RNAⅢ抑制肽抑制葡萄球菌对Hela细胞的黏附

doi:10.3969/j.issn.2095-4344.2014.44.024

摘要/Abstract

摘要：

背景：葡萄球菌导致的细菌感染和生物被膜形成可在骨科植入物或创口愈合时发生。受细菌群体感应机制调控，葡萄球菌RNAⅢ抑制肽可以干预葡萄球菌的群体感应系统，阻断葡萄球菌细胞间的信号传导通路，从而抑制葡萄球菌的生物被膜形成，防止葡萄球菌感染。
目的：观察RNAⅢ抑制肽抑制表皮葡萄球菌对人宫颈癌上皮细胞黏附的效果。
方法：体外培养人宫颈癌上皮细胞，实验分4组，空白组每孔加入DMSO的生理盐水，RNAⅢ抑制肽组加含RNAⅢ抑制肽的DMSO溶液，左氧组加入左氧氟沙星的水溶液，联合组用药剂量参照上述两组联合干预。通过组间对照的方法，对比研究表皮葡萄球菌在生理盐水、RNAⅢ抑制肽、左氧氟沙星及两药联合作用下对Hela 细胞的黏附情况。
结果与结论：空白组Hela细胞层表面有大量细菌黏附，而各用药组细菌黏附的数量均显著低于空白组 (P < 0.001)，左氧组光点计数明显低于RNAⅢ抑制肽组(P < 0.05)，而联合组Hela细胞层表面黏附的细菌数量进一步降低(P < 0.01)。结果证实，RNAⅢ抑制肽可以有效抑制表皮葡萄球菌对宿主细胞表面的黏附，并且与抗生素有协同作用。

中国组织工程研究杂志出版内容重点：人工关节；骨植入物；脊柱；骨折；内固定；数字化骨科；组织工程

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关键词: 植入物, 人工假体, 表皮葡萄球菌, RNAⅢ抑制肽, 黏附, HeLa细胞, 群体感应, 生物被膜, 实验研究, 国家自然科学基金

Abstract:

BACKGROUND: Staphylococcal infections and its biofilm formation can occur when orthopedic implants or wound is healing, and are regulated by bacterial population sensing mechanism. RNAIII inhibiting peptide intervenes the quorum-sensing system of staphylococcal and blocks the signal transduction among staphylococcal cells, and inhibits staphylococcal biofilm formation, and then prevents staphylococcal infections.
OBJECTIVE: To investigate the influence of RNAIII inhibiting peptide on the adhesion of staphylococcus epidermis to the Hela cells.
METHODS: The Hela cells were cultured in vitro. There were four groups in this study. In the blank group, saline with dimethyl sulfoxide was added in each well. In the RNAIII inhibiting peptide group, dimethyl sulfoxide solution containing RNAIII inhibiting peptide was added. In the levofloxacin group, levofloxacin was added. In the combination group, the dose was in accordance with above methods. Using intergroup control method, the adhesion of staphylococcus epidermis to the Hela cells was compared under the effects of saline, RNAIII inhibiting peptide and levofloxacin and their combination.
RESULTS AND CONCLUSION: In the blank group, abundant bacterial adhered to Hela cells. The number of adhered bacteria was significantly lower in each medicine group than in the blank group (P < 0.001). The spot count was significantly lower in the levofloxacin group than in the RNAIII inhibiting peptide group (P < 0.05). In the combination group, the number of bacteria adhered to Hela cells was decreased (P < 0.01). Results verified that RNAIII inhibiting peptide effectively suppressed the adhesion of staphylococcus epidermis to the host cells, and showed synergistic effects on antibiotics.

中国组织工程研究杂志出版内容重点：人工关节；骨植入物；脊柱；骨折；内固定；数字化骨科；组织工程

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Key words: tissue engineering, bacteria, infection, RNA, staphylococcus

中图分类号:

R318

邢庆昌，郝立波，王继芳. RNAⅢ抑制肽抑制葡萄球菌对Hela细胞的黏附[J]. 中国组织工程研究, 2014, 18(44): 7183-7187.

Xing Qing-chang, Hao Li-bo, Wang Ji-fang. RNAIII inhibiting peptide suppresses the adhesion of staphylococcus epidermis on the Hela cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(44): 7183-7187.

