Mack D R, Cheng P W, Perini F, Wei S, Hollingsworth M A
Department of Pediatrics, University of Nebraska Medical Center, Omaha 69198, USA.
Glycoconj J. 1998 Dec;15(12):1155-63. doi: 10.1023/a:1006924208135.
To determine whether cell growth conditions impacted carbohydrate expression, HT29 cells were gradually transferred from a conventional glucose-containing media to a glucose-free galactose containing media. Indirect immunofluorescence on acetone fixed cells showed increased expression of sialyl Lewis A antigen (CA19-9), sialyl Lewis C (DUPAN2) and Tn/sialyl-Tn on the surface of HT29 cells grown in the glucose-free galactose containing media compared to those grown in the glucose containing media. Sialyltransferases responsible for the synthesis for these sialylated epitopes were Increased in the galactose-fed HT29 cells. Media overlying the cells was subjected to isopycnic ultracentrifugation in cesium chloride and the fractions derived from both glucose and galactose media with equivalent buoyant densities of 1.56 g/L, which are predicted to contain mucin glycoforms, were further separated by HPLC using a Mono-Q anion exchange column. The chromatograph of eluent from the sample derived from the cells growing in the galactose containing media showed an increased peak that reacted with the anti-sialyl Lewis A antibody, CA19-9. These results show that alteration of in vitro culture conditions may cause HT29 colonic carcinoma cells to alter the expression of sialylated carbohydrates.
为了确定细胞生长条件是否会影响碳水化合物的表达，将HT29细胞从传统的含葡萄糖培养基逐渐转移至不含葡萄糖但含半乳糖的培养基中。对丙酮固定的细胞进行间接免疫荧光检测显示，与在含葡萄糖培养基中生长的HT29细胞相比，在不含葡萄糖但含半乳糖的培养基中生长的HT29细胞表面，唾液酸化路易斯A抗原（CA19-9）、唾液酸化路易斯C（DUPAN2）和Tn/唾液酸化-Tn的表达增加。在以半乳糖喂养的HT29细胞中，负责合成这些唾液酸化表位的唾液酸转移酶增加。将覆盖细胞的培养基在氯化铯中进行等密度超速离心，来自葡萄糖和半乳糖培养基、具有1.56 g/L等效浮力密度（预计含有粘蛋白糖型）的级分，使用Mono-Q阴离子交换柱通过高效液相色谱进一步分离。来自在含半乳糖培养基中生长的细胞的样品洗脱液的色谱图显示，与抗唾液酸化路易斯A抗体CA19-9反应的峰增加。这些结果表明，体外培养条件的改变可能导致HT29结肠癌细胞改变唾液酸化碳水化合物的表达。
