6A8 α-甘露糖苷酶表达抑制致人鼻咽癌细胞CNE-2L2与层粘连蛋白粘附减弱及片状伪足减少

目的探讨6A8α-甘露糖苷酶表达抑制对CNE-2L2细胞与层粘连蛋白的粘附及细胞表面片状伪足的影响。方法Western印迹检测6A8α-甘露糖苷酶的表达。置细胞于层粘连蛋白(laminin)包被的培养板孔中,37℃孵育1h后,用结晶紫染色细胞,用0.1mol/L枸橼酸钠/50%乙醇溶解结合到细胞中的染料。测定540nm处的吸光度值(AT),按R=AT/A100×100%计算粘附率(A100为100%粘附值)。扫描电镜观察细胞表面的片状伪足。结果6A8α-甘露糖苷酶表达抑制的2个克隆细胞株(AS1和AS2)对层粘连蛋白的相对粘附率分别为0.447±0.096与0.533±0.065,而6A8α-甘露糖苷酶表达正常的对照细胞(W、M和S)的相对粘附率分别为0.78±0.035、0.7±0.05和0.80±0.04。两者的差别有生物学意义(P<0.01)。6A8α-甘露糖苷酶表达正常的细胞表面片状伪足丰富,6A8α-甘露糖苷酶表达抑制的细胞表面的片状伪足基本上消失。结论6A8α-甘露糖苷酶表达抑制可导致人鼻咽癌细胞CNE-2L2与层粘连蛋白粘附减弱及片状伪足减少。

Inhibition of 6A8 alpha-manosidase expression induces decrease of adhesion to laminin and reduction of lamellipodia of human nasopharyngeal carcinoma cell CNE-2L2

OBJECTIVE: To investigate the inhibitory effect of 6A8 alpha-manosidase expression on the adhesiveness of CNE-2L2 cells to laminin and the lamellipodia on cell surface. METHODS: 6A8 alpha-manosidase expression was detected by Western blotting. For assaying the adhesion of cells to laminin, cells were incubated in laminin-coated plate at 37 degrees C for 1 h, the adhered cells were stained with crystal purple dissolved in 0.1 mol/L Sodium Citrate/50% ethanol. Absorbance 540 nm was measured. Adhesion rate (R) was calculated according to formula R = AT/A100 x 100%. Here A100 represents 100% adhesion. lamellipodia on cell surface was observed upon a scanning electron microscopy. RESULTS: The adhesion rate of two clones (AS1 and AS2) with inhibition of 6A8 alpha-manosidase expression to laminin was 0.447 +/- 0.096 and 0.533 +/- 0.065 respectively. The adhesion rate of three controls with normal expression of 6A8 alpha-manosidase to laminin was 0.78 +/- 0.035, 0.7 +/- 0.05 and 0.80 +/- 0.04 respectively. The difference was significant (P < 0.01). CNE-2L2 cells with normal expression of 6A8 alpha-manosidase was rich in lamellipodia on their surface. Lamellipodia nearly disappeared on the cells with inhibition of 6A8 alpha-manosidase expression. CONCLUSIONS: Inhibition of 6A8 alpha-manosidase expression results in decrease of adhesion to laminin and reduction of lamellipodia of human nasopharyngeal carcinoma cell CNE-2L2.