流体力学介导的RNA干扰对小鼠肝脏线粒体融合素基因-2、空腹血糖和血清甘油三酯水平的影响

Effects of hydrodynamics-mediated RNAi on Mfn2 expression, blood sugar and fat levels in mice

目的 观察流体力学介导的RNA干扰（RNAi）对小鼠肝脏线粒体融合素基因-2（Mfn2）、空腹血糖（FBS）和血清甘油三酯（TG）水平的影响.方法 将56只雄性BALB/c小鼠随机分为空白对照组（NC组,n=8）、阴性对照组（HK组,n=24）和Mfn2质粒干扰组（Mfn2组,n=24）.HK组小鼠利用流体力学注射75μg阴性对照质粒溶液1.5 ml,Mfn2组小鼠利用流体力学注射75 μg Mfn2 shRNA质粒溶液1.5 ml.应用逆转录-聚合酶链反应和Western blot方法分别测定注射24、72、120 h后,小鼠肝脏Mfn2的mRNA和蛋白质表达;同时分别取血测定小鼠FBS和TG水平.多组间比较采用单因素方差分析,两两比较采用Scheffe＇s t检验.结果 质粒注射72、120 h后,Mfn2组小鼠肝组织Mfn2 mRNA相对表达量分别为1.00±0.03和1.01±0.053,较HK组（分别为1.14±0.07和1.18±0.07）明显下降（f值分别为4.027和4.234,P值均＜0.01）; Mfn2蛋白分别为7.81±0.80和8.05±0.15,较HK组（分别为8.01±0.08和8.56±0.01）也明显下降（f值分别为2.941和4.883,P值均＜0.05）.注射24 h后,Mfn2组小鼠FBS低于HK组[（2.65±0.70）mmol/L比（5.28±0.82）mmol/L,t=6.879,P＜0.01],TG高于HK组[（1.96±0.32）mmol/L比（1.12±0.16）mmol/L,t=-6.711,P＜0.01）],HK组与NC组之间FBS和TG差异无统计学意义（F值分别为1.412和2.711,P值均＞0.05）;注射72、12h后,Mfn2组小鼠FBS高于HK组（7.23±0.82）mmol/L比（5.18±0.69）mmol/L,t=2.050,P＜0.01;（7.00±0.67）mmol/L比（6.05±0.76）mmol/L,t=3.57,P＜0.05）],血清TG高于HK组,但差异无统计学意义[（1.53±0.27）mmol/L比（1.37±0.18）mmol/L,t=0.160,P＞0.05;（1.84±0.30）mmol/L比（1.52±0.37）mmol/L,t=0.330,P＞0.05）].结论 流体力学介导的RNAi在干扰72、120h后可有效抑制肝脏目的基因的表达,抑制Mfn2表达可导致小鼠葡萄糖和脂肪代谢异常.

Objective To investigate the effects of hydrodynamics-mediated RNAi for Mfn2 gene expression in liver and the levels of blood sugar and fat in mice. Methods Fifty-six male BALB/c mice were randomly divided into normal control group （NC, n = 8）, negative control group （HK, n = 24） and transfection group （Mfn2, n = 24） according to random digits table. 1.5 ml plasmid （negative control or Mfn2 shRNA, 75 μg for each mouse） diluted into phosphate buffered solution （PBS） was injected into the HK and Mfn2 groups mice via hydrodynamic intravascular injection. Mfn2 mRNA and protein expression in hepatic tissue was detected by RT-PCR and Western-blot 24 hours, 72 hours and 120 hours respectively after injection.At the same time, the levels of fasted blood sugar （FBS） and triglyceride （TG） were measured. Results Compared with HK mice, the expressions of Mfn2 mRNA （1.00 ± 0.03 vs 1.14 ± 0.07, t = 4.027, P = 0.007;1.01 ± 0.053 vs 1.18 ± 0.07, t = 4.234, P = 0.006） and protein （7.81 ± 0.80 vs 8.01 ± 0.08, t = 2.941, P =0.042; 8.05 ± 0.15 vs 8.56 ± 0.014, t = 4.883, P = 0.039） decreased markedly in Mfn2 mice in 72 and 120 hours after injection. In the fasting state, in 24 hours after injection, FBS in Mfn2 group was significantly lower than that in HK group [（2.65 ± 0.70 vs 5.28 ± 0.82） mmol/L, t = 6.879, P 〈 0.01] and TG was also significantly higher than that in HK group [（1.96 ± 0.32 vs 1.12 ± 0.16） mmol/L, t = -6.711, P 〈 0.01 ]. No statistical differences found between the NC and HK groups for FBS and TG （F = 1.412, P = 0.26; F = 2.711,P = 0.14）. The plasma glucose level in Mfn2 mice was significantly higher than that in HK mice [（7.23 ±0.82 vs 5.18 ± 0.69） mmol/L, t = 2.050, P 〈 0.01; （7.00 ± 0.67 vs 6.05 ± 0.76） mmol/L, t = 3.57, P = 0.023]in 72 and 120 hours after injection. However, no differences found between the two groups for blood TG [（1.53 ± 0.27 vs 1.37 ± 0.18） mmol/L