摘要
目的:应用RNA干扰技术沉默小鼠RAW 264.7巨噬细胞盐皮质激素受体(MR)基因,建立稳定干扰细胞株,并观察其对细胞增殖和凋亡的影响。方法:针对MR基因设计合成重组MR shRNA质粒,脂质体法转染质粒至RAW 264.7细胞,经G418筛选后获得稳定表达细胞株。细胞分为3组:野生型(WT)组、阴性对照(NC)组和干扰(shMR)组。荧光显微镜下观察确定细胞的转染效率;实时定量PCR法检测细胞中MR mRNA的表达;CCK-8方法检测细胞增殖活性;流式细胞术分析细胞周期分布和凋亡情况。结果:(1)MR shRNA能明显抑制RAW264.7细胞的MR基因表达,抑制率70%以上。(2)从第3 d开始,shMR组细胞的生长速度明显低于NC和WT组(P<0.05),说明MR shRNA能明显抑制细胞增殖。(3)WT、NC和shMR组的增殖指数分别为(37.2±0.5)%、(37.5±1.6)%和(31.0±1.3)%,shMR组的细胞周期出现改变,S期和G2/M期比例明显下降,增殖指数下降(P<0.05)。(4)WT、NC和shMR组的细胞凋亡率分别为(2.18±0.36)%、(6.65±0.81)%和(7.70±1.34)%,shMR组略高于NC组,但二者的差异无统计学意义(P>0.05)。结论:本研究成功构建了稳定干扰MR基因表达的RAW 264.7细胞株,MR shRNA能够明显抑制RAW 264.7细胞增殖,但对其凋亡无明显影响。
AIM： To establish stable knockdown of mineralocorticoid receptor （MR） expression through short hairpin RNA （shRNA） -mediated silencing in murine RAW 264.7 macrophages. METHODS： Stable MR silencing in RAW 264.7 cells was achieved by recombinant shRNA plasmid targeting murine MR gene via liposome - mediated transfec- tion, followed by G418 selection. The efficacies of plasmid transfection and MR silencing in G418 - resistant ceils were ver- ified by immunofluorescent microcopy and real - time PCR, respectively. Proliferative activity of MR - silencing cell line was analyzed by CCK -8 assay. Cell cycle and apoptosis were evaluated by flow cytometry. RESULTS： MR gene expres- sion was down - regulated by 70% compared with the negative control （NC） plasmid transfection. In addition, MR - silen- cing cells exhibited lower proliferative activity compared with NC and wide type RAW 264.7 cells （ P 〈 0.05 ）, along with reduced proliferation index of 31.0%±1.3% （P 〈 0.05 ）, compared with the wide type cells （37.2%± 0.5% ） and the NC cells （37.5%±1.6% ）. In resting state, the apoptotic rate in wide type, NC and MR - silencing cells were 2.18% ± 0.36%, 6.65% ±0.81% and 7.70%± 1.34%, respectively, and no statistical difference was observed between NC and MR - silencing cells （P 〉 0.05 ）. CONCLUSION： MR gene silencing inhibits the proliferation of RAW 264.7 macropha- ges, but has no obvious effect on the apoptosis of the resting state cells.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2012年第12期2261-2265,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81102088)
天津市自然科学基金一般项目(No.12JCYBJC16600)
武警医学院科研项目(No.WYB201108)