图/表（结果） 3

参考文献

[1]Patterson JL, Stull-Lane A, Girerd PH, et al. Analysis of adherence, biofilm formation and cytotoxicity suggests a greater virulence potential of Gardnerella vaginalis relative to other bacterialvaginosis-associated anaerobes. Microbiology. 2010;156(2):392-399.
[2]Kjelleberg S, Molin S. Is there a role for quorom sensing signals in bacterial biofilm? Current Opin Microb 2002;5: 254-258.
[3]Dunne WM. Bacterial adhesion: seen any good biofilms lately? Clin Microbiol Rev. 2002;15:55-66.
[4]Gov Y, Bitler A, Dell'Acqua G, et al. RNAⅢ inhibiting peptide (RIP), a global inhibitor of Staphylococcus aureus pathogenesis: strucure and function analysis. Peptides. 2001; 22(10): 1609-1620.
[5]Balaban N, Giacometti A, Cirioni O. Use of the quorum-sensing inhibitor RNAⅢ-inhibiting peptide to prevent biofilm formation in vivo by drug-resistant Staphylococcus epidermidis. J Infect Dis. 2003;187(4):625-630.
[6]邢庆昌,郝立波,王继芳.固相合成法合成葡萄球菌RNAⅢ抑制肽的鉴定[J].中国组织工程研究与临床康复,2009,13(13): 2419-2422.
[7]Lowy FD. Staphylococcus aureus infections. N Engl J Med. 1998;339:520-532.
[8]Giacometti A, Cirioni O, Gov Y, et al. RNA Ⅲ inhibiting peptide inhibits in vivo biofilm formation by drug-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2003; 47(6):1979-1983.
[9]Balaban N, Giacometti A, Cirioni O. Use of the quorum-sensing inhibitor RNAⅢ-inhibiting peptide to prevent biofilm formation in vivo by drug-resistant Staphylococcus epidermidis. J Infect Dis. 2003;187(4):625-630.
[10]Vieira-da-Motta O, Ribeiro PD, Dias da Silva W, et al. RNAⅢ inhibiting peptide (RIP) inhibits agr-regulated toxin production. Peptides. 2001;22(10):1621-1627.
[11]邢庆昌,郝立波,王继芳.应用RNAⅢ抑制肽( RIP)抑制葡萄球菌毒素产生的实验研究[J].科学技术与工程,2011,11(2):317- 320.
[12]Yang G, Cheng H, Liu C, et al. Inhibition of Staphylococcus aureus pathogenesis in vitro and in vivo by RAP-binding peptides. Peptides. 2003;24(11):1823-1828.
[13]Dell'Acqua G, Giacometti A, Cirioni O, et al. Suppression of drug-resistant Staphylococcal Infections by the quorum-sensing inhibitor RNAⅢ-inhibiting peptide. J Infect Dis. 2004;190(2):318-320.
[14]Balaban N, Gov Y, Giacometti A, et al. A chimeric peptide composed of a dermaseptin derivative and an RNA Ⅲ-inhibiting peptide prevents graft-associated infections by antibiotic-resistant staphylococci. Antimicrob Agents Chemother. 2004; 48(7):2544-2550.
[15]郝立波,邢庆昌,王继芳,等.RNAⅢ抑制肽抑制葡萄球菌在人工关节材料表面黏附的实验研究[J].中国矫形外科杂志,2008,16(23): 1814-1817.
[16]Balaban N, Singh B, Goldkorn RT, et al. Activation and inhibition of the staphylococcal agr system. Technical comment: Response. Science. 2000;287-391.
[17]Gustafsson E, Nilsson P, Karlsson S, et al. Characterizing the Dynamics of the Quorum-Sensing System in Staphylococcus aureus. J Mol Microbiol Biotechnol. 2004; 8(4):232-242.
[18]Park MK, Park JS, Park EM, et al. Halofuginone ameliorates autoimmune arthritis in mice by regulating the balance between Th17 and Treg cells and inhibiting osteoclastogenesis.Arthritis Rheumatol. 2014;66(5): 1195-207.
[19]Lee J, Park DY, Park do Y, et al. Angiopoietin-1 suppresses choroidal neovascularization and vascular leakage. Invest Ophthalmol Vis Sci. 2014;55(4):2191-2199.
[20]Ariyoshi W, Okinaga T, Knudson CB, et al. High molecular weight hyaluronic acid regulates osteoclast formation by inhibiting receptor activator of NF-κB ligand through Rho kinase. Osteoarthritis Cartilage. 2014;22(1):111-120.
[21]Hamm A, Veschini L, Takeda Y, et al. PHD2 regulates arteriogenic macrophages through TIE2 signalling. EMBO Mol Med. 2013;5(6):843-857.
[22]Naidu VG, Dinesh Babu KR, et al. RANKL targeted peptides inhibit osteoclastogenesis and attenuate adjuvant induced arthritis by inhibiting NF-κB activation and down regulating inflammatory cytokines. Chem Biol Interact. 2013;203(2): 467-479.
[23]Rodrigues WF, Madeira MF, da Silva TA, et al. Low dose of propranolol down-modulates bone resorption by inhibiting inflammation and osteoclast differentiation. Br J Pharmacol. 2012;165(7):2140-2151.
[24]Campa C, Harding SP. Anti-VEGF compounds in the treatment of neovascular age related macular degeneration. Curr Drug Targets. 2011;12(2):173-181.
[25]Lee YS, Kim YS, Lee SY, et al. AMP kinase acts as a negative regulator of RANKL in the differentiation of osteoclasts. Bone. 2010;47(5):926-937.
