中文摘要：

目的：应用RNA干扰技术沉默小鼠RAW264．7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RAW264．7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3d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RAW264．7细胞株，MR shRNA能够明显抑制RAW264．7细胞增殖，但对其凋亡无明显影响。

英文摘要：

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.