论文部分内容阅读
目的 在不同的生物学过程中观察磷脂酰胆碱特异性磷脂酶C(PCPLC)的活性变化,以及PCPLC在肝癌细胞中的作用。方法 采用亚硝胺诱发大鼠肝癌,全反式视黄酸为肝癌细胞的诱导分化剂,PCPLC酶活性采用间接比色法测定,大鼠PCPLC酶蛋白采用亲和层析法进行部分纯化。结果 在肝癌细胞中观察到了两种不同活性的磷脂酰胆碱特异的磷脂酶C,一种是非钙依赖性,另一种是钙依赖性的。这两种酶活性在不同的生物学过程中都有显著变化。当视黄酸诱导大鼠肝癌细胞CBRH7919细胞分化成熟时,非钙依赖性PCPLC无论是膜颗粒部分还是胞质部分均显著升高,钙依赖性PCPLC活性则显著下降。在佛波酯刺激增殖和化学诱发大鼠肝癌过程中,非钙依赖性PCPLC活性则显著下降,而钙依赖性PCPLC活性则逐步升高。经过亲和层柱分离,获得了部分纯化的酶蛋白,聚丙酰胺电泳位置为60×103和64×103,而且酶的催化活性不依赖钙离子。结论 肝癌细胞同时存在非钙依赖性和钙依赖性PCPLC活性,他们可能具有不同的生物学功能,并在肝癌细胞发生发展过程中起重要作用。我们认为这种部分纯化的酶蛋白可能含有非钙依赖性的PCPLC酶蛋白,而钙依赖性的PCPLC可能存在于另外的组份中
Objective To observe the changes in the activity of phosphatidylcholine-specific phospholipase C (PCPLC) and the role of PCPLC in hepatoma cells in different biological processes. Methods Rat hepatocellular carcinoma was induced by nitrosamine. All-trans retinoic acid was the differentiation inducer of hepatocellular carcinoma cells. PCPLC enzyme activity was measured by indirect colorimetry. Rat PCPLC enzyme protein was partially purified by affinity chromatography. Results Two differently active phosphatidylcholine-specific phospholipase Cs were observed in hepatoma cells, one being non-calcium-dependent and the other calcium-dependent. Both of these enzyme activities have changed significantly in different biological processes. When retinoic acid induced differentiation of rat hepatocarcinoma cells CBRH7919 cells, the non-calcium-dependent PCPLC was significantly increased in both the granular and cytoplasmic fractions, and calcium-dependent PCPLC activity was significantly decreased. In the process of phorbol ester stimulated proliferation and chemically induced rat liver cancer, the non-calcium-dependent PCPLC activity was significantly decreased, while the calcium-dependent PCPLC activity was gradually increased. After affinity column separation, a partially purified enzyme protein was obtained. The polyacrylamide electrophoresis positions were 60×103 and 64×103, and the catalytic activity of the enzyme was independent of calcium ions. Conclusion Liver cancer cells have both non-calcium-dependent and calcium-dependent PCPLC activities. They may have different biological functions and play an important role in the development of hepatocellular carcinoma cells. We believe that this partially purified enzyme protein may contain non-calcium-dependent PCPLC enzyme proteins, whereas calcium-dependent PCPLC may be present in other components.