在ts-RSV LA90细胞转化过程中钙流变化的研究

STUDIES ON THE CHANGES IN CALCIUM FLUXES IN ts-RSV LA 90 CELLS TRANSFORMATION

本文以ts-RSV LA90细胞为模型,用放射性同位素示踪技术测定了通过细胞质膜的^(45)Ca^(2+)流水平;同时用钙指示剂Indo-1 AM和光学多道分析仪测定了胞内[Ca^(2+)]_i,初步研究了Ca^(2+)流和[Ca^(2+)]_i在v-src基因引起细胞转化过程中的动态变化。结果表明LA90细胞质膜上^(45)Ca^(2+)流的改变是细胞转化过程中可以检测到的早期事件之一,转化状态(33℃)细胞的^(45)Ca^(2+)流大于正常状态(40℃)的,细胞从正常到转化(40℃→33℃)的25分钟内^(45)Ca^(2+)流就有明显增大。TMB-8可以抑制转化引起的^(45)Ca^(2+)流出的增大,小牛血清可以刺激正常状态细胞的^(45)Ca^(2+)流出增大,^(45)Ca^(2+)流出与温度有一定依赖关系;细胞转化引起的^(45)Ca^(2+)流入增大,可被异博定抑制,^(45)Ca^(2+)流入不受温度的影响。LA90细胞[Ca^(2+)]_i在转化早期有明显升高,并维持在较正常细胞高2—3倍的水平,A23187-Br可提高正常LA90细胞[Ca^(2+)]_i,[Ca^(2+)]_i不受温度的影响。从质膜上^(45)Ca^(2+)流和[Ca^(2+)]_i的增大说明转化细胞虽然对胞外Ca^(2+)浓度依赖性下降,但维持增殖及转化状态仍然需要一定的胞外Ca^(2+),并通过提高质膜Ca^(2+)流入和释放内源性Ca^(2+),使转化细胞[Ca^(2+)]_i维持在较高水平上。LA90细膜质膜上^(45)Ca^(2+)流和[Ca^(2+)]_i的增大在细胞转化中起着重大作用。

To study the role of Ca^(2+) fluxes and [Ca^(2+)]_i in cell transformation by the v-src gene, ts-RSV LA 90 cells was used in this experiment. Ca^(2+) fluxes across the plasma membrane was measured with radioisotopes. The relative [Ca^(2+)]_i in LA 90 cells loaded Indo-1AM was measured by computer-based Optical Multichannel Analyzer connected with fluorescence microscopy.It was observed that changes in rate of Ca^(2+) fluxes across the plasma membrane are one of the earliest detectable changes of LA 90 cells transformation, Rates of Ca^(2+) fluxes in transformed LA 90 cells (40℃) is higher than that in normal LA 90 cells (33℃), and rates of Ca^(2+) fluxes increased in 25 minutes when LA90 cells shifted from nonpermissive (40%℃) to permissive (33℃) temperature. TMB-8 inhibited increases in rate of Ca^(2+) efflux induced by Pp 60^(v-src), and increase in rate of Ca^(2+) efflux in normal LA 90 cells was stimulated by calf serum. The rate of Ca^(2+) efflux was related to the changes in temperature. The increase in rate of Ca^(2+) influx induced by pp 60^(v-src) could be blocked by verapamil. The rate of Ca^(2+) influx was not affected by the changes in temperature. The increase in relative [Ca^(2+)]_i induced by pp 60^(v-src) is one of the early events in the transformation process. The level of [Ca^(2+)]_i in transformed LA 90 cells was about 2--3 times as much as that in normal LA 90 cells. The increase in relative [Ca^(2+)]_i of normal LA 90 cells was induced by A23187-Br, and the level of [Ca^(2+)]_i was not influenced by the changes in temperature.The increases in rates of Ca^(2+) fluxes and in [Ca^(2+)]_i indicated that the proliferation of transformed cells still need some extracellular Ca^(2+), and the increase in relative[Ca^(2+)]_i induced by pp 60^(v-src) was due to increased both net influx of Ca^(2+) across the plasma membrane and the release of Ca^(2+) from intracellular Ca^(2+) resources, although transformed cells have a much (about 100 folds) reduced need for extracellular Ca^(2+) for cell growth. The present results suggested that the increases in Ca^(2+) fluxes and in relative [Ca^(2+)]_i have the vital role in LA 90 cells transformation by the v-src gene.