【作者】 孟静茹；

【导师】 罗晓星；

【作者基本信息】 第四军医大学 ， 药理学， 2008， 博士

【摘要】 目的:耐甲氧西林金黄色葡萄球菌(MRSA)和耐甲氧西林表皮葡萄球菌(MRSE)是医院内感染最常见、最重要的致病菌,对所有β-内酰胺类抗生素和其他多种抗菌药物耐药,因此由MRSA和MRSE引起的感染,治疗十分困难,病死率很高。近年来MRSA和MRSE的感染率逐年上升,特别是MRSA,其传播已经不止局限于医院内,社区获得性MRSA(CA-MRSA)的检出率日益增多,并且呈现广泛传播和扩大流行的趋势。CA-MRSA能够在健康人群中引起严重的甚至致命的感染,它的流行已经成为严重的公共健康问题,并引起了医学界的高度关注。目前治疗MRSA和MRSE最有效的药物是万古霉素,然而对万古霉素耐药的MRSA的出现,迫使人们必须寻找一种更有效的控制MRSA和MRSE感染的策略。MRSA和MRSE耐药性的产生主要是由于生成了由mecA编码的新的青霉素结合蛋白PBP2a。我们实验室前期的研究结果显示,阻断耐药基因mecA的上游调控基因mecR1的表达能够部分逆转MRSA的耐药性。但由于缺乏有效的递药系统;同时mecA的上游调控基因在临床菌株中容易发生突变或缺失,因此抗mecR1的PS-ODN对mecR1表达的抑制作用有限,而且只对部分携带完整SCCmec元件的临床菌株有效。基于以上问题,本研究中我们的目的有二:1.根据细菌细胞壁以及PS-ODN的特性,我们通过改良配方制备了新型的高效转染PS-ODN的脂质体;2.以SCCmec元件中的下游耐药基因mecA为靶基因,观察体内体外实验中PS-ODN能否完全恢复β-内酰胺类抗生素对MRSA和MRSE的抗菌活性。方法:1.新型高效包裹反义脱氧寡核苷酸的纳米微粒脂质体的制备:以mecA mRNA为靶基因设计合成硫代反义寡核苷酸;将25KD的PEI与PS-ODN通过静电相互作用制备成荷正电荷的纳米微粒,选用蛋黄卵磷脂(EPC)、二肉豆蔻酰磷脂酰甘油(DMPG)和聚乙二醇2000-二硬脂酰磷脂酰乙醇胺(PEG2000-DSPE)以薄膜分散法制备包裹PS-ODN/PEI纳米微粒的脂质体;用粒径分析仪测定纳米微粒和脂质体的平均粒径;用葡聚糖凝胶过滤法分离未被包封的PS-ODN/PEI纳米微粒和脂质体;紫外法测A260nm值计算脂质体的包封率;通过体外释放实验评估脂质体的稳定性。2.脂质体包裹的抗mecA mRNA的PS-ODN010、PS-ODN853逆转MRSA耐药性的体内体外实验研究:以脂质体为载体将包裹的PS-ODN010或PS-ODN853转染到MRSA菌体内。通过平板克隆形成实验计数菌落数(CFU)并测定的MRSA生长曲线,观察给予PS-ODN010、PS-ODN853后苯唑西林对MRSA生长的影响;观察给予PS-ODN853后抗生素对MRSA的最小抑菌浓度(MIC)的变化;通过荧光实时定量PCR法检测PS-ODN853对靶基因mecA表达变化的影响;构建BALB/c小鼠的败血症模型,并观察给予PS-ODN853后,苯唑西林是否能够提高小鼠的存活率。在体外实验中包裹PS-ODN010或PS-ODN853的脂质体分别设0.7、2、6、18μM四个组,同时设PBS对照组、包裹PBS脂质体组、包裹随机对照链PS-ODN203(18μM)脂质体组、游离PEI(0.2μM)组、裸PS-ODN010或PS-ODN853(18μM)组。在动物实验中设生理盐水对照组、苯唑西林组、空白脂质体加苯唑西林组、裸PS-ODN853加苯唑西林组以及脂质体包裹PS-ODN853(2.5、5、10 mg/kg)加苯唑西林组,上述分组中的苯唑西林给药量均为100mg/kg、2/日、连续7天,裸PS-ODN853为10mg/kg/日、连续3天,脂质体为1/日、连续3天。3.脂质体包裹的抗mecA mRNA的PS-ODN853逆转MRSE耐药性的研究:用脂质体将包裹的PS-ODN853转染到MRSE菌体内。通过平板克隆形成实验计数CFU并测定MRSE的生长曲线,观察给予PS-ODN853后苯唑西林对MRSE生长的影响;观察给予PS-ODN853后抗生素对MRSE的最小抑菌浓度(MIC)的变化;通过荧光实时定量PCR法检测PS-ODN853对靶基因mecA表达变化的影响。实验中包裹PS-ODN853的脂质体分别设0.7、2、6、18μM四个组,同时设PBS对照组、包裹PBS脂质体组、包裹随机对照链PS-ODN203(18μM)脂质体组、游离PEI(0.2μM)组和裸PS-ODN853(18μM)组。结果:1.新型高效包裹反义脱氧寡核苷酸的纳米微粒脂质体的制备: PS-ODN/PEI纳米微粒和脂质体的粒径分别为(84.8±21.7)nm和(217.1±78.6)nm,脂质体的平均包封率为(79.7±2.69)%。稳定性分析结果显示,将脂质体混悬液放置于4℃和室温14 d后,脂质体的载药量分别为初始载药量的76.32%和70.1%,但与4℃相比脂质体放置于室温时其载药量的变化波动较大。脂质体混悬液于37℃放置时,放置2 d后脂质体的载药量仅为初始载药量的61.69%,约40%的PS-ODN渗漏到脂质体外,表明脂质体在体内37℃时有较好的释药率,能缓慢的将包裹的药物释放。2.脂质体包裹的抗mecA mRNA的PS-ODN010、PS-ODN853逆转MRSA耐药性的体内体外实验研究:⑴与空白对照组相比,脂质体包裹PS-ODN010、PS-ODN853组的MRSA(WHO-2或MRSA071001)的菌落数均明显减少(P<0.01),而空白脂质体组、随机链对照组和游离PEI组的菌落数无显著性的减少(P>0.05),裸PS-ODN组MRSA的菌落数也明显减少(P<0.01),但与脂质体包裹PS-ODN组相比,脂质体包裹PS-ODN组的CFU减少的更加明显(P<0.01)。⑵与空白对照组相比,脂质体包裹PS-ODN010、PS-ODN853组苯唑西林明显的抑制了MRSA(WHO-2或MRSA071001)的生长,并且PS-ODN的浓度越大,苯唑西林的抑制作用越明显。而空白脂质体组、随机链对照组、游离PEI组和裸PS-ODN组MRSA的生长与空白对照组相比没有明显的差异。⑶给予18μM脂质体包裹的PS-ODN853后,苯唑西林等6种抗生素对WHO-2及5株MRSA临床菌株的MIC值均明显降低。⑷荧光实时定量PCR结果显示脂质体包裹的PS-ODN853能明显抑制基因mecA mRNA的表达,并具有浓度依赖性抑制效应。而随机对照链PS-ODN203对mecA mRNA的表达没有明显影响。⑸动物实验结果显示脂质体包裹的2.5、5、10 mg/kg PS-ODN853治疗组小鼠的存活率分别为26.7%、46.7%和53.3%,而其它对照组的小鼠全部死亡;脂质体包裹的PS-ODN853治疗组的血液培养物中的菌落数明显少于对照组。3.脂质体包裹的抗mecA mRNA的PS-ODN853逆转MRSE耐药性的研究:⑴与空白对照组相比,脂质体包裹PS-ODN853组的MRSE070901的菌落数明显减少(P<0.01),而空白脂质体组、随机链对照组和游离PEI组的菌落数无显著性的减少(P>0.05),裸PS-ODN853组MRSE的菌落数也明显减少(P<0.01),但与脂质体包裹PS-ODN853组相比,脂质体包裹PS-ODN853组的CFU减少的更加明显(P<0.01)。