[26]Levitin A, Yanofsky C. Positions of Trp codons in the leader peptide-coding region of the at operon influence anti-trap synthesis and trp operon expression in Bacillus licheniformis. J Bacteriol. 2010;192(6):1518-1526.
[27]Bandhakavi S, Kim YM, Ro SH, et al. Quantitative nuclear proteomics identifies mTOR regulation of DNA damage response. Mol Cell Proteomics. 2010;9(2):403-414.
[28]Yu M, Moreno JL, Stains JP, et al. Complex regulation of tartrate-resistant acid phosphatase (TRAP) expression by interleukin 4 (IL-4): IL-4 indirectly suppresses receptor activator of NF-kappaB ligand (RANKL)-mediated TRAP expression but modestly induces its expression directly. J Biol Chem. 2009;284(47):32968-32979.
[29]Barakat MR, Kaiser PK. VEGF inhibitors for the treatment of neovascular age-related macular degeneration. Expert Opin Investig Drugs. 2009;18(5):637-646.
[30]Geng W, Hill K, Zerwekh JE, et al. Inhibition of osteoclast formation and function by bicarbonate: role of soluble adenylyl cyclase. J Cell Physiol. 2009;220(2):332-240.
[31]Burton JB, Priceman SJ, Sung JL, et al. Suppression of prostate cancer nodal and systemic metastasis by blockade of the lymphangiogenic axis. Cancer Res. 2008;68(19): 7828-7837.
[32]Ma WW, Jimeno A. Strategies for suppressing angiogenesis in gynecological cancers.Drugs Today (Barc). 2007;43(4): 259-273.
[33]Balaban N, Cirioni O, Giacometti A, et al. Treatment of Staphylococcus aureus biofilm infection by the quorum-sensing inhibitor RIP. Antimicrob Agents Chemother. 2007;51(6):2226-2229.
[34]Kim K, Kim JH, Lee J, et al. MafB negatively regulates RANKL-mediated osteoclast differentiation. Blood. 2007; 109(8):3253-3259.
[35]Christensen EN, Mendelsohn ME. Cyclic GMP-dependent protein kinase Ialpha inhibits thrombin receptor-mediated calcium mobilization in vascular smooth muscle cells.J Biol Chem. 2006;281(13):8409-8416.
[36]Bundschu K, Gattenlöhner S, Knobeloch KP, et al. Tissue-specific Spred-2 promoter activity characterized by a gene trap approach. Gene Expr Patterns. 2006;6(3):247-255.
[37]Murray JL, Mavrakis M, McDonald NJ, et al. Rab9 GTPase is required for replication of human immunodeficiency virus type 1, filoviruses, and measles virus. J Virol. 2005;79(18): 11742-11751.
[38]Mangashetti LS, Khapli SM, Wani MR. IL-4 inhibits bone-resorbing activity of mature osteoclasts by affecting NF-kappa B and Ca2+ signaling. J Immunol. 2005;175(2): 917-925.
[39]Mei ZZ, Zheng XF, Dong Y, et al. Inhibiting expression of human telomerase reverse transcriptase promotes degradation of survivin protein. Ai Zheng. 2005;24(5): 525-530.
[40]Hu ZW, Shen ZY, Huang JH. Experimental study on effect of epimedium flavonoids in protecting telomere length of senescence cells HU. Zhongguo Zhong Xi Yi Jie He Za Zhi. 2004;24(12):1094-1097.
[41]Biswas SK, McClure D, Jimenez LA, et al. Curcumin induces glutathione biosynthesis and inhibits NF-kappaB activation and interleukin-8 release in alveolar epithelial cells: mechanism of free radical scavenging activity. Antioxid Redox Signal. 2005;7(1-2):32-41.
[42]Yanofsky C. The different roles of tryptophan transfer RNA in regulating trp operon expression in E. coli versus B. subtilis. Trends Genet. 2004;20(8):367-374.
[43]Chen G, Yanofsky C. Features of a leader peptide coding region that regulate translation initiation for the anti-TRAP protein of B. subtilis. Mol Cell. 2004;13(5):703-711.
[44]Oh KO, Kim SW, Kim JY, et al. Effect of Rehmannia glutinosa Libosch extracts on bone metabolism. Clin Chim Acta. 2003; 334(1-2):185-195.
[45]Wulff C, Wilson H, Rudge JS, et al. Luteal angiogenesis: prevention and intervention by treatment with vascular endothelial growth factor trap (A40). J Clin Endocrinol Metab. 2001;86(7):3377-3386.
[46]Balaban N, Goldkorn T, Gov Y, et al. Regulation of Staphylococcus aureus pathogenesis via target of RNAIII-activating Protein (TRAP). J Biol Chem. 2001;276(4): 2658-2667.
[47]Laitala-Leinonen T, Löwik C, Papapoulos S, et al. Inhibition of intravacuolar acidification by antisense RNA decreases osteoclast differentiation and bone resorption in vitro. J Cell Sci. 1999;112( Pt 21):3657-3666.

