Kataoka H, Kobayashi T K, Amano S, Yamada E, Ishida M, Kushima R, Okabe H
Clinical Laboratory, Hikone Municipal Hospital, Hikone, Shiga, Japan.
Cytopathology. 2012 Aug;23(4):237-41. doi: 10.1111/j.1365-2303.2011.00888.x. Epub 2011 Jul 8.
Primary culture of CD34 positive stem cells collected from human peripheral blood was performed with and without supplementation with concentrated ascitic fluid; morphological and immunocytochemical pictures of cultured cells were taken chronologically and compared.
CD34-positive stem cells collected from peripheral blood were cultured for 1, 24 and 48 hours. Concentrated ascitic fluid was added to the plates for the 24-and 48-hour cultures. For immunocytochemical studies, CD34, AE1/AE3, Ber-Ep4 (EA), EMA, EGFR, CD31, CA125 and D2-40 monoclonal antibodies were used.
After culture, small round cells with naked nuclei began to enlarge and to exhibit various changes in the cytoplasm and nucleus. Supplementation with concentrated body cavity fluid enhanced these changes. CD34-positive cells with small round cell features were detected 1 hour after culture and these had no epithelial or mesothelial markers. After 24 hours, CD34-positive cells had disappeared and cells weakly positive for EGFR, EMA, CA125 and D2-40 were detected. Cells with strong and moderate positive reactions for EGFR, AE1/AE3, EA, EMA, D2-40 and CA125 were detected after 48 hours. Supplementation with concentrated body cavity fluid increased the intensity and number of positive cells for these markers compared with the control group. The positive reaction, not only for the epithelial markers such as EGFR and AE1/AE3, but also for mesothelial markers such as CA125 and D2-40, was found to be increased in small numbers of cells in direct proportion to the duration of the primary culture of the peripheral blood cells. CD31, characteristically expressed in endothelial cells, was negative in the cultured cells.
Supplementation of peripheral blood CD34-positive stem cells with body cavity fluid in vitro enhanced their differentiation toward cells of an epithelial or mesothelial phenotype, concomitant with loss of immunoreactivity for CD34. It is assumed that the routine cytological observation of cells obtained from body cavity fluid might cause possible cytomorphological and immunophenotypical changes due to the action of the growth factors contained in the body cavity fluid.
对从人外周血中收集的CD34阳性干细胞进行原代培养，一组添加浓缩腹水，另一组不添加；按时间顺序拍摄培养细胞的形态学和免疫细胞化学图片并进行比较。
将从外周血中收集的CD34阳性干细胞培养1、24和48小时。在24小时和48小时培养时向培养板中添加浓缩腹水。进行免疫细胞化学研究时，使用了CD34、AE1/AE3、Ber-Ep4（EA）、EMA、EGFR、CD31、CA125和D2-40单克隆抗体。
培养后，裸核小圆形细胞开始增大，并在细胞质和细胞核中呈现各种变化。添加浓缩体腔液增强了这些变化。培养1小时后检测到具有小圆形细胞特征的CD34阳性细胞，这些细胞没有上皮或间皮标记物。24小时后，CD34阳性细胞消失，检测到EGFR、EMA、CA125和D2-40弱阳性细胞。48小时后检测到EGFR、AE1/AE3、EA、EMA、D2-40和CA125呈强阳性和中等阳性反应的细胞。与对照组相比，添加浓缩体腔液增加了这些标记物阳性细胞的强度和数量。发现不仅对于EGFR和AE1/AE3等上皮标记物呈阳性反应，而且对于CA125和D2-40等间皮标记物呈阳性反应的细胞数量与外周血细胞原代培养时间成正比增加。在内皮细胞中特异性表达的CD31在培养细胞中呈阴性。
体外向外周血CD34阳性干细胞中添加体腔液可增强其向上皮或间皮表型细胞的分化，同时伴有CD34免疫反应性的丧失。据推测，对从体腔液中获得的细胞进行常规细胞学观察可能会由于体腔液中所含生长因子的作用而导致可能的细胞形态学和免疫表型变化。
