肝细胞表面去唾液酸糖蛋白受体的流式细胞分析

Flow Cytometric Evaluation of Asialoglycoprotein Receptor on the Surface of Hepatocytes

建立肝细胞表面去唾液酸糖蛋白受体 (ASGPR)的流式细胞分析方法 (FCM ) ,对正常及损伤鼠肝细胞、肝癌细胞 (BEL 740 2 )表面的ASGPR作同步比较分析 .以异硫氰酸荧光素标记的新半乳糖白蛋白 (FITC NGA)为ASGPR的特异性配体 ,以培养的正常肝细胞 (L 0 2 )为靶细胞 ,建立肝细胞表面ASGPR的FCM .测定并计算正常及损伤鼠肝细胞 ,BEL 740 2细胞与同一浓度的FITC NGA同步反应后的平均荧光强度 (MIF)值 .FITC NGA与L 0 2细胞表面ASGPR趋近饱和结合的浓度为 0 4mg/L ,该浓度下正常及损伤鼠肝细胞 ,BEL 740 2细胞的MIF值分别为 2 2 8 7、 5 81、 1 13.该结合可以被至少 5 0倍于FITC NGA的NGA或10mmol/L的EDTA完全抑制 .FCM能够良好地揭示FITC NGA同ASGPR之间的受配体结合特性 .该方法证实BEL 740 2细胞表面几乎没有ASGPR ,损伤鼠肝细胞表面ASGPR的数量较正常鼠肝细胞显著减少 .

To establish a flow cytometric method (FCM) for simultaneous evaluation of asialoglycoprotein receptor (ASGPR) on the surface of normal rat hepatocytes, injured rat hepatocytes and hepatoma cells (BEL 7402). FCM for ASGPR was established using normal hepatocytes (L 02) and FITC conjugated galactosyl neoglycoalbumin (FITC NGA), the specific ligand for ASGPR. The mean intensity of fluorescence (MIF) of the three different hepatocytes were determined and calculated after simultaneously incubated with FITC NGA at the same concentration. The concentration of FITC NGA for saturating ASGPR on the surface of L 02 was 0 4 mg/L , at which the MIF of the three different hepatocytes were 228 7,5 81 and 1 13 respectively . The saturated combination can be completely inhibited by 50 fold NGA or 10mmol/L EDTA . FCM can display soundly the characteristic of the receptor ligand combination between ASGPR and FITC NGA. Compared with normal rat hepatocytes, there is no ASGPR on the surface of BEL 7402, and the quantities of ASGPR on the surface of injured rat hepatocytes decreases significantly.