摘要
目的：探讨RNA干扰沉默小鼠p16基因表达对137Csγ射线诱导的小鼠胚胎成纤维细胞（MEF）衰老的影响。方法构建p16 RNA干扰载体，采用实时荧光定量PCR、Western blot和衰老相关β-半乳糖苷酶（SA-β-Gal）染色观察RNA干扰前后辐射诱导衰老的MEF细胞mRNA水平、蛋白水平和衰老细胞百分率等的变化。结果辐射诱导衰老的MEF细胞RNA干扰后24 h，照射＋p16 siRNA-1和照射＋p16 siRNA-2组p16 mRNA 相对表达水平较照射组显著降低（t=16.52、16.13，P均〈0.05），蛋白水平较照射组显著降低；干扰后5 d，照射＋p16 siRNA-1和照射＋p16 siRNA-2组SA-β-Gal染色阳性细胞百分比[（34.17±2.08）%和（33.83±1.75）%]明显低于照射组[（68.83±1.26）%]（t=37.72、38.09，P均〈0.01）。结论 RNA干扰沉默小鼠p16基因表达能够抑制MEF细胞的衰老，为其在细胞衰老相关研究中的应用奠定了基础。
Objective To explore the effects of silencing p16 gene by RNA interference （RNAi） mediated onγ-irradiation-induced mouse embryonic fibroblast（MEF）. Methods p16 RNAi vector was constructed by using pcDNATM6.2-GW/EmGFPmiR plasmid. p16 mRNA and protein levels of the irradia-tion-induced MEF before and after RNAi were detected by real-time PCR, Western blot and the percent-age of aging cells was detected by senescence associated-β-galactosidase（SA-β-Gal） staining. Results 24 h after RNAi, p16 mRNA levels of the irradiation-induced MEF decreased significantly in the irradiation＋p16 siRNA-1 and irradiation＋p16 siRNA-2 group as compared with the irradiation group （t=16.52 and 16.13, both P〈0.05）, protein levels of the irradiation＋p16 siRNA-1 and irradiation＋p16 siRNA-2 group also decreased as compared with the irradiation group. 5 days after irradiation the percentages of SA-β-Gal positive cells in the irradiation＋p16 siRNA-1 and irradiation＋p16 siRNA-2 group were（34.17±2.08）%and（33.83±1.75）%, significantly lower than that of the irradiation group[（68.83±1.26）%]（t=37.72 and 38.09, both P〈0.01）. Conclusion MEF aging can be effectively controlled by RNAi which can shut down the expression of silencing p16 gene and lay the foundations for its application in the related studies on cells senescence.
出处
《国际放射医学核医学杂志》
2014年第5期281-284,303,共5页
International Journal of Radiation Medicine and Nuclear Medicine
基金
国家自然科学基金（81072237）
国家重点基础研究发展计划（2011CB964800-G）
天津市自然科学基金（08JCY-BJC07300）
协和青年基金（3332013044）
中央高校基本科研业务费专项资金（3332013044）
中国医学科学院放射医学研究所学科发展专项基金（1340）