⑵与空白对照组相比,脂质体包裹的PS-ODN853组苯唑西林明显抑制了MRSE070901的生长,并且PS-ODN853的浓度越大,苯唑西林的抑制作用越明显。而空白脂质体组、随机链对照组、游离PEI组和裸PS-ODN853组MRSE070901的生长与空白对照组相比没有明显的差异。⑶给予18μM脂质体包裹的PS-ODN853后,苯唑西林等6种抗生素对MRSE070901的MIC值明显降低。⑷荧光实时定量PCR结果显示脂质体包裹的PS-ODN853能明显抑制基因mecA mRNA的表达,并具有浓度依赖性抑制效应,而随机对照链PS-ODN203对mecA mRNA的表达没有影响。结论:1.脂质体包裹的抗mecA mRNA的反义硫代脱氧寡核苷酸(PS-ODN853和PS-ODN010)能够选择性抑制MRSA标准菌株WHO-2及临床菌株的耐药基因mecA的表达,进而能够有效的逆转MRSA对β-内酰胺类抗生素的耐药性,恢复其敏感性;2.我们首次在小鼠感染模型上观察到脂质体包裹的PS-ODN853能够恢复WHO-2对苯唑西林的敏感性,部分恢复了苯唑西林对WHO-2的杀菌作用,感染小鼠的存活率提高到53.3%。3.脂质体包裹的抗mecA mRNA的反义硫代脱氧寡核苷酸(PS-ODN853)能够选择性抑制MRSE临床菌株耐药基因mecA的表达,从而能够有效的逆转MRSE对β-内酰胺类抗生素的耐药性,恢复其敏感性;4.我们首次制备了新型的包裹PS-ODN/PEI纳米微粒的脂质体,该脂质体易于制备,包封率高,性质稳定。药效学实验证明了我们制备的新型脂质体能将其包裹的PS-ODN/PEI纳米微粒有效的转染入MRSA和MRSE菌体内,从而使PS-ODN能够有效的抑制靶基因mecA的表达,表明该新型脂质体可以作为转染PS-ODN进入细菌体内的有效载体;5.以细菌的耐药基因为靶点,应用反义技术抑制其表达,从而逆转耐药细菌的耐药性,恢复对β-内酰胺类抗生素的敏感性,使失效的抗生素重新发挥其疗效,这种方法可能成为人们对抗耐药细菌的新策略。更多还原

【Abstract】 AIM: Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) are common and important pathogens causing hospital infections. Treatment of staphylococcal infections is becoming very difficult and mortality rates are on-going high because of the increasing emergence of resistance to virtually all of theβ-lactams and a wide variety of antimicrobials. In recent years the number of MRSA and MRSE infections is gradually on the rise. Especially the epidemiology of MRSA may be undergoing a change because MRSA is isolated not only in hospital but also in community. Furthermore community-acquired MRSA (CA-MRSA) prevails and spreads wide with high isolating rate, and a particularly serious aspect of CA-MRSA is that it is capable of causing serious infectious illness in healthy individuals, sometimes even fatalities. The prevalence of CA-MRSA is becoming a serious public health problem, and arousing great concern in the medical profession. Now vancomycin is most effective to MRSA and MRSE, but isolation of vancomycin-resistant S. aureus has been documented. So it is evident that we need to seek a new defending strategy against MRSA and MRSE. Alteration of membrane-bound enzymes known as penicillin-binding proteins (PBPs) has been identified as a primary mechanism of“intrinsic”methicillin resistance of MRSA and MRSE. PBP2a is known to be encoded by a chromosomal gene known as mecA. Our previous study had proved that antisense oligonucleotide targeting mecR1, upstream gene of mecA, can partially reverse the resistance of MRSA to oxacillin. But because we lacked effective drug delivery system, inhibition of PS-ODN was limited. In addition, upstream gene of mecA readily mutates or deletes in clinical isolates of S. aureus, therefore PS-ODN targeting mecR1 is only suitable for MRSA strains harboring complete SCCmec elements. Therefore, the present investigation was designed for two key purposes: 1. According to the characteristics of bacterial cell wall and PS-ODN, we prepare a novel kind of liposome with high efficient transfection of PS-ODN through an improved formulation. 