Hollmer Cara, Essmann Michael, Ault Kevin, Larsen Bryan
Office of University Research, Des Moines University, Des Moines, IA 50312, USA.
Infect Dis Obstet Gynecol. 2006;2006:98218. doi: 10.1155/IDOG/2006/98218.
Pathogenesis of mucosal microorganisms depends on adherence to the tissues they colonize and infect. For Candida albicans, cell surface hydrophobicity may play a significant role in tissue binding ability.
A continuous cell line of vaginal epithelial cells (VEC) was grown in keratinocyte serum-free medium (KSFM) with supplements and harvested by trypsinization. VEC were combined with yeast cells to evaluate adherence and inhibition of adherence. In this experimental setup, yeast stained with fluorescein isothiocyanate were allowed to attach to VEC and the resulting fluorescent VEC were detected by flow cytometry.
VEC were cultured and examined daily after plating and showed morphology similar to basal epithelial cells. Culture media supplemented with estradiol showed increased VEC proliferation initially (first 24 h) but cell morphology was not altered. Fluorescinated Candida cells bound effectively to the cultured VEC. Using fresh cells exposed to various preparations of K-Y, we showed that all formulations of the product reduced Candida binding to VEC by 25% to 50%. While VEC were generally harvested for use in experiments when they were near confluent growth, we allowed some cultures to grow beyond that point and discovered that cells allowed to become overgrown or stressed appeared to bind yeast cells more effectively.
Flow cytometry is a useful method for evaluating binding of stained yeast cells to cultured VEC and has demonstrated that commercially available products have the ability to interfere with the process of yeast adherence to epithelial cells.
黏膜微生物的发病机制取决于其对所定植和感染组织的黏附。对于白色念珠菌而言,细胞表面疏水性可能在组织结合能力方面发挥重要作用。
阴道上皮细胞(VEC)连续细胞系在添加了补充剂的角质形成细胞无血清培养基(KSFM)中培养,通过胰蛋白酶消化收获细胞。将VEC与酵母细胞混合以评估黏附及黏附抑制情况。在此实验设置中,使异硫氰酸荧光素染色的酵母附着于VEC,然后通过流式细胞术检测产生的荧光VEC。
接种后每日对VEC进行培养和检查,其形态与基底上皮细胞相似。添加雌二醇的培养基最初(最初24小时)显示VEC增殖增加,但细胞形态未改变。荧光标记的念珠菌细胞能有效结合至培养的VEC。使用暴露于各种K-Y制剂的新鲜细胞,我们发现该产品的所有配方均可使念珠菌与VEC的结合减少25%至50%。虽然VEC通常在接近汇合生长时收获用于实验,但我们让一些培养物生长超过该点,发现过度生长或受应激的细胞似乎能更有效地结合酵母细胞。
流式细胞术是评估染色酵母细胞与培养的VEC结合的有用方法,并且已证明市售产品有能力干扰酵母黏附上皮细胞的过程。