摘要
【目的】构建稳定表达腺苷酸活化蛋白激酶(adenosine 5′-monophosphate-activated protein kinase,AMPK)/AMPK-T172D的NIH3T3细胞株,探讨AMPK对过氧化氢(hydrogen peroxide,H 2O 2)诱导的细胞衰老的影响。【方法】采用PCR扩增AMPK基因及其突变形式AMPK-T172D,将其克隆至慢病毒载体pLVX-IRES-Puro中,构建pLVX-IRES-AMPK/AMPK-T172D重组质粒并进行双酶切验证。将构建好的重组质粒包装成慢病毒,感染NIH3T3细胞,并利用嘌呤霉素筛选获得NIH3T3稳转细胞株。采用实时荧光定量PCR及Western blotting检测稳转细胞株中AMPK的mRNA和蛋白表达情况。利用8.8 mmol/L H 2O 2处理AMPK/AMPK-T172D过表达NIH3T3细胞株,培养2 d后,采用衰老相关β-半乳糖苷酶(senescence-associated-β-galactosidase,SA-β-Gal)染色检测细胞衰老情况,利用实时荧光定量PCR检测p 53、p 21及IL-6基因的表达情况。【结果】双酶切验证结果显示,pLVX-IRES-AMPK/AMPK-T172D表达质粒构建成功。实时荧光定量PCR和Western blotting结果显示,与对照组相比,AMPK/AMPK-T172过表达的NIH3T3细胞中AMPK和AMPK-T172D mRNA和蛋白表达水平均极显著或显著升高(P<0.01;P<0.05),肉毒碱棕榈酰转移酶1(carnitine palmitoyltransferase-1,CPT-1)和脂肪酸合酶(fatty acid synthase,FAS)mRNA表达水平显著或极显著升高(P<0.05;P<0.01),SA-β-Gal细胞阳性率极显著降低(P<0.01);p 53、p 21和IL-6基因mRNA表达水平显著或极显著降低(P<0.05;P<0.01)。【结论】试验成功获得AMPK/AMPK-T172D过表达NIH3T3稳转细胞株,激活AMPK能降低H 2O 2诱导的衰老细胞中SA-β-Gal细胞阳性率和衰老相关因子p53、p21和IL-6的表达。试验结果为研究AMPK在衰老中的作用、可能的分子机制及抗衰老治疗策略提供依据。
【Objective】This study was aimed to construct a stable expression of adenosine 5′-monophosphate-activated protein kinase(AMPK)/AMPK-T172D NIH3T3 cell lines,and investigate the effect of AMPK on hydrogen peroxide(H 2O 2)-induced cell senescence.【Method】AMPK gene and its mutant form AMPK-T172D were amplified by PCR and cloned into lentiviral vector pLVX-IRES-Puro.The pLVX-IRES-AMPK/AMPK-T172D recombinant plasmids were constructed and double digestion was verified.The constructed recombinant plasmid was packaged into Lentivirus,infected with NIH3T3 cells,and puromycin screening was used to obtain stable cell lines.Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of AMPK in stable cell lines,respectively.The AMPK/AMPK-T172D overexpression of NIH3T3 cell lines was treated with 8.8 mmol/L H 2O 2,and after 2 days of culture,cellular senescence was detected by senescence-associated-β-galactosidase(SA-β-Gal)and the expression of p 53,p 21 and IL-6 genes were detected by Real-time quantitative PCR.【Result】The results of double digestion verification showed that compared with control group,the pLVX-IRES-AMPK/AMPK-T172D expression plasmid was successfully constructed.Real-time quantitative PCR and Western blotting results showed that the expressions of mRNA and protein of AMPK and AMPK-T172D were extremely significantly or significantly increased(P<0.01 or P<0.05).The mRNA expression of carnitine palmitoyltransferase-1(CPT-1)and fatty acid synthase(FAS)in AMPK/AMPK-T172D overexpressing NIH3T3 cells were significantly or extremely significantly increased(P<0.05 or P<0.01).The cell positive rate of SA-β-Gal was extremely significantly decreased(P<0.01).The mRNA expression of p 53,p 21 and IL-6 genes were significantly or extremely significantly decreased in AMPK/AMPK-T172D overexpressed NIH3T3 cells(P<0.05 or P<0.01).【Conclusion】The stable expression of AMPK/AMPK-T172D overexpression NIH3T3 stable cell lines was successfully obtained,and AMPK activation could reduce the cell positive rate of SA-β-Gal and the expression of aging-related factors(p53,p21 and IL-6)in H 2O 2-induced senescent cells.The results would provide a basis for the research on the role of AMPK in aging,possible molecular mechanisms and anti-aging treatment strategies.
作者
陈翠
龚蕾
徐晢
汪小波
CHEN Cui;GONG Lei;XU Zhe;WANG Xiaobo(Institute of Function and Morphology,Qujing Medical College,Qujing 655000,China;School of Basic Medicine,Dali University,Dali 671000,China;Key Laboratory of University Cell Biology Yunnan Province,Dali 671000,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2023年第6期2255-2264,共10页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金地区科学基金项目(81860158)
曲靖医学高等学校校级项目(2022XF003)。