2. To evaluate whether PS-ODN targeting mecA, the downstream gene of SCCmec elements, could absolutely restore the susceptibility of MRSA and MRSE toβ-lactam antibiotics in vitro and in vivo.METHODS:1. Preparation and characterization of a new kind of encapsulating PS-ODN/PEI nanometer particle liposome: Choose mecA mRNA as target gene to design and synthesis PS-ODN. At first we prepared condensed PS-ODN/PEI nanometer particle, and then the liposome was prepared by thin film-dispersion technique with EPC, DMPG and PEG2000-DSPE. The mean diameter of PS-ODN/PEI nanometer particle and liposome were determined by a laser light scattering particle-size analyzer. A gel chromatography was used to separate the non-encapsulated drug from the liposomal dispersion. And then the amount of PS-ODN was monitored by UV absorbance at 260 nm (A260nm) using an UV/Visible Spectrophotometers to calculate encapsulation efficiency of liposome. In vitro release experiments were carried out at different temperature to evaluate the stability of liposome.2. Reversal of resistance of MRSA by anti-mecA mRNA PS-ODN010/PS-ODN853 encapsulated in liposome in vitro and in vivo: Different concentrations (0.7、2、6、18μM) of PS-ODN010 or PS-ODN853 were introduced into MRSA by liposome. The total colony forming unit (CFU) per sample was determined by correcting the colony count for the dilution and the change of MRSA growth rates in the broth medium was monitored by A630 measurements at different time points. Drug-resistant characters of MRSA were evaluated by measuring minimal inhibitory concentration (MIC) of different antibiotics. To determine whether the expression of mecA was inhibited after anti-mecA PS-ODN treatment, real-time PCR was used. Establish BALB/c mouse intraperitoneal model of WHO-2 infection and observe the effect on the survival rate of mouse by PS-ODN853 encapsulated into liposome treatment. In vitro experiments, MRSA stains were treated by PBS, encapsulated PBS liposome, encapsulated random chain PS-ODN203 liposome (18μM), free PEI (0.2μM), free PS-ODN (18μM) and encapsulated PS-ODN liposome (PS-ODN at a concentration of 0.7, 2, 6 or 18μM), respectively. In vivo study, BALB/c mouse respectively received isotonic sodium chloride solution (control group), 100 mg/kg oxacillin (twice daily, for 7 days), encapsulated PBS liposome (once daily, for 3 days) combined with 100 mg/kg oxacillin(twice daily, for 7 days), 10 mg/kg free PS-ODN853 (once daily, for 3 days) combined with 100 mg/kg oxacillin (twice daily, for 7 days) or 2.5, 5, 10 mg/kg PS-ODN853 encapsulated into liposome (once daily, for 3 days) combined with 100 mg/kg oxacillin(twice daily, for 7 days).3. Reversal of resistance of MRSE by anti-mecA mRNA PS-ODN853 encapsulated in liposome: Different concentrations (0.7、2、6、18μM) of PS-ODN853 were introduced into MRSE by liposome. The total colony forming unit (CFU) per sample was determined by correcting the colony count for the dilution and the change of MRSE growth rates in the broth medium was monitored by A630 measurements at different time points. Drug-resistant characters of MRSE were evaluated by measuring minimal inhibitory concentration (MIC) of different antibiotics. To determine whether