨基卟啉类化合物,体外实验表明：该五种卟啉类化合物PAl-PA5均具有较缓慢的光漂白率和较强的单线态氧产生能力,其中PA1的单线态氧产生率最高(0.69),高于四苯基卟啉(0.64),其余化合物PA1-PA4单线态氧产率在0.15-0.37之间。细胞吞噬实验表明MRSA,E.coli, P.aeruginosa在30min即对上述五种化合物的吞噬量达到最大。激光共聚焦显微成像显示上述三种菌株对五种化合物的吸收具有选择性,对化合物PAl-PA2吞噬较强,而对PA3-PA5吞噬却较弱。光敏抗菌实验表明PA1产生的灭菌效果较其他四种化合物好,对三种菌的MIC均为16μM,对MRSaA,E.coli的MBC为31μM,对P.aeruginosa的MBC为62.5μM。PAl的暗毒性也低(MBC>500μM)。利用菌落计数法检测了PA1-PA5对三种细菌的光反应、暗反应浓度曲线。结果表明随着浓度的增加,PA1表现出明显的杀菌效果,对MRSA的杀菌效果最强,当浓度为15.6μM时,全部杀死革兰氏阳性菌MRSA.当浓度上升为31.25μM时,PA1几乎能全部杀死E.coli,P.aeruginosa。但在此浓度范围内对3T3成纤维细胞的损伤却很小。以上结果表明PA1对革兰氏阳性菌及阴性菌均有很好的抗菌性能,可作为一种新型广谱的抗菌光敏剂使用,具有广阔的前景。2卟啉类抗菌光敏剂在皮肤感染类疾病中的光敏抗菌效果评价2.1光敏抗菌对创伤感染的效果评价皮肤破损性疾病是一种临床常见病、多发病,由于多重耐药细菌的出现,临床治疗面临很大的难度。以筛选出的苗头化合物PA1为研究对象,重点探讨PA1对由MRSA,E.coli,P.aeruginosa混合感染的大鼠创面模型的PACT作用,结果表明：PA1介导的PACT能促进大面积创伤感染的大鼠皮肤愈合.其机制可能与诱导bFGF,TGF-β1的高表达,促进创面纤维合成和重新分布有关。造模后4,10及14天,高剂量PACT组的bFGF和TGF-β1的表达最高,模型对照组最低(P<0.01).PACT能显著改善创面感染组织炎症状况,降低组织中炎症相关因子TNF-a和IL-6含量。减少创面组织中细菌的数量,加快创面愈合的速率。PACT的效果与PA1的浓度呈正相关,表现出较好的剂量依赖关系。大面积创伤发生时,由于屏障系统被破坏,细菌很容易穿透屏障,造成败血症。尽管PACT可以外用,体内却只能靠自身免疫力清除细菌,如果感染程度超过自身免疫力则容易引起败血症。基于这种考虑将PACT和抗生素的优势结合起来,PACT可以清除外表细菌,而体内细菌则由抗生素清除。基于大鼠创伤模型的实验表明：PACT联合抗生素治疗能显著降低大鼠大面积重度感染组织的炎症表达；减少创面组织中细菌数量,促进创面愈合,结果优于PACT治疗组和抗生素治疗组。PACT联合抗生素治疗大鼠大面积重度感染,死亡率为20%,而模型对照组的死亡率是60%,表明PACT联合抗生素治疗的有效性。免疫组化分析表明PACT联合抗生素治疗可显著提高创面组织中bFGF(?)TGF-β1的含量表达,促进创面纤维合成和重新分布,加快创面愈合的速率,同时显著降低创面组织中炎症相关因子TNF-α和IL-6含量,减少创伤组织的炎症反应,有助于创伤组织的修复。2.2光敏抗菌作用在烧伤感染中的效果评价烧烫伤(bums/scalds)是皮肤创伤的一种常见形式,为临床常见病和多发病。细菌感染是烧伤患者的主要并发症,发病率高,是造成病人死亡的主要因素,因此抗感染成为治疗烧伤病人的重要环节。PACT联合抗生素治疗大鼠Ⅲ度烧伤感染结果表明,PACT联合抗生素治疗能显著降低烧伤感染组织的炎症表达；减少创面组织中细菌数量,促进创面愈合,结果优于PACT治疗组和抗生素治疗组。(?)PACT联合抗生素治疗大鼠Ⅲ度烧伤感染,死亡率为0%,而模型对照组的死亡率是50%。免疫组合分析表明PACT联合抗生素治疗可显著提高创面组织中bFGF和新生血管数CD31的含量表达,促进血管新生和成纤维细胞的增殖,促进了创面肉芽组织的生长,从而促进创面愈合,同时可以提高机体的自身免疫能力显著降低烧伤创面组织中炎症相关因子TNF-α和IL-6含量,减少创伤组织的炎症反应,有助于创伤组织的修复。综上所述,PA1是一种具有良好抗菌效果的广谱光敏剂,体内、体外实验表明,该光敏剂具有高效、低毒的性质,可作为一种新型广谱的抗菌光敏剂使用,具有广阔的前景。更多还原

【Abstract】 In recent years, the overuse of antibiotics has led to the emergence and spread of superbug. The so-called superbugs refer to the bacteria which are multidrug-resistant to present antibiotics. This has added to a lot of difficulties for the treatment of wound infection in clinic. Although there is only a potential likelihood for the explosive prevalence of multiantibiotics-resistant superbugs, what terror it will cause has brought great panics in many countries around the world. Therefore, the development of new anti-infection strategy is urgent. Photodynamic antimicrobial chemotherapy (PACT) is one of the most promising new antimicrobial therapies in treating infections caused by bacteria, Fungi and viruses, especially in treating infections by multidrug-resistant bacteria Photodynamic antimicrobial therapy is an oxidative damage mechanism relying on a combined action of three factors----light, photosensitizer and oxygen, which will not bring some drug-resistant problems related with such factors as single-drug administration, the concentration of photosensitizer, inadequate exposure time etc.Additionally, superbugs are most likely to be found in the infection site of the wound, in the infection of the lungs, the blood and the urinary tract. Except for blood infection, PACT is relatively easy to implement in the other parts’ infections. Hence, in terms of drug-resistance, bactericidal effect and convenience, PACT has its great advantages. Currently, a lot of research in foreign countries has been done into the photodynamic inactivation of different bacteria with different photosensitizers, which aims at discovering a desired photosensitizer. A desired antimicrobial photosensitizer should have the following characteristics-----high efficacy yet low toxicity, good selectivity, strong permeability of the cell wall and high effectiveness in microorganism inactivation while doing little harm to the normal tissues. In previous reports, photosensitive drugs as antibacterial agents have no highlighted selectivity, with some effective to Gram-positive bacteria and some effective to Gram-negative bacteria We expect that the desired photosensitive drugs can be effective in treating the above two bacteria and the potential bacteria Therefore, it is of great practical significance to achieve the promotion of PACT. Based on the above objectives, we were screened antibacterial sensitive drugs in vitro and in vivo, the major contents of paper includes the following two parts:1. The design and screening of high efficacy antimicrobial photosensitizer1.1The photodynamic antibacterial