蛆虫分泌物抑制金黄色葡萄球菌入侵成骨细胞的机制探讨

doi:10.19438/j.cjoms.2020.05.003

中国口腔颌面外科杂志 ›› 2020, Vol. 18 ›› Issue (5): 395-400.doi: 10.19438/j.cjoms.2020.05.003

蛆虫分泌物抑制金黄色葡萄球菌入侵成骨细胞的机制探讨

朱方兴, 徐晓峰, 司家文, 廖倩, 徐兵

上海交通大学医学院附属第九人民医院·口腔医学院口腔颅颌面科,国家口腔疾病临床医学研究中心, 上海市口腔医学重点实验室,上海市口腔医学研究所,上海 200011

收稿日期:2019-12-04 修回日期:2020-03-24 出版日期:2020-09-20 发布日期:2020-10-28
通讯作者: 徐兵,E-mail：bingxu568@hotmail.com
作者简介:朱方兴（1994-）,男,硕士研究生,E-mail：sp_terry94@163.com
基金资助:
国家自然科学基金（81600827）; 上海市科学技术委员会生物医药处重点项目（15411951300）

Mechanism of maggot excretion/secretion in anti-intracellular Staphylococcus aureus internalization of osteoblast: an in vitro study

ZHU Fang-xing, XU Xiao-feng, SI Jia-wen, LIAO Qian, XU Bing

Department of Oral and Craniomaxillofacial Surgery, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology. Shanghai 200011, China;

Received:2019-12-04 Revised:2020-03-24 Online:2020-09-20 Published:2020-10-28

摘要/Abstract

摘要： 目的：通过体外实验探讨蛆虫分泌物抑制金黄色葡萄球菌入侵成骨细胞的机制。方法：提取蛆虫分泌物,用糜蛋白酶抑制剂VR23、丝氨酸蛋白酶抑制剂（PMSF）预处理,将未处理的蛆虫分泌物、与抑制剂预处理后的蛆虫分泌物、PBS分别加入小鼠前成骨细胞细胞系MC 3T3-E1进行培养,24、48、72 h后,以蛋白免疫印迹法检测纤连蛋白含量,以CCK-8法检测细胞活性。与金黄色葡萄球菌共培养后,通过细菌涂板计数及免疫荧光法检测胞内菌数量。实验数据采用SPSS 25.0软件包进行统计学分析。结果：蛆虫分泌物可以降解纤连蛋白,不对细胞活性产生影响。细菌涂板计数可见,经蛆虫分泌物处理的小鼠前成骨细胞细胞系中,胞内菌含量较经糜蛋白酶抑制剂或丝氨酸蛋白酶抑制剂预处理的蛆虫分泌物处理的小鼠前成骨细胞细胞系中胞内菌含量低,而经PBS处理的小鼠前成骨细胞细胞系中胞内菌含量最高。免疫荧光法检测亦得到相似结果。结论：蛆虫分泌物可用于减少金黄色葡萄球菌对小鼠前成骨细胞细胞系的内化作用,为慢性难治性骨髓炎的治疗提供了新的思路。

关键词: 蛆虫, 骨髓炎, 金黄色葡萄球菌, 纤连蛋白, 胞内菌

Abstract: PURPOSE: This study was aimed to explore the mechanism of excretion/secretion (E/S) in protecting osteoblast being internalized by Staphylococcus aureus through in vitro experiments. METHODS: Excretion/secretion(E/S), E/S pre-treated with serine protease inhibitors(PMSF), E/S pre-treated with chymotrypsin inhibitors VR23 and PBS were respectively added to the mouse pre-osteoblast cell line (MC 3T3-E1) for culture. After 24, 48, 72 hours, Western blot was used to detect the fibronectin degradation, and CCK-8 was used to detect cell viability. After 24 hours, the cell line was co-cultured with Staphylococcus aureus to form intracellular bacteria to observe whether intracellular bacteria had formed in different groups. Flat colony counting method and immunofluorescence method were used to observe intracellular bacteria. The data were analyzed by SPSS 25.0 software package. RESULTS: E/S degraded fibronectin in extracellular matrix without influencing cell viability. Through flat colony counting method, the least intracellular bacteria were observed in the cell line treated with E/S. In cell line treated with E/S pre-treated with PMSF or the chymotrypsin protease inhibitor, more intracellular bacteria were observed. In PBS treated cell line, the most intracellular bacteria were observed. These results showed statistically significant(P<0.05) and similar results were obtained from immunofluorescence photography. CONCLUSIONS: E/S can be used to reduce the effect of Staphylococcus aureus on the internalization of MC 3T3-E1, which provides a new idea for the treatment of chronic refractory osteomyelitis.

