反义寡核苷酸-聚乙烯亚胺纳米微粒逆转耐甲氧西林金黄色葡萄球菌耐药性的研究

中国临床药理学与治疗学 ›› 2008, Vol. 13 ›› Issue (1): 31-35.

反义寡核苷酸-聚乙烯亚胺纳米微粒逆转耐甲氧西林金黄色葡萄球菌耐药性的研究

王慧¹, 孟静茹¹, 陈涛^2,3, 贾敏¹, 何功浩¹, 马雪¹, 罗晓星¹

¹第四军医大学药学系药理教研室, 西安 710032, 陕西;
²西北工业大学生命科学院, 西安 710072, 陕西;
³陕西省脂质体工程中心, 西安 710075, 陕西

收稿日期:2007-09-30 修回日期:2007-12-12 出版日期:2008-01-26 发布日期:2020-10-13
通讯作者: 罗晓星,男, 教授, 博士生导师, 研究方向:分子药理学及新药研究。Tel:029-84774591 　E-mail:xxluo3@fmmu.edu.cn
作者简介:王慧, 女, 硕士研究生, 助教, 研究方向:分子药理学。Tel:13572161299 E-mail: wangdlx@fmmu.edu.cn
基金资助:
国家自然科学基金项目(30271556); 全军医药卫生科研基金项目(02M008); 全军医药卫生科研基金项目(06G100)

Reversal of antibiotic resistance methicillanin-resistant staphylococcus aureus by polyethyleneimine-phosphothioate oligodeoxynucleotide nanometer particle

WANG Hui¹, MENG Jing-ru¹, CHEN Tao^2,3, JIA Ming¹, HE Gong-hao¹, MA Xue¹, LUO Xiaoxing¹

¹Department of Pharmacology, Fourth Military Medical University, Xi'an 710032, Shaanxi, China;
²Faculty of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, Shaanxi, China;
³Shaanxi Liposome Research Center, Xi 'an 710075, Shaanxi, China

Received:2007-09-30 Revised:2007-12-12 Online:2008-01-26 Published:2020-10-13

摘要/Abstract

摘要： 目的以聚乙烯亚胺(polyethyleneimine, PEI) 为载体, 将硫代寡聚脱氧核苷酸(PS-ODNs) 转染到耐甲氧西林金黄色葡萄球菌(methicillin resistant staphylococcus aureus, MRSA) 体内, 通过药效学观察PS-ODNs 逆转MRSA 对β-内酰胺类抗生素的耐药性。方法制备PEI 与PS-ODNs 结合的纳米微粒(PEI-ODNs 纳米微粒) ;PEI-ODNs 纳米微粒的粒径分析及结合率的测定;平板克隆形成实验计数菌落数(CFU) ;微量法测定细菌生长曲线;液体稀释法测定细菌的最小抑菌浓度(MIC)。结果 PEI-ODNs 纳米微粒的粒径为(85±22) nm, PEI与PS-ODNs 的结合率最高为(97.3±1.1) %。含苯唑西林(6 mg L) 的M-H 琼脂板上, 30 μg/mL 的PEI-ODNs 纳米微粒组MRSA 的菌落数为6.4×108 mL, 而空白对照组的菌落数为3.3×109 mL,二者相比给药组菌落数明显减少, 差异具有统计学意义(P<0.01), 而其他对照组与空白对照组比较差异无统计学意义。实验结果显示PEIODNs纳米微粒组的苯唑西林对MRSA 生长有抑制作用, 30 μg/mL 的PEI-ODNs 纳米微粒可将苯唑西林对MRSA 最小抑菌浓度由1 024 μg/mL 降低至16 μg/mL。结论 PEI 与PS-ODNs 的结合率很高且粒径较小, PS-ODNs 可以部分逆转MRSA 对β-内酰胺类抗生素的耐药性。药效学结果显示PEI 可以有效地将反义寡核苷酸转入MRSA 体内, 可考虑将其作为反义寡核苷酸进入细菌的载体。