effect of a-D-galactopyranosyl Zinc PhthalocyaninesPhthalocyanines photosensitizers have its own advantages but there is limitation to the use of Phthalocyanines because of its poor water solubility and easily gathering. Considering the rapid metabolism and reproduction of bacteria, galactose is used as modification to increase its water solubility and its biocompatibility. So,drugs that bacteria selectively take in are well designed so that photosensitive drugs are preferentially concentrated in bacteria rather than in the surrounding tissues or cells so as to achieve selective sterilization. The novel a-D-galactopyranosyl zinc phthalocyanines (T1, T2, T3, T4) have been synthesized to study the biological properties and antibacterial properties. The study showed that the bacterial uptake to four phthalocyanine compounds (T1-T4) reached maximum in30minutes. With increased irradiation time, the number of colonies decreased and when the energy density reached6J/cm2, the inactivation rate of bacteria reached maximum, which no longer increased with increased laser energy density. Of the four a-D-galactopyranosyl zinc phthalocyanines compounds (T1-T4), the irradiation of T1had effective inactivation to Gram-positive drug-resistant bacteria MRSA with low dark toxicity, but the inactivation effect to Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) was very poor. The laser confocal showed that MRSA had maximum uptake of T1and T1had the strongest fluorescent.1.2The antibacterial effect of Porphyrin compoundsPhthalocyanines photosensitizers are effective in fighting against Gram-positive bacteria but ineffective to Gram-negative bacteria, which greatly limit the use of such photosensitizers. Porphyrins is another kind of natural photosensitizer, But its parent nucleus of photosensitive efficiency and selectivity are not ideal. Considering the reproduction of bacteria need huge amounts of carbon and nitrogen sources, double ethylenediamine is used as the modification of the original porphyrin compounds, and obtained a new type of antibacterial photosensitizer. Amino heme was taken as an example and a study of PACT was conducted against Gram-negative and Gram-positive bacteria, showing that it had good antibacterial effect in vitro. Experiments on rats with acute trauma infection in vivo indicated that PACT with Amino heme could obviously kill P. aeruginosa and promote the wound healing. Amino heme photosensitizer has its unique antibacterial advantages, but its limitation is unstability and easy decomposition to light, which greatly limits its use. To further study the effect of the amino-substituted porphyrins on the stability and the biological activity of photosensitizers, Amino heme was further designed and five amino porphyrin compounds were obtained. The experiments in vitro showed that the five compounds PA1-PA5were all slower in photobleaching and a strong ability to generate singlet oxygen, of which PA1had the highest rate of generating singlet oxygen (0.69), higher than that of the tetraphenyl porphyrin (0.64), and the singlet oxygen yield of PA2-PA4was0.15-0.37. The uptake of PA1-PA5by MRS A, E.coli, P.aeruginosa showed that the uptake reached maximum in30minutes incubation. The laser confocal microscopy revealed that three strains were selective in taking in5compounds. The uptake of PA1-PA2by three strains was higher whereas the uptake of PA3-PA5was relatively low. The irradiation bacterial effect of PA1was better than the other four compounds. The MIC of the three strains was16μM, the MBC for MRS A, E.coli’s was31μM and the MBC for P.aeruginosa was62.5uM. The dark toxicity of PA1was very low (MBC>500μM). The colony counting method was used to detect the concentration curves of PA1-PA5in the light and dark reactions to three strains. The results showed that with increasing concentration, PA1had significant bactericidal effect and the greatest bactericidal effect against MRSA happened in the concentration of15.6uM. When the concentration reached31.25μM, PA1almost completely killed MRSA. However, within this concentration range, PA1had minor inactivation to3T3fibroblasts.These results indicate that PA1has good antibacterial properties against Gram-positive and negative bacteria, and it can be used as a novel broad-spectrum antimicrobial photosensitizers, which will have a broad and bright prospects.2Antibacterial effect evaluation of porphyrin photosensitizers in the skin-infection diseases2.1Effects