Key words: Larva, Osteomyelitis, Staphylococcus aureus, Fibronectins, Intracellular bacteria

中图分类号:

R782.3

朱方兴, 徐晓峰, 司家文, 廖倩, 徐兵. 蛆虫分泌物抑制金黄色葡萄球菌入侵成骨细胞的机制探讨[J]. 中国口腔颌面外科杂志, 2020, 18(5): 395-400.

ZHU Fang-xing, XU Xiao-feng, SI Jia-wen, LIAO Qian, XU Bing. Mechanism of maggot excretion/secretion in anti-intracellular Staphylococcus aureus internalization of osteoblast: an in vitro study[J]. China J Oral Maxillofac Surg, 2020, 18(5): 395-400.

参考文献

[1] Baer WS.The classic: the treatment of chronic osteomyelitis with the maggot (larva of the blow fly)[J]. Clin Orthop Relat Res, 2011, 469(4): 920-944.
[2] Horobin AJ, Shakesheff KM, Woodrow S, et al.Maggots and wound healing: an investigation of the effects of secretions from Lucilia sericata larvae upon interactions between human dermal fibroblasts and extracellular matrix components[J]. Br J Dermatol, 2003, 148(5): 923-933.
[3] Horobin AJ, Shakesheff KM, Pritchard DI.Promotion of human dermal fibroblast Migration, matrix remodelling and modification of fibroblast morphology within a novel 3D model by lucilia sericata larval secretions[J]. J Invest Dermatol, 2006, 126(6): 1410-1418.
[4] Polakovicova S, Polák Š, Kuniaková M, et al.The effect of salivary gland extract of lucilia sericata maggots on human dermal fibroblast proliferation within collagen/hyaluronan membrane in vitro: transmission electron microscopy study[J]. Adv Skin Wound Care, 2015, 28(5): 221-226.
[5] Pöppel AK, Kahl M, Baumann A, et al.A Jonah-like chymotrypsin from the therapeutic maggot Lucilia sericata plays a role in wound debridement and coagulation[J]. Insect Biochem Mol Biol, 2016, 70: 138-147.
[6] Chambers L, Woodrow S, Brown AP, et al.Degradation of extracellular matrix components by defined proteases from the greenbottle larva Lucilia sericata used for the clinical debridement of non-healing wounds[J]. Br J Dermatol, 2003, 148(1): 14-23.
[7] Ghasemian A, Peerayeh SN, Bakhshi B, et al.The microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) genes among clinical isolates of Staphylococcus aureus from hospitalized children[J]. Iran J Pathol, 2015, 10(4): 258-264.
[8] Pritchard DI, Brown AP.Degradation of MSCRAMM target macromolecules in VLU slough by Lucilia sericata chymotrypsin 1 (ISP) persists in the presence of tissue gelatinase activity[J]. Int Wound J, 2015, 12(4): 414-421.
[9] Reilly SS, Hudson MC, Kellam JF, et al.In vivo internalization of Staphylococcus aureus by embryonic chick osteoblasts[J]. Bone, 2000, 26(1): 63-70.
[10] Ahmed S, Meghji S, Williams RJ, et al.Staphylococcus aureus fibronectin binding proteins are essential for internalization by osteoblasts but do not account for differences in intracellular levels of bacteria[J]. Infect Immun, 2001, 69(5): 2872-2877.
[11] Kalinka J, Hachmeister M, Geraci J, et al.Staphylococcus aureus isolates from chronic osteomyelitis are characterized by high host cell invasion and intracellular adaptation, but still induce inflammation[J]. Int J Med Microbiol, 2014, 304(8): 1038-1049.
[12] Josse J, Velard F, Gangloff SC.Staphylococcus aureus vs. osteoblast: relationship and consequences in osteomyelitis[J]. Front Cell Infect Microbiol, 2015, 5: 85.
[13] de Mesy Bentley KL, Trombetta R, Nishitani K, et al. Evidence of Staphylococcus aureus deformation, proliferation, and migration in canaliculi of live cortical bone in murine models of osteomyelitis[J]. J Bone Miner Res, 2017, 32(5): 985-990.
