摘要
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。
To observe the activity changes of phosphatidylcholine specific phospholipase C(PC*.PCL)in different biological processes and to determine the role of PC*.PCL in hepatocarcinoma cells. Methods N nitrosodiethylamine was used as hepatocarcinogen and all trans retinoic acid as differentiation inducer.Co lorimeter assay was employed to determine the activity of PC*.PLC.The protein of rat hepatocarcinoma PC*. PLC was partially purified by affinity chromatograph. Results We observed two activities of PC*.PLC in hepatocarcinoma cells.One was Ca 2+ independent,the other was Ca 2+ dependent.Both activities changed significantly during different biological processes.When CBRH*.7919 cells were induced by retinoic acid to differentiate,the activity of Ca 2+ independent PC*.PLC increased significantly both in particulated and cytosolic fractions.The activity of Ca 2+ dependent PC*.PLC decreased significantly.During the courses of phorbol 12 myristate 13 acetate stimulated proliferation or n nitrosodiethylamine induced hepatocarcinogenesis,Ca 2+ independent activity of PC*.PLC significantly decreased,while Ca 2+ dependent activity of PC*.PLC increased gradually throughout the courses.Through an affinity chromatography column linked with the antibody against Perfringens PC*.PLC,we isolated and partially purified the PC*.PLC protein from rat hepatocarcinoma cells.The isolated protein appeared at the bands of 60×10 3,64×10 3 by SDS*.PAGE electrophoresis and showed an activity of PC*.PLC which was independent on calcium. Conclusions Our results has provided the evidence supporting the opinion that both Ca 2+ independent and dependent PC*.PLC exist in hepatocarcinoma cells and play an important role in their development.We consider that our partially purified protein might contribute to the activity of Ca 2+ independent PC*.PLC,while the Ca 2+ dependent PC*.PLC might be in other fractions of protein.
出处
《上海医科大学学报》
CSCD
1999年第5期321-325,共5页
Journal of Fudan University(Medical Science)
基金
上海市教委重点学科基金!资助 (B980 80 1 )
关键词
钙离子
肝肿瘤
肝细胞癌
PC-PLC
phosphatidylcholine specific phospholipase C
hepatocarcinoma
calcium