of Photodynamic antimicrobial therapy of porphyrin photosensitizer on wound infectionSkin trauma is a common disease, and it is more difficult to treat in clinic. The PA1compounds is taken as the study object, mainly focusing on the effect of PA1on the trauma model in rats with mixed wound infection of MRSA, E.coli, P.aeruginosa. The results showed that PACT could greatly promote the skin healing in rats with large areas of wound infections. Its mechanism lay in inducing the high expression of BFGF, TGF-β1and promoting the synthesis and redistribution of fibrolos in the wounds. The expression of BFGF and TGF-β1in the PACT group is the highest while the expression of the control group is the lowest.(p<0.01). PACT could significantly downregulate inflammation condition of the infected tissues in the burns, reducing the amount of TNF-and IL-6and the number of bacteria and promoting the healing speed of the wounds. The concentration change of PA1was positively correlated with PACT effect, showing good dose-dependent relation.Trauma tend to damage the barrier system of the body, which makes it easy for bacteria to penetrate the barrier and cause sepsis. While PACT can be used in vitro, bacteria in vivo can only be removed by immunity in itself. The combined advantage of PACT and antibiotics is that PACT can remove bacteria in vitro while bacteria in vivo can be cleaned through antibiotics, small amount of which can work. Therefore, the combined therapy of PACT and antibiotics has been used to treat trauma models in seriously-infected rats. The results showed that PACT combined with antibiotics could significantly reduce the inflammation expression of the seriously-infected tissues in rats with severe wounds, greatly reducing the number of bacteria in the wound tissues and promoting the wound healing. The result was far better than that of the PACT group and the antibiotics group. The combined therapy of PACT and antibiotics was used to treat rats with a large area of severe infections, with the mortality20%, whereas the mortality of the control group was60%, which indicated the effectiveness of the combined therapy. The explanation to the treatment of a large area of trauma infections with the combined therapy of PACT and antibiotics was as follows:It could significantly improve the amount of BFGF and TGF-β1, promoting the fiber composition and redistribution of the wound and thus accelerating the healing speed. At the same time, it could greatly reduce the secretion of inflammation-related factors TNF-a and IL-6, reducing the inflammation of the infected tissues in trauma, thus contributing to the healing of the skin lesion.2.2Effects of photodynamic antimicrobial chemotherapy on burn infection model.Scalds/Burns is a common form of skin wounds and frequently-occurring disease in clinical. Bacterial infection, the main complications of burn patients, is the dominating factor of death. So, Anti-infection treatment is the important link for burn patients. The results showed that PACT combined with antibiotics could significantly reduce the inflammation expression of the burned tissues in rats with severe burns, greatly reducing the number of bacteria in the wound tissues and promoting the wound healing. The result was far better than that of the PACT group and the antibiotics group. The combined therapy of PACT and antibiotics was used to treat rats with burned severe infections, with the mortality0%, whereas the mortality of the control group was50%, which indicated the effectiveness of the combined therapy. The explanation to the treatment of degree Ⅲ burns infection in rats with the combined therapy of PACT and antibiotics was as follows: It could significantly improve the expression of bFGF and CD31in trauma, facilitating the proliferation of new capillaries and fibroblast cells in the early stage and the growth of wound granulation tissues, and enhancing the wound healing. It could greatly improve the rats’immunity system, regulating the secretion of inflammation-related factors TNF-a and IL-6and reducing the inflammation of burn tissues, thus contributing to the healing of the skin lesion.In summary, PA1is a broad-spectrum photosensitizer with good antimicrobial effect. The experiments in vitro and in vivo reveal that the photosensitizer has the naure of high efficiency and low toxicity. Therefore, it can be used as a novel broad-spectrum photosensitizer against bacteria, with broad prospects.更多还原