the expression of mecA was inhibited after anti-mecA PS-ODN853 treatment, real-time PCR was used. In above experiments, MRSE stains were treated by PBS, encapsulated PBS liposome, encapsulated random chain PS-ODN203 liposome (18μM), free PEI (0.2μM), free PS-ODN853 (18μM) and encapsulated PS-ODN853 liposome (PS-ODN853 at a concentration of 0.7, 2, 6 or 18μM), respectively.RESULTS:1. Preparation and characterization of a new kind of liposome encapsulating PS-ODN/PEI nanometer particle: The encapsulation efficiencies of liposomes were found to be 79.7%±2.69%. The representative particle size of PS-ODN/PEI nanometer particle and liposomes were 82.0±21.0 nm and 217.1±78.6nm, respectively. The stability study of liposomes showed that, the liposomes retained 76.32% and 70.1% of the encapsulated drug until 14 days at 4℃and room temperature, respectively. Though the results obtained at 4℃and at room temperature were statistically comparable, larger fluctuations were observed with liposome-encapsulated PS-ODN maintained at room temperature than at 4℃. Analysis of the samples from liposomes preparations collected at different time points for 2 days maintained at 37℃showed sustained significant release of PS-ODN, and at second day, about 40% of the drug was released from the liposomes, which indicated PS-ODN could slowly release from liposomes at 37℃.2. Reversal of resistance of MRSA by anti-mecA mRNA PS-ODN010/PS-ODN853 encapsulated in liposome in vitro and in vivo:⑴Compared to the untreated group (control), the number of MRSA colonies (WHO-2 or MRSA071001) was decreased significantly in free anti-mecA PS-ODN010/PS-ODN853 treated group and all encapsulated anti-mecA PS-ODN010/PS-ODN853 liposome treated groups (P < 0.01). However, compared to control, the CFU counts of MRSA were not influenced in encapsulated PBS liposome, encapsulated random chain PS-ODN203 liposome and free PEI treated group (P > 0.05). Compared with free PS-ODN, PS-ODN encapsulated into liposome (at different concentration) could decreased the CFU significantly (P < 0.01), although free PS-ODN also decreased the CFU.⑵Results showed marked growth inhibition of MRSA cells by oxacillin treated with PS-ODN010/PS-ODN853 encapsulated into liposomes as compared to cells grown in control, encapsulated PBS liposome, encapsulated random chain PS-ODN203 liposome, free PS-ODN and free PEI treated group. At the same time, the growth of MRSA was inhibited in a concentration-dependent manner when cells were treated with different concentrations PS-ODN encapsulated in liposomes.⑶Compared to untreated group, The MICs of six antibiotics for all MRSA strains were reduced obviously in encapsulated PS-ODN853 liposome treated groups.⑷PS-ODN853 encapsulated into liposome significantly inhibited the expression of mecA mRNA concentration-dependently. The PS-ODN203 showed no effects on the expression of mecA.