Slp2反义真核表达载体的构建及功能研究

Construction of antisense Slp2 gene and its effects on growth and proliferation of lung cancer cell lines

背景与目的肺癌相关基因的研究一直是研究的热门课题,Slp2基因是通过cDNA微阵列技术筛选出的肺癌差异表达基因之一,本研究旨在对Slp2基因在肺癌细胞系中的表达及其功能进行初步研究。方法提取肺癌细胞系A549、GLC-82、NCI-H446、NCI-H460的细胞总RNA,用RT-PCR方法检测肺癌细胞系中Slp2基因mRNA水平的表达;构建Slp2反义基因,通过离子脂质体将克隆的Slp2反义基因导入肺癌细胞系GLC-82、NCI-H446、NCI-H460,用半定量RT-PCR检测转染目的基因对肺癌细胞系Slp2表达的影响;通过四唑盐比色法绘制细胞生长曲线以及流式细胞分析、软琼脂细胞克隆形成试验,观测转染Slp2反义基因对肺癌细胞系GLC-82、NCI-H446、NCI-H460生长速度、细胞周期以及细胞增殖能力的影响。结果在4个肺癌细胞系中,Slp2基因均为阳性表达;转染Slp2反义基因后的肺癌细胞系GLC-82、NCI-H446、NCI-H460的细胞生长速度明显减慢;在流式细胞分析直方图上表现为G2期的细胞增多,并曾在转染目的基因的NCI-H446细胞流式直方图中观察到了位于G0/G1期左侧的“亚G1峰”的出现;转染Slp2反义基因后肺癌细胞系的软琼脂培养细胞克隆形成率明显低于未转染细胞。结论Slp2基因与肺癌细胞的生长、增殖力密切相关,有必要进行深入研究和探讨。

Background and objective S1p2 gene is one of the differential expression genes in lung cancer tissues and normal controls, the aim of this study is to observe the effects of antisense S1p2 gene on growth and proliferation in lung cancer cell lines. Methods Total RNAs were extracted from 4 lung cancer cell lines （A549, GLC-82, NCI-H446, NCI-H460）, S1p2 gene expressions were detected at mRNA level by reverse transcription-polymerase chain reaction （RT-PCR）; antisense Slp2 gene plasmid was constructed and trans-fected into GLC-82, NCI-H446 and NCI-H460 cells with lipofectinmin. The expressions of S1p2 gene were analyzed by RT-PCR in these cells before and after transfection; Flow cytometry, MTT assay and soft agar culture were used to investigate the effects of antisense S1p2 gene transfection on DNA content changes in cell cycle, proliferation and colony formation ability of these cells. Results S1p2 gene was positive expressed in A549, GLC-82, NCI-H446 and NCI-H460 cell lines. The proliferation of GLC-82, NCI-H446 and NCI-H460 cells were inhibited by antisertse Slp2 gene conspicuously. The flow cytometry experiments showed accumulation of transfected cells in G2-phase, for one time concomitant appearance of apoptotic cells of sub-G1 （hypodiploid） DNA content was observed in transfected NCI-H446 cell. Dramatic inhibition of colony formation in soft agar was observed in those transfected lung cancer cell lines. Conclusion The results suggest that Slp2 gene might be a key gene in lung cancer cell growth and proliferation.