AMPK对过氧化氢诱导细胞衰老的阻遏研究

doi:10.16431/j.cnki.1671-7236.2023.06.009

摘要/Abstract

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，H₂O₂）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 H₂O₂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p53、p21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）；p53、p21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H₂O₂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。

关键词: AMPK基因; 持续激活型; 稳转细胞株; NIH3T3细胞; 衰老

Abstract: 【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 (H₂O₂)-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 H₂O₂, and after 2 days of culture, cellular senescence was detected by senescence-associated-β-galactosidase (SA-β-Gal) and the expression of p53, p21 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 p53, p21 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 H₂O₂-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.

Key words: AMPK gene; sustained activation type; stable cell line; NIH3T3 cell; aging

中图分类号:

Q255

陈翠, 龚蕾, 徐晢, 汪小波. AMPK对过氧化氢诱导细胞衰老的阻遏研究[J]. 中国畜牧兽医, 2023, 50(6): 2255-2264.

CHEN Cui, GONG Lei, XU Zhe, WANG Xiaobo. Study on the Inhibition of Hydrogen Peroxide-induced Cellular Aging by AMPK[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2255-2264.

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