摘要
[目的]初步探讨RPS12基因对于胃癌细胞生长、增殖,细胞周期,凋亡状态等生物学特性及浸润转移能力等影响。[方法]RPS12基因特异的RNA抗体(RNAi)载体通过脂质体介导法转染RPS12基因高表达MKN45细胞,后经G418压力筛选法获得稳定转染的细胞系,经过RT-PCR法及Western-blot法证实。而后使用流式细胞仪、生长曲线法、平板克隆形成实验法、细胞迁徙实验法等方法分析稳定转染株相关生物学特性的变化,每种检测实验均设立RNAi载体转染细胞实验组、点突变对照组、MKN45细胞空白对照组。[结果]稳定转染的RPS12基因RNAi细胞系经过免疫细胞化学法、RT-PCR法及Western-blot法证实,表达抑制率可达68%左右。与对照组细胞相比,实验组细胞株生长减慢,各时间点细胞计数显著低于对照组(P<0.05);各对照组之间无显著差异,流式细胞仪检测细胞周期显示实验组G2-M期比例显著低于对照组,S期比例显著高于对照组(P<0.05),实验组细胞凋亡率显著高于对照组(P<0.05)。平板克隆形成实验结果显示RPS12RNA干扰组克隆形成率显著低于对照组(P<0.05);细胞迁徙实验结果提示实验组穿膜率与对照组相比差异无统计学意义(P>0.05)。[结论]RPS12基因可能具有促进细胞生长增殖作用,同时可影响细胞周期,降低RPS12基因表达可能抑制胃癌细胞的恶性生物学行为。
[Objective]To investigate the influence of RPS12 gene on the growth, proliferation, apopatosis,invasion and cell cycle of the gastric cancer line MKN45. [Methods]The specific silengcing sequences to RPS12 were selected,designed and synthesized based on the sequence of RPS12 mRNA. They were separately inserted into the plasmid of IMG800 containing U6 promoter. The RNA interference eukaryotic expression vector specific to RPS12 gene were constructed, followed by transfection in MKN45 by using liposome. Then stable expression clones were selected and appraised. The apoptosis and cell cycles were detected by using flow cytometry. The growth and proliferation were analyzed by making cell growth curves and colony formation assay respectively. The ability of invasion was tested by using cancer cell migration assay. The experimental group and two control groups were detected. [Results]The expression of RPS12 in the stable transfected cell line was suppressed by about 68 % appraised by immunocytochemistry,RT-PCR and Westen-blot methods. The RPS12 RNAi cell line grew slower significantly than its control groups. The cell counts in the fifth, sixth and seventh days were significantly different with those of control groups（P〈 0.05）. Cell cycle analysis showed that the RPS12 RNAi cell line proliferated slower,proportions of cells in S were higher and proportions in G2-M were lower significantly than those of control groups（P〈0.05）. Cell apoptosis analysis showed that proportions of apoptosis cells were higher than those of control groups （P〈0.05）. [Results]of colony formation assay showed that the colony formation rate of RPS12 RNAi cell line was lower than those of control groups（P〈0.05）. Further more,the cell migration rate of RPS12 RNAi cell line was not different significantly with those of control groups （P〉0.05）. [Conclusion] RPS12 could pr promote omote the growth and proliferation or restrain apoptosis of gastric invasion and ameliorate malignant metastasis of gastric cancer cells. Knockd
出处
《临床消化病杂志》
2017年第6期357-361,共5页
Chinese Journal of Clinical Gastroenterology
