金黄色葡萄球菌超抗原与人耳部瘢痕疙瘩形成的相关性研究

Correlation study of Staphylococcus aureus superantigens and formation of human ear keloid

目的探讨金黄色葡萄球菌超抗原与人耳部瘢痕疙瘩形成的相关性。方法采用回顾性病例对照研究方法。2017年6月—2018年3月,取南通大学附属医院收治的10例(女9例、男1例,年龄19~59岁)耳部瘢痕疙瘩患者行耳部瘢痕疙瘩核心切除术后弃用瘢痕组织及3例(均为女性,年龄20~24岁)色素痣患者手术后弃用正常皮肤组织。取耳部瘢痕疙瘩表面分泌物,培养细菌并进行鉴定。取瘢痕疙瘩和正常皮肤组织,采用蛋白质印迹法检测金黄色葡萄球菌肠毒素A+肠毒素B+毒性休克综合征毒素1(TSST-1)的蛋白表达,并根据蛋白表达情况将瘢痕疙瘩分为超抗原阳性组和超抗原阴性组。蛋白质印迹法检测2组瘢痕疙瘩T细胞受体(TCR)Vβ蛋白表达。Masson、苏木精-伊红(HE)染色观察2组瘢痕疙瘩真皮内胶原纤维形成和炎症细胞浸润情况。酶联免疫吸附测定法检测超抗原阳性瘢痕疙瘩中金黄色葡萄球菌肠毒素A、肠毒素B、TSST-1表达。对数据行配对样本t检验。结果耳部瘢痕疙瘩表面分泌物培养出细菌,优势菌培养24 h可见细菌周围出现溶血现象,菌落呈白色或金黄色,鉴定为金黄色葡萄球菌。3例患者正常皮肤肠毒素A+肠毒素B+TSST-1蛋白表达阴性,蛋白表达量为0.267±0.016。4例患者瘢痕疙瘩金黄色葡萄球菌肠毒素A+肠毒素B+TSST-1蛋白表达阳性,蛋白表达量为0.472±0.016,纳入超抗原阳性组;6例患者瘢痕疙瘩金黄色葡萄球菌肠毒素A+肠毒素B+TSST-1蛋白表达阴性,蛋白表达量为0.255±0.004,纳入超抗原阴性组。超抗原阳性组瘢痕疙瘩金黄色葡萄球菌肠毒素A+肠毒素B+TSST-1蛋白表达量明显高于超抗原阴性组瘢痕疙瘩和正常皮肤(t=15.22、8.63,P<0.01)。超抗原阳性组瘢痕疙瘩TCR Vβ蛋白表达量为0.389±0.023,明显高于超抗原阴性组的0.169±0.014(t=8.62,P<0.01)。Masson染色显示,2组瘢痕疙瘩真皮内均见大量胶原纤维增生。HE染色显示,超抗原阴性组瘢痕疙瘩真皮可见血管周围少量炎症细胞浸润,超抗原阳性组瘢痕疙瘩血管周围可见大量炎症细胞浸润。4例超抗原阳性瘢痕疙瘩患者中金黄色葡萄球菌肠毒素A阳性2例、金黄色葡萄球菌肠毒素B阳性2例,其中1例患者检测出肠毒素A和肠毒素B双阳性,未有患者检出金黄色葡萄球菌TSST-1。结论金黄色葡萄球菌分泌的超抗原是耳部瘢痕疙瘩众多发病原因中的一种,可能与金黄色葡萄球菌超抗原激活瘢痕疙瘩信号通路有关。

Objective To investigate the correlation of Staphylococcus aureus superantigens and human ear keloid formation.Methods The retrospective case-control study method was used.The discarded keloid tissue was collected from 10 patients(9 females and 1 male,aged 19-59 years)with ear keloid after core excision of ear keloid,and the discarded normal skin tissue was collected from 3 female patients(aged 20-24 years)with pigmented nevus after operation,who were admitted to affiliated Hospital of Nantong University from June 2017 to March 2018.The exudation from ear keloid surface was collected for bacteria culture and identification.The keloid and normal skin tissue were collected to detect the protein expression of Staphylococcus aureus enterotoxin A+enterotoxin B+toxic shock syndrome toxin-1(TSST-1)by Western blotting,and the keloid was divided into superantigen positive group and superantigen negative group according to the protein expression of Staphylococcus aureus enterotoxin A+enterotoxin B+TSST-1.The protein expressions of T cell receptor(TCR)Vβof keloid in 2 groups were detected by Western blotting.Collagen fibers formation and inflammatory cells infiltration in keloid dermis of 2 groups were observed by Masson and hematoxylin eosin staining,respectively.Enzyme-linked immunosorbent assay method was used to detect expression of Staphylococcus aureus enterotoxin A,enterotoxin B,and TSST-1 in keloids with superantigen positive.Data were statistically analyzed with paired sample t test.Results Bacteria were cultured from the exudation on ear keloid surface,hemolysis was observed around the dominant bacteria after being cultured for 24 hours,and the colony was white or golden yellow,which was identified as Staphylococcus aureus.The protein expression of Staphylococcus aureus enterotoxin A+enterotoxin B+TSST-1 of normal skin was negative in 3 patients,and the protein expression was 0.267±0.016.The protein expression of Staphylococcus aureus enterotoxin A+enterotoxin B+TSST-1 of keloid was positive in 4 patients,with the protein expression of 0.472±0.016,which was included into superantigen positive group,and the protein expression of Staphylococcus aureus enterotoxin A+enterotoxin B+TSST-1 of keloid was negative in 6 patients,with the protein expression of 0.255±0.004,which was included into superantigen negative group.The protein expression of Staphylococcus aureus enterotoxin A+enterotoxin B+TSST-1 of keloid in superantigen positive group was obviously higher than that of keloid in superantigen negative group and that in normal skin(t=15.22,8.63,P<0.01).The protein expression of TCR Vβin keloid of superantigen positive group was 0.389±0.023,which was significantly higher than 0.169±0.014 in superantigen negative group(t=8.62,P<0.01).Masson staining showed that a large number of collagen fibers existed in dermis of keloid in 2 groups.HE staining showed that there were a small number of inflammatory cells infiltration around the blood vessels of keloid dermis in superantigen negative group and a large number of inflammatory cells infiltration around the blood vessels of keloid in superantigen positive group.Among the 4 patients with superantigen positive keloid,Staphylococcus aureus enterotoxin A was positive in 2 patients,Staphylococcus aureus enterotoxin B was positive in 2 patients,and among that Staphylococcus aureus enterotoxin A and enterotoxin B were both positive in 1 patient,with no TSST-1 detected in any patient.Conclusions The superantigen secreted by Staphylococcus aureus is one of the many causes of ear keloid,which may be related to the activation of keloid signal pathway by superantigen of Staphylococcus aureus.