摘要
目的研究转染反义Bmi-1RNA对人肺腺癌细胞A549的体外增殖及细胞周期和凋亡的影响。方法将表达反义Bmi-1RNA的真核表达载体稳定转染A549细胞株，G418筛选挑取阳性克隆。应用即时荧光定量RT-PCR及Westernblot检测转染后Bmi-1基因在mRNA水平及蛋白水平的表达；应用M1Tr比色法及平板克隆形成实验检测A549细胞体外生长能力；用PI单染及Annexin-VPI双染技术在流式细胞仪上检测细胞周期及凋亡情况。结果Westernblot结果显示转染反义Bmi-1RNA使A549细胞中Bmi-1蛋白表达量减少了95％；MTT检测细胞生长曲线表明反义Bmi．1RNA使A549细胞生长速度明显减慢；平板克隆形成实验结果显示转染反义Bmi．1RNA的A549细胞在平板中形成集落的数目（0．67±0．50）明显低于未转染组（73．04±4．1）及转染空质粒组（67．04±4．0，P〈0．01）；流式细胞仪结果显示反义Bmi-1使A549细胞阻滞在G0／G1期，但对细胞凋亡没有影响。结论反义Bmi-1RNA在体外对A549细胞增殖有明显的抑制作用，其抑制作用是通过使A549细胞阻滞在G0／G1期实现的。
Objective To study the effects of antisense Bmi-1 （ B cell-specific moloney murine leukemia virus insertion site 1 ） RNA on the growth, cell cycle and apoptosis of lung cancer cell line 3.549. Methods Recombinant plasmids carrying antisense Bmi-1 RNA were transfected into A549 cells, which expressed a high level of endogenous Bmi-1. The mRNA level of A549 cell was analyzed by real time quantitative RT-PCR and the protein level was determined using Western blot. MTT growth curve and plate colony forming assay were used to measure the effect of antisense Bmi-1 RNA expression on the growth of A549. Flow cytometry was used to analyze cell cycle and apoptosis. Results Antisense Bmi-1 RNA reduced the Bmi-1 expression at the protein level, but did not alter the mRNA level in A549 ceils. Compared with the control cells, A549 cells transfected with antisense Bmi-1 RNA showed a strong inhibition of the cell growth . The number of plate colony formation of the antisense Bmi-1 transfected cells （0. 67±0. 50） was less than those of the control （73. 0 ± 4. 1 ） and cells transfected with empty vector （67.0 ±4. 0,P 〈 0. 01 ）. Transfection of antisense Bmi-1 RNA arrested the A549 ceils at G1/G0 phase of the cell cycle and did not increase the apoptosis. Conclusion Antisense Bmi-1 RNA expression inhibits A549 cells proliferation, likely through the interference of Bmi-1 leading to an arrest of the proliferating cells at the G0/G1 phase.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2009年第12期829-832,共4页
Chinese Journal of Pathology
基金
辽宁省自然科学基金（20072169）
