基因干扰Sirtuin3后对缺氧预处理的影响

Effect of Sirtuin3 on Hypoxic Preconditioning with Its Gene Knocked Down

【目的】探讨基因干扰Sirtuin3(SIRT3)后对缺氧预处理的影响。【方法】使用空白载体pGCSIL-GFP进行预实验,筛选适合的感染复数(MOI)。正式实验使用pGCSIL-PUR-SIRT3转染PC12细胞。成功构建空白载体(empty vector)及SIRT3-/-稳定细胞株后,将两种细胞分别分为对照组(control);缺氧预处理+缺氧组(Hyp+OGD);单纯缺氧组(OGD);MTT测定细胞活性。ATP荧光检测试剂盒测定胞内的ATP水平。Western Blot法测定各组细胞内SIRT3、过氧化物酶体增生激活受体-γ的共刺激因子-1α(PGC-1α),锰超氧化物歧化酶(MnSOD)的蛋白表达水平。【结果】OGD后SIRT3-/-组(0.430±0.009)与Vector组(0.562±0.140)相比,细胞活性下降更明显(P<0.05),经预处理后SIRT3-/-组细胞活性增加,且与Vector组间差异无统计学意义(P=0.311>0.05)。OGD后SIRT3-/-组(0.334±0.006)与Vector组(0.453±0.015)相比,ATP下降更明显(P<0.05)。经预处理后SIRT3-/-组ATP水平增加,且与Vector组间差异无统计学意义(P=0.866>0.05)。SIRT3-/-3组细胞间PGC-1α及MnSOD蛋白的表达与Vector组对应的3组比较明显下降(P<0.05),Hyp+OGD及OGD相对于各自control组均上调(P<0.05),且预处理组的上调更明显(与OGD组比较,P<0.05)【结论】干扰SIRT3基因后PGC-1α及MnSOD的表达下降,SIRT3为预处理或缺氧处理上调PGC-1α及MnSOD的其中一条途径,干扰SIRT3后其他的途径仍然在预处理中发挥重要的细胞保护作用。

[ Objective] To investigate the effect of Sirtuin3 （SIRT3） on hypoxic preconditioning （Hyp） with its gene knocked down. [Methods] Empty vectors （pGCSIL-GFP） were used to select the suitable MOI. pGCSIL-PUR-SIRT3 -vectors were used in formal experiments. Cells transfected with empty vectors or SIRT3-RNAi-LV were divided into control, hypoxic preconditioning ＋ Oxygen-glucose deprivation （Hyp＋OGD） and OGD respectively. MTF assay was used for testing the cells viability. The ATP level was evaluated using ATP Fluorometric Assay Kit. The expression of SIRT3, Peroxisome proliferator-activated receptor-γcoactivator-la （PGC-la） and Manganese superoxide dismutase （MnSOD） were assessed at the protein level by Western blot. [Results] After OGD, MTY values decreased more in SIRT3-/- compared with Vector （0.430 ± 0.009 vs 0.562 ±0.140, P 〈 0.01 ）, but were increased in Hyp ＋ OGD respectively （P 〈 0.05）, resulting in no significant difference between SIRT3-/- and Vector in Hyp＋OGD （P = 0.311 〉 0.05）. After OGD. The ATP levels decreased more in SIRT3-/- compared with Vector （0.334 ±0.006 vs 0.453 ±0.0O15.P〈0.01 but were increased in Hyp＋OGD respectively（P 〈 0.05）, resuhing in no significant difference between SIRT3-/- and Vector in Hyp＋ OGD （P = 0.866 〉 0.05）. The expression of PGC-lc~ and MnSOD were declined significantly in the three groups of SIRT3-/- compared with that in Vector respectively （P 〈 0.05）, but were both increased in Hyp＋OGD and OGD compared with control （P 〈 0.05）, and were increased more in Hyp＋OGD compared with OGD （P 〈 0.05）. [ Conclusion ] The expression of PGC-lcL and MnSOD were decreased with SIRT3 gene knocked down and SIRT3 was one of the ways that hypoxic preconditioning or hypoxia up regulated the expression of MnSOD through PGC-lc pathway.