Jauregui H O, McMillan P N, Hevey K, Naik S
Department of Pathology, Rhode Island Hospital, Providence.
In Vitro Cell Dev Biol. 1988 May;24(5):401-12. doi: 10.1007/BF02628491.
A quantitative evaluation of lectin binding to adult rat hepatocyte cell surfaces was done using cells isolated by two different collagenase perfusion methodologies and cultured as monolayers with two different tissue culture media formulations (protocol I vs. protocol II). The presence of alpha-D-mannosyl and alpha-D-glucosyl groups was detected by the binding of Concanavalin A (Con A), Lens culinaris agglutinin (LCA), and Pisum sativum agglutinin (PSA) to freshly isolated cells. Furthermore, beta-D-galactose [Ricinus communis agglutinin (RCA)] and sialic acid residues [wheat germ (WGA)] were also found. Protocols I and II served as models for evaluation of: a) the stripping effect of collagenase separation procedures, b) the restoration in culture of collagenase-stripped sugar residues, c) the effect of the culture environment on cell viability [as measured by lactic acid dehydrogenase (LDH) leakage] and the protein content of hepatocytes, and d) the presence of cell surface sugar residues as a function of culture duration. The ultrastructural morphology of freshly isolated and cultured hepatocytes was also evaluated. These studies indicated that a decline in lectin binding invariably occurred earlier than a massive leakage of LDH and a decrease in the protein content of the cells in culture. Ultrastructurally, autophagocytosis was an early phenomenon in cells isolated and cultured by protocol I, which was also inferior to protocol II regarding the preservation of hepatocyte glycocalyces. Sugar residues lost due to the collagenase-stripping effect were restored, as shown by lectin binding, within the first 24 h of culture. This stripping effect was confirmed by quantitative evaluations of lectin binding to hepatocytes in culture after an incubation with collagenase. This study shows that the binding of peroxidase-labeled lectins is a useful tool for quantitative evaluation of the sugar composition of hepatocyte cultures.
使用通过两种不同胶原酶灌注方法分离的细胞，并在两种不同的组织培养基配方（方案I与方案II）中培养为单层，对凝集素与成年大鼠肝细胞表面的结合进行了定量评估。通过伴刀豆球蛋白A（Con A）、扁豆凝集素（LCA）和豌豆凝集素（PSA）与新鲜分离的细胞结合，检测α-D-甘露糖基和α-D-葡萄糖基的存在。此外，还发现了β-D-半乳糖[蓖麻凝集素（RCA）]和唾液酸残基[麦胚凝集素（WGA）]。方案I和方案II用作评估以下方面的模型：a）胶原酶分离程序的剥离效果；b）胶原酶剥离糖残基在培养中的恢复情况；c）培养环境对细胞活力[通过乳酸脱氢酶（LDH）泄漏测量]和肝细胞蛋白质含量的影响；d）细胞表面糖残基作为培养持续时间的函数的存在情况。还评估了新鲜分离和培养的肝细胞的超微结构形态。这些研究表明，凝集素结合的下降总是比培养中细胞的LDH大量泄漏和蛋白质含量降低更早发生。在超微结构上，自噬是通过方案I分离和培养的细胞中的早期现象，在肝细胞糖萼的保存方面也不如方案II。如凝集素结合所示，因胶原酶剥离作用而丢失的糖残基在培养的最初24小时内得以恢复。在用胶原酶孵育后，通过对培养的肝细胞中凝集素结合的定量评估证实了这种剥离作用。这项研究表明，过氧化物酶标记的凝集素的结合是定量评估肝细胞培养物糖组成的有用工具。