⑸In vivo study, 26.7%, 46.7% and 53.3% mouse survived in encapsulated anti-mecA PS-ODN853 (2.5, 5, 10 mg/kg) liposome treated group, while 100% mouse died in other control groups. Compared to control group, the CFU of WHO-2 strain in blood samples reduced significantly in encapsulated anti-mecA PS-ODN853 (2.5, 5, 10 mg/kg) liposome treated group.3. Reversal of resistance of MRSE by anti-mecA mRNA PS-ODN853 encapsulated in liposomes in vitro:⑴Compared to the untreated group (control), the number of MRSE070901 colonies was decreased significantly in free anti-mecA PS-ODN853 treated group and all encapsulated anti-mecA PS-ODN853 liposome treated groups (P < 0.01). However, compared to control, the CFU counts of MRSE070901 were not influenced in encapsulated PBS liposome, encapsulated random chain PS-ODN203 liposome and free PEI treated group (P > 0.05). Compared with free PS-ODN853, PS-ODN853 encapsulated into liposome (at different concentration) could decreased the CFU significantly (P < 0.01), although free PS-ODN853 also decreased the CFU.⑵Results showed marked growth inhibition of MRSE070901 cells by oxacillin treated with PS-ODN853 encapsulated into liposomes as compared to cells grown in control, encapsulated PBS liposome, encapsulated random chain PS-ODN203 liposome, free PS-ODN853 and free PEI treated group. At the same time, the growth of MRSE070901 was inhibited in a concentration-dependent manner when cells were treated with different concentrations PS-ODN853 encapsulated in liposomes.⑶Compared to untreated group, The MICs of six antibiotics for MRSE070901 were reduced obviously in encapsulated anti-mecA PS-ODN853 liposome treated groups.⑷PS-ODN853 encapsulated into liposomes significantly inhibited the expression of mecA mRNA concentration-dependently. The PS-ODN203 showed no effects on the expression of mecA. CONCLUSION:1. Anti-mecA mRNA PS-ODN010 and PS-ODN853 encapsulated into liposome selectively inhibited the expression of mecA mRNA, and increased the killing effect ofβ-lactam antibitics against MRSA standard strain WHO-2 and clinical strains, as well as conversing phenotype of antibiotic resistance of MRSA to drug sensitivity.2. We first observed that anti-mecA mRNA PS-ODN853 encapsulated into liposome could restore WHO-2 strain sensitivity to oxacillin in mouse intraperitoneal model of WHO-2. Therefore oxacillin rescued 53.3% of infected animals.3. Anti-mecA mRNA PS-ODN853 encapsulated into liposomes selectively inhibited the expression of mecA mRNA, and increased the killing effect ofβ-lactam antibitics against a MRSE clinical strain MRSE070901, as well as conversing phenotype of antibiotic resistance of MRSE to drug sensitivity.4. We first prepared a novel liposome which encapsulated PS-ODN/PEI nanometer particle. This kind of liposome was stable and easy to obtain with high encapsulation efficiency. We certified liposome could availably delivery PS-ODN/PEI nanometer particle into MRSA and MRSE cells, and inhibited the expression of mecA mRNA. This kind of liposome encapsulating PS-ODN/PEI nanometer particle could to be an efficient vector in delivering PS-ODN into bacteria.5. The blockade of resistant gene of bacteria by antisense drug might be a new viable strategy to preserve the efficacies of existingβ-lactam antibiotics to resistant bacteria.更多还原