[14] Kalinka J, Hachmeister M, Geraci J, et al.Staphylococcus aureus isolates from chronic osteomyelitis are characterized by high host cell invasion and intracellular adaptation, but still induce inflammation[J]. Int J Med Microbiol, 2014, 304(8): 1038-1049.
[15] Tuchscherr L, Kreis CA, Hoerr V, et al.Staphylococcus aureus develops increased resistance to antibiotics by forming dynamic small colony variants during chronic osteomyelitis[J]. J Antimicrob Chemother, 2016, 71(2): 438-448.
[16] Hamza T, Dietz M, Pham D, et al.Intra-cellular Staphylococcus aureus alone causes infection in vivo[J]. Eur Cell Mater, 2013, 25: 341-350.
[17] 高磊, 尹叶锋, 王寿宇, 等. 纯化蛆虫分泌物抗菌肽对糖尿病大鼠溃疡创面的抗菌作用[J]. 中国组织工程研究, 2012, 16(24): 4437-4440.
[18] Hamad MA, Skeldon AM, Valvano MA.Construction of aminoglycoside-sensitive burkholderia cenocepacia strains for use in studies of intracellular bacteria with the gentamicin protection assay[J]. Appl Environ Microbiol, 2010, 76(10): 3170-3176.
[19] Dupieux C, Assant ST, Camus C, et al.Intraosteoblastic activity of daptomycin in combination with oxacillin and ceftaroline against MSSA and MRSA[J]. J Antimicrob Chemother, 2017, 72(12): 3353-3356.
[20] Josse J, Velard F, Alami SM, et al.Increased internalization of Staphylococcus aureus and cytokine expression in human Wharton's jelly mesenchymal stem cells[J]. Biomed Mater Eng, 2014, 24(1 Suppl): 27-35.
[21] Sherman RA, Hall MJ, Thomas S.Medicinal maggots: an ancient remedy for some contemporary afflictions[J]. Annu Rev Entomol, 2000, 45: 55-81.
[22] 夏效泳, 范媛媛, 史宗新, 等. 蛆虫清创疗法在难愈性感染创面的临床应用[J]. 现代生物医学进展, 2014, 14(36): 7186-7189.
[23] 蒋克春, 王爱萍, 王伟, 等. 蛆虫治疗糖尿病足溃疡的临床研究[J]. 实用糖尿病杂志, 2009, 5(6): 49-50.
[24] 王江宁, 王寿宇, 赵贵庆, 等.蛆虫疗法修复严重感染创面的应用体会[J]. 中国美容整形外科杂志, 2004, 15(6): 294-296.
[25] van der Plas MJA, Andersen AS, Nazir S, et al. A novel serine protease secreted by medicinal maggots enhances plasminogen activator-induced fibrinolysis[J]. PLoS One, 2014, 9(3): e92096.
[26] Brown A, Horobin A, Blount DG, et al.Blow fly Lucilia sericata nuclease digests DNA associated with wound slough/eschar and with Pseudomonas aeruginosa biofilm[J]. Med Vet Entomol, 2012, 26(4): 432-439.
[27] Telford G, Brown AP, Seabra RAM, et al.Degradation of eschar from venous leg ulcers using a recombinant chymotrypsin from Lucilia sericata[J]. Br J Dermatol, 2010, 163(3): 523-531.
[28] 朱方兴, 祁磊, 徐兵. 蛆虫清创治疗机制的研究进展[J]. 中国口腔颌面外科杂志, 2019, 17(1): 85-89.
[29] Henderson B, Nair S, Pallas J, et al.Fibronectin: a multidomain host adhesin targeted by bacterial fibronectin-binding proteins[J]. FEMS Microbiol Rev, 2011, 35(1): 147-200.
[30] Burke FM, McCormack N, Rindi S, et al. Fibronectin-binding protein B variation in Staphylococcus aureus[J]. BMC Microbiol, 2010, 10: 160.
[31] Symersky J, Patti JM, Carson M, et al.Structure of the collagen-binding domain from a Staphylococcus aureus adhesin[J]. Nat Struct Biol, 1997, 4(10): 833-838.
[32] Mcdevitt D, Francois P, Vaudaux P, et al.Identification of the ligand-binding domain of the surface-located fibrinogen receptor (clumping factor) of Staphylococcus aureus[J]. Mol Microbiol, 1995, 16(5): 895-907.
[33] Downer R, Roche F, Park PW, et al.The Elastin-binding protein of Staphylococcus aureus(EbpS) is expressed at the cell surface as an integral membrane protein and not as a cell wall-associated protein[J]. J Biol Chem, 2002, 277(1): 243-250.

蛆虫分泌物抑制金黄色葡萄球菌入侵成骨细胞的机制探讨

Mechanism of maggot excretion/secretion in anti-intracellular Staphylococcus aureus internalization of osteoblast: an in vitro study

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