CAJ | 학술논문

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。
목적：탐토RNA간우침묵소서p16기인표체대137Csγ사선유도적소서배태성섬유세포（MEF）쇠로적영향。방법구건p16 RNA간우재체，채용실시형광정량PCR、Western blot화쇠로상관β-반유당감매（SA-β-Gal）염색관찰RNA간우전후복사유도쇠로적MEF세포mRNA수평、단백수평화쇠로세포백분솔등적변화。결과복사유도쇠로적MEF세포RNA간우후24 h，조사+p16 siRNA-1화조사+p16 siRNA-2조p16 mRNA 상대표체수평교조사조현저강저（t=16.52、16.13，P균<0.05），단백수평교조사조현저강저；간우후5 d，조사+p16 siRNA-1화조사+p16 siRNA-2조SA-β-Gal염색양성세포백분비[（34.17±2.08）%화（33.83±1.75）%]명현저우조사조[（68.83±1.26）%]（t=37.72、38.09，P균<0.01）。결론 RNA간우침묵소서p16기인표체능구억제MEF세포적쇠로，위기재세포쇠로상관연구중적응용전정료기출。
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.