引言

材料方法

设计：细胞学实验。

时间及地点：于2007年1至6月在解放军总医院药理研究所细菌实验室完成。

材料：

菌株：菌株表皮葡萄球菌ATCC35984(上海复旦大学范长胜教授馈赠)。

细胞：HeLa细胞购自协和医科大学细胞室。

方法:

细菌培养与染色：将表皮葡萄球菌ATCC35984接种到50 mL TSB培养基，在37 ℃、150 r/min培养箱中摇晃过夜。取20 mL菌液。通过离心从上清液收集细菌，再悬浮于含有10 mg FITC 的由1.5 mL PBS和10.5 mL碳酸氢钠 (0.5 Mp，pH 9.5)组成的混合溶液中，菌液大幅搅拌4 ℃过夜培养。菌液用0.5 mol/L的碳酸氢钠溶液离心洗涤3次，用比浊仪调配成0.5麦氏单位(McFarland，Mc)的PBS菌液备用。

Hela细胞的培养与爬片制备：将冻存的Hela细胞放入37-42 ℃水浴中快速解冻；将其全部吸出到离心管内，并加入等体积的培养液稀释(含体积分数10%胎牛血清的DMEM培养液)，1 000 r/min离心5 min；弃去上清液，加入适量的培养液并轻轻吹散细胞，然后将其全部吸入到 25 cm²的培养瓶内并使其内含有 5 mL的培养液，放入 37 ℃、体积分数5% CO₂孵箱培养。待细胞生长铺满80%的培养瓶底面积或有较多的浮游细胞时，将培养瓶内的培养液吸出；加入体积分数0.25%胰蛋白酶1 mL消化1 min，显微镜下观察，当细胞皱缩变圆甚至脱落时，加入含体积分数10%胎牛血清的DMEM培养液终止消化；用弯头吸管吹打培养瓶底使细胞脱落，并吸出细胞悬液到离心管中， 1 500 r/min离心5 min；弃去上清液，加入1.0-1.5 mL的冻存液(含体积分数10%的二甲亚砜及50%的胎牛血清的DMEM)轻轻吹散细胞使其成细胞悬液。取无菌24孔板，每孔加入1×10⁷ L^-1细胞悬液(以含体积分数10%胎牛血清的DMEM配制)，于37 ℃、体积分数5% CO₂孵箱培养18 h，培养第2天开始进行细菌黏附实验，以保证Hela细胞能够在暴露到细菌前结合到培养板表面。

分组及细菌对Hela细胞黏附试验：FITC标记的细菌按1∶10用PBS稀释，按10⁶个细菌/孔(90 μL PBS)将细菌滴入到上述铺好Hela细胞的96孔培养板内。实验随机分4组：空白组、RNAⅢ抑制肽组、左氧组和联合组。干预5 min后，向空白组每孔加入50 μL含有0.75%DMSO的生理盐水，RNAⅢ抑制肽组每孔加入含50 μg RNAⅢ抑制肽的DMSO溶液(体积分数0.75%)，左氧组每孔加入含100 μg左氧氟沙星的水溶液50 μL，联合组组用药剂量参照上述两组。含有细菌和Hela细胞的24孔板在37 ℃下孵育30 min，用PBS冲洗，用荧光显微镜(×400)在400 nm处进行检测、拍照，每组检测6个孔，每孔随机拍摄6张图像，图像采用Image pro plus 6.0软件进行荧光光点计数，取每孔的平均值。

