摘要
MARVELD1基因是在非小细胞肺癌细胞系中存在差异表达的一个新基因，本文中我们将对MARVELD1蛋白的亚细胞定位及其功能作用做进一步的研究。运用免疫胶体金技术和重组荧光蛋白定位对MARVELD1蛋白进行亚细胞定位，观察发现MARVELD1蛋白在细胞核的核质中具有明显的标记信号，在胞浆中主要是在线粒体中有金颗粒存在。根据转染后细胞表面超微结构的变化结果，用FITC-鬼笔环肽对F—actin染色，激光共聚焦扫描显微镜观察结果显示，NIH3T3-MARVELD1细胞的微丝结构出现了明显的改变：微丝减少，细胞间通讯连接消失或降低，细胞膜边缘卷翘、皱缩，微丝变得紊乱、松散。说明该基因的表达影响了微丝的组装情况。应用间接免疫荧光技术对微管的分析表明在形态学上没有明显改变。根据微丝变化的形态学特征，采用半定量RT—PCR、western—blot进一步分析转染MARVELD1前后NIH3T3细胞中的纽蛋白（vinculin）和连接蛋白43（connexin43，Cx43）的表达情况。结果表明，转染后细胞中vinculin与Cx43的表达显著降低。表明MARVELD1基因的表达影响了细胞的粘附与细胞间通讯连接。鉴于MARVELD1亚细胞定位结果，提示MARVELD1蛋白的功能作用是通过核内调控vinculin与Cx43的表达机制，影响了微丝的结构，细胞粘附和细胞间通讯。
MARVELD1, one of the significantly over-expressed cDNA fragments in Anip973, was identified to be an unknown sequence. In this paper, we use immune colloidal gold technique and MARVELD1-DsRed recombination protein to investigate the subcellular localization of MARVELD1. The results show MARVELD1 is located in the cell nucleus and cytoplasm. We stained the Factiul with FITC-phalloidin. Result from laser confocal scanning microscope observing reveals that, the microfilament structure of NIH3T3-MARVELD1 cells has changed significantly. While the mircotube have nothing changed morphologically. With semi-quantity RT-PCR and western blot, we further analyzed the expression of vinculin and connexin43 in the cells before and after transfection. Results show that the expression of vinculin and connexin43 has decreased significantly after transfection. Associated with the subcellular location results of MARVELD 1, it indicates MARVELDI may regulate the expression of vineulin and connexin43 in nuclear, and therefore affects microfilament structure, cell adherence and intercellular communication.
出处
《生命科学仪器》
2008年第1期22-27,共6页
Life Science Instruments
