国家自然科学基金资助项目(9388002、39200024和39430080).
通过DNA重组构建蛋白激酶Cγ(PKCγ)亚类的重组质粒并经基因转染技术和DNA印迹、蛋白质印迹与PKC活性分析,获得了过表达PKCγ的C3H10T1/2细胞——NCP4.NCP4细胞生长速率提高,流式细胞光度术检测表明,NCP4细胞G1期百分率下降,S期和G2+M期百分率升高,与对照组细胞相比,血清依赖性明显下降,贴壁依赖性降低,在软琼脂中形成小集落,出现部分转化表型.进一步检测,首次观察到NCP4细胞中癌基因c-sis表达明显增强,这可能是NCP4细胞血清依赖性下降的分子机理之一.实验表明,在正常C3H10T1/2细胞中PKCγ的过表达可直接导致细胞增殖加速并可诱导出现部分转化特征.
A eukaryotic vector highly expressing PKCγ,was constructed by DNA recombination and transfected into C3H10T1/2 cells by gene transfection. The NCP4 cells stably expressing PKCγ was isolated successfully by Southern blot,Western blot and PKC activity assay.The NCP4 cells displayed an enhanced growth rate,especially under low serum conditions, apparently reduced dependence on serum. By using FCM, it was shown that NCP4 cells exhibited decreased percentage in G1 phase, increased percentage in S phase and G2+M phase. In contrast to control cells, NCP4 cells overexpressing PKCγ decreased anchorage dependence and formed small colonies in soft agar. Furthermore, it was observed that the expression of oncogene c-sis increased obviously in NCP4 cells, it may be one of the molecular mechanisms of the reduced dependence on serum in NCP4 cells. The results indicated that specific elevation of the PKCγ level directly affected the increase of growth rate and lead to some transforming phenotypes in C3H10T1/2 cells.
赵霞,柳惠图. PKCγ过表达诱导C3H10T1/2细胞生长失控的初探[J].生物化学与生物物理进展,1999,26(6):569-573
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