主要观察指标：荧光显微镜下图像分析及荧光光点计数。

统计学分析：计量资料以x(_)±s表示，采用SPSS 13.0软件对组间数据进行两样本t 检验分析，P < 0.05为差异有显著性意义。

中国组织工程研究杂志出版内容重点：人工关节；骨植入物；脊柱；骨折；内固定；数字化骨科；组织工程

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讨论

细菌耐药性日益成为一个世界性的难题。葡萄球菌(金黄色葡萄球菌和表皮葡萄球菌)作为人类最常见的致病菌，同时也是院内感染的最为常见的原因^[7]。当前，多数葡萄球菌对抗生素具有不同程度的耐药性。葡萄球菌耐药的原因之一在于它们能够黏附在宿主细胞或内植物表面，形成生物被膜，以抵御抗生素的攻击。因此，能够抑制、减少细菌的黏附应是新型生物抑菌、抗菌制剂必备的功效之一。 RNAⅢ抑制肽是一种葡萄球菌感染的全面抑制剂^[4]。研究显示它可以预防葡萄球菌生物被膜形成^[8]、预防葡萄球菌生物被膜相关感染^[9]、抑制金黄色葡萄球菌毒素的产生^[10-11]、抑制耐药葡萄球菌感染^{[10, 12]}、预防移植物相关的耐药葡萄球菌感染^[13]、增强抗生素治疗葡萄球菌感染的效果^[14]。但是，有关RNAⅢ抑制肽抑制细菌黏附的实验数据比较缺乏。

2006年，实验首次通过固态法合成得到了RNAⅢ抑制肽^[6]，并且证实，RNAⅢ抑制肽能够抑制葡萄球菌在人工关节材料表面的黏附^[15]。为了进一步验证RNAⅢ抑制肽能否抑制细菌对宿主细胞的黏附，本次实验实验选取表皮葡萄球菌ATCC35984为实验菌株，以人类宫颈癌上皮细胞(Hela)为黏附对象，以左氧氟沙星为对照药物，通过荧光显微镜对细菌在细胞培养板上Hela细胞层的黏附情况进行观测、拍照，并采用专业的图像处理软件对获得的数字图像进行量化分析。与既往国外研究相比，实验采用的Hela细胞更加容易培养，体积更大，有利于实验的顺利进行^[4]。同时在设计中选取左氧氟沙星为对照药，实验设计更加严密合理，也对RNAⅢ抑制肽与传统抗生素之间的相互作用进行了探索。

实验结果显示，RNAⅢ抑制肽确实可以抑制表皮葡萄球菌对Hela细胞的黏附，这与以往的研究结果相同^[4]。说明RNAⅢ抑制肽能够抑制葡萄球菌黏附分子的形成，阻止细菌在宿主细胞表面聚集形成生物被膜。

同时，实验结果也显示，RNAⅢ抑制肽与抗生素同时存在，更加有效地抑制了细菌对宿主细胞的黏附，说明RNAⅢ抑制肽与抗生素有协同作用。尽管RNAⅢ抑制肽对细菌黏附、聚集的抑制作用与抗生素对细菌的杀灭作用机理不同，但RNAⅢ抑制肽的存在影响了细菌生物被膜的形成，使抗生素能够充分发挥对散在细菌的杀灭作用。这也证明了RNAⅢ抑制肽，这种葡萄球菌生物被膜抑制剂的潜在药用价值。

天然的RNAⅢ抑制肽由凝固酶阴性葡萄球菌菌株RN833(ATCC 55619产生，它具有和RAP极为相似的NH2-末端序列(分别是YSPXTNF和YKPITN)^[16-47]，能够优先与TRAP结合，阻止其磷酸化，从而阻断葡萄球菌的群体感应(QS)机制^[7^，^17]。实验中所用的RNAⅢ抑制肽系实验自主合成^[6]，采用的是与国外不同的固态合成的方法，实验的有效性也进一步证明了实验自主合成的RNAⅢ抑制肽的生物活性。

实验中仍存在偏倚和不足。实验的设计源于对葡萄球菌QS机制的理解，实验结果对RNAⅢ抑制肽的功效进行了验证，但并没有从分子生物学水平对RNAⅢ抑制肽在葡萄球菌QS系统中的作用机制进行揭示，缺乏对RNAⅢ抑制肽抑制细菌黏附作用的细节论证，今后实验将不断完善。

中国组织工程研究杂志出版内容重点：人工关节；骨植入物；脊柱；骨折；内固定；数字化骨科；组织工程
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