关键词: 硫代寡聚脱氧核苷酸, 聚乙烯亚胺, 纳米微粒, 耐甲氧西林金黄色葡萄球菌, 转染

Abstract: AIM: This study used polyethyleneimine (PEI) and phosphothioate oligodeoxynucleotide (PS-ODNs) to prepare PEI-ODNs nanometer particle and transmit it into methicillin resistant staphylococcus aureus (MRSA) in vitro by pharmacodynamic evaluation. METHODS: The PEI-ODNs nanometer particle was prepared and its diameter was analyzed. The PEI and PS-ODNs binding rate by fluorospectrophotometer was evaluated. The total colony forming unit (CFU) was counted. The cell growth curve was drawn by measuring A630 nm at different time points with microplate reader. The minimal inhibitory concentration (MIC) was determined by fluid dilution method. RESULTS: The diameter of PEI-ODNs nanometer particle was (85±22) nm and the highest binding rate was (97.3±1.1) %. In M-H agar plate(including 6 μg/mL oxacillin), the number of MRSA colonies was 6.4×108 mL in 30 μg/mL PEI-ODNs nanometer particle treated group, while the number of MRSA colonies in blank control was 3.3×109 mL. The number of MRSA colonies was significantly decreased in PEI-ODNs nanometer particle treated group (P<0.01), while CFU of MRSA was not influenced in control group (P >0.05). Significant growth inhibition of cells treated with PEI-ODNs nanometer particle was observed as compared with those cells in control. The MIC of oxacillin to MRSA was decreased from 1 024 μg/mL to 16 μg/mL when treated with 30 μg/mL PEI-ODNs nanometer particle. CONCLUSION: PEI binds PS-ODNs with high binding rate and has little diameter. The susceptibility of MRSA to β-lactam antibiotics is significantly restored. The results indicate that the PEI can transmit PS-ODNs into MRSA and it is considered as a new vector for PS-ODNs into bacteria.

Key words: phosphothioate oligodeoxynucleotide, polyethyleneimine, nanometer particle, methicillin resistant staphylococcus aureus, transfection

中图分类号:

R966

王慧, 孟静茹, 陈涛, 贾敏, 何功浩, 马雪, 罗晓星. 反义寡核苷酸-聚乙烯亚胺纳米微粒逆转耐甲氧西林金黄色葡萄球菌耐药性的研究[J]. 中国临床药理学与治疗学, 2008, 13(1): 31-35.

WANG Hui, MENG Jing-ru, CHEN Tao, JIA Ming, HE Gong-hao, MA Xue, LUO Xiaoxing. Reversal of antibiotic resistance methicillanin-resistant staphylococcus aureus by polyethyleneimine-phosphothioate oligodeoxynucleotide nanometer particle[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2008, 13(1): 31-35.

参考文献

[1] Hiramatsu K, Hanaki H, Ino T, et al. Methicillin- resistant staphylococcus aureus clinical strain with reduced vancomycin susceptibity[J]. J Antimicrob Chemother, 1997,40(2): 135-136.
[2] Archer GL, Bosilevac JM. Signaling Antibiotic Resistance in Staphylococci[J]. Science, 2001, 291(9): 1915-1916.
[3] Crooke ST. Oligonucleotide analogs might be designed to bind to mRNA[J]. Anticancer Drug Des, 1997, 12(5): 311-313.
[4] 孟静茹, 罗晓星, 扈本荃, 等. 特异性阻断MecR1-MecI-MecA 通路逆转耐甲氧西林金黄色葡萄球菌耐药性[J]. 第四军医大学学报, 2005, 26(15): 1352-1355.
[5] Meng JR, Hu BQ, Liu J, et al. Restoration of oxacillin susceptibility in methicillin-resistant staphylococcus aureus by blocking the MecR1-mediated signaling pathway[J]. J Chemother, 2006, 18(4): 88-93.
[6] Zhou Z, Meng JR, Niu C, et al. Restoration of antibiotic susceptibility in methicillinresistant staphylococcus aureus by targeting mecr1 with a phosphorothioate deoxyribozyme[J]. Clin Exp Pharmacol Physiol, 2007, 34(11): 1160-1164.
[7] Lungwitz U, Breuning M, Blunk T, et al. Polyethyleneimine-based non-viral gene delivery system[J]. Eur J Pharm Biopharm, 2005, 60(2): 247-266.
[8] ThomasM, Klibanov AM. Non-viral gene therapy: polycation-mediated DNA delivery[J]. Appl Microbiol Biotechnol,2003, 62(1): 27-34.
[9] Boussif O, Lezoualc'h F, Zanta MA, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethyleneimine[J]. Proc Natl Acad Sci USA, 1995, 92(16): 7297-7301.
[10] Torres Viera C, Tsiodras S, Gold HS, et al. Restoration of Vancomycin susceptibility in Enterococcus faecalis by antiresistance determinant gene transfer[J]. Antimicrob Agents Chemother, 2001, 45(3): 973-975.
[11] Harth G, Zamecnik PC, Tang JY, et al. Treatment of Mycobaterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-L-glutamate glutamine cell wall struture, and bacterial replication[J]. Proc Natl Acad Sci USA, 2000, 97(1): 418-423.

[1]	乔红亮, 丁宁, 邓凯红, 刘彬彬, 董传江, 陈晓波, 邹黎黎. 耐甲氧西林金黄色葡萄球菌感染新型控制策略研究新进展[J]. 中国临床药理学与治疗学, 2023, 28(6): 676-687.
[2]	罗胜勇，贾德武，李小羿，戴向荣. 抗血小板溶栓素对大鼠脑缺血再灌注损伤保护作用机制研究[J]. 中国临床药理学与治疗学, 2018, 23(10): 1109-1115.
[3]	陈旺, 俞竟, 屈强, 张伟, 李智, 吴兰香, 温纯洁, 周宏灏. CYP2C19基因多态性真核表达载体的构建及稳定转染细胞系的建立[J]. 中国临床药理学与治疗学, 2012, 17(3): 263-268.
[4]	王伟, 王玉, 王若宁, 徐苏颖, 丁杨, 周建平. 新型阳离子聚合物非病毒载体介导野生型p53基因体外转染效果的研究[J]. 中国临床药理学与治疗学, 2011, 16(11): 1227-1233.
[5]	华俊彦, 田伟强, 朱雅艳, 虞惠康. 万古霉素治疗耐甲氧西林金黄色葡萄球菌的疗效和血药浓度分析[J]. 中国临床药理学与治疗学, 2010, 15(6): 673-677.
[6]	林丙来, 姜兰兰, 方五旺. 转染p21基因对人动脉平滑肌细胞的影响[J]. 中国临床药理学与治疗学, 2009, 14(9): 1013-1017.
[7]	梁蓓蓓, 王睿. 利奈唑胺、去甲万古霉素等抗菌药物对耐甲氧西林金黄色葡萄球菌的抗菌活性[J]. 中国临床药理学与治疗学, 2008, 13(9): 1044-1047.
[8]	江贤朋, Robert L.Elliott, Jonathan F.Head. 转染IL-6基因增强MCF-7人乳腺癌细胞肿瘤相关抗原的表达[J]. 中国临床药理学与治疗学, 2007, 12(3): 275-281.
[9]	宦梦蕾, 罗晓星, 孟静茹, 衡亮. 脂质体包裹的反义寡核苷酸逆转耐甲氧西林金黄色葡萄球菌耐药性的研究[J]. 中国临床药理学与治疗学, 2006, 11(9): 983-986.
[10]	游捷, 赵蓉, 刘礼斌, 林建银. 晚期糖化终产物受体、核因子-κB 双基因反义RNA抑制糖化终产物刺激的炎症因子表达[J]. 中国临床药理学与治疗学, 2006, 11(7): 784-788.
[11]	柳军, 张陆勇. 胰高血糖素受体拮抗剂筛选模型的建立[J]. 中国临床药理学与治疗学, 2006, 11(10): 1129-1132.
[12]	刘杰, 罗晓星, 孟静茹, 扈本荃, 王海芳, 孟嘉. 脱氧核酶抑制耐甲氧西林金黄色葡萄球菌耐药基因的表达[J]. 中国临床药理学与治疗学, 2004, 9(6): 641-645.