头皮银屑病与生物膜:电子显微镜

A. Stepanova, V. Kornisheva, K. Raznatovskiy, O. A. Smolina
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摘要

用透射电子显微镜(TEM) -磷钨酸(FVA)负对比-直接鉴定表皮鳞片上的微生物,以描述它们的发生频率和形态。为了进行透射电子显微镜(TEM) -用微生物环对FVА染色的阴性对比,从患者头皮受影响和未受影响的区域收集一部分表皮鳞片,将其转移到无菌塑料管中,用于含有生理溶液(0.1ml)的Eppendorf微探针(1.5ml),持续10分钟。将得到的悬浮液用自动移液器涂在直径为3mm的电子显微镜下的铜网格上,上面覆盖一层透明的模具清漆基底膜。然后,将1.5%的FVA溶液(pH 6.7)涂在带有材料的网格上10分钟,将物体染成黑色,使TEM清晰地显示其超微结构组织的细节。用FVA染色后,用蒸馏水清洗网格,转移到培养皿中的干燥滤纸上,干燥10分钟,在Jem 100-SX (Jeol, Japan)中进行TEM检查。银屑病是一种免疫介导的炎症性皮肤病。在检查银屑病患者头皮穿刺活检时,我们首次使用扫描电子显微镜(SEM)结合透射电子显微镜(TEM)的表达方法-磷酸钨(FVA)负对比。该研究比较了受牛皮癣影响和未受牛皮癣影响的头皮活检。研究表明,银屑病的头皮区域覆盖着一层生物膜,由球状细菌和真菌的酵母细胞组成,由薄而混乱的线连接。患牛皮癣的皮肤上的毛发数量比未受影响的皮肤少2倍。病变部位的头发角质层营养不良。透射电镜方法的使用只显示了头皮鳞片中的酵母细胞,从大小、形状(宽疤痕的存在)和单极芽殖来看,它们属于马拉色菌属。与未受影响的皮肤区域相比,马拉色菌属真菌在受影响的皮肤区域的鳞片中不太常见。这种结果上的差异可以用这样一个事实来解释:由于细胞外基质的存在,形成的细菌-真菌生物膜影响了真菌的重要活动,并阻止了它们在鳞片之间自由沉降。最有可能的是,细菌和真菌以细菌-真菌膜的形式结合在皮肤上,引发银屑病爆发的出现,在进一步的研究中,考虑细菌和真菌的结合是可取的。有必要找出在生物膜形成过程中促成这种关联形成的因素,这将有助于找到新的治疗方法。
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Scalp psoriasis and biofilms: electron microscopy
of transmission electron microscopy (TEM) - negative contrasting with phosphoric-tungstic acid (FVA) - was tested to identify microorganisms directly in epidermal scales in order to describe the frequency of their occurrence and their morphology. To carry out transmission electron microscopy (TEM) - negative contrast with the staining of the FVА with a microbiological loop, a part of the epidermal scales collected from the affected and unaffected areas of the patient’s scalp was transferred into sterile plastic tubes for Eppendorf microprobes (1.5ml) containing physiological solution (0.1ml) for 10 minutes. The resulting suspensions were applied with an automatic pipette on electron microscopic copper grids with a diameter of 3mm, covered with a transparent mold-varnished substrate-film. Then, a 1.5% solution of FVA (pH 6.7) was applied to the grids with the material for 10 minutes, which stained the objects negatively in black and allowed TEM to clearly reveal the details of their ultrastructural organization. After staining with FVA, the grids were washed with distilled water, transferred to dry filter paper in a Petri dish, dried for 10 minutes, and examined in TEM Jem 100-SX (Jeol, Japan). Abstract Psoriasis is an immune-mediated inflammatory skin disease. When examining punch biopsies of a patient’s scalp with psoriasis, we for the first time used scanning electron microscopy (SEM) in combination with the express method of transmission electron microscopy (TEM) - negative contrasting with phosphoric tungsten acid (FVA). The study compared biopsies from scalp affected and unaffected by psoriasis. It was shown that psoriatic areas of the scalp are covered with bio films consisting of clusters of coccoid bacteria and yeast cells of fungi, connected by thin chaotically located strands. The number of hairs on the affected skin area with psoriasis was 2 times less than on the unaffected one. and the hair cuticle at the lesions was dystrophic. The use of the TEM method revealed only yeast cells in the scalp scales, which, in terms of size, shape (the presence of a wide scar) and monopolar budding, allowed them to be attributed to the genus Malassezia . Fungi of the genus Malassezia were less common in the scales of the affected skin area, as compared to the unaffected one. This discrepancy in the results can be explained by the fact that the formed bacterial-mycotic biofilms, due to the extracellular matrix, affect the vital activity of fungi and prevent them from freely settling between the scales. Most likely, it is the combination of bacteria and fungi in the form of bacterial-mycotic films on the skin that initiates the appearance of psoriatic eruptions, and in further studies it is advisable to consider bacteria and fungi in combination. It is necessary to find out what factors contribute to the formation of such associations during the formation of biofilms, which will allow finding new methods of therapy.
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