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-7402)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-02)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-7402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-02细胞表面ASGPR趋近饱和结合的浓度为0.4 mg/L,该浓度下正常及损伤鼠肝细胞,BEL-7402细胞的MIF值分别为228.7、5.81、1.13.该结合可以被至少50倍于FITC-NGA的NGA或10 mmol/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-7402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.
李文新,张荣军,谭成,陶永辉,金坚.肝细胞表面去唾液酸糖蛋白受体的流式细胞分析[J].生物化学与生物物理进展,2000,27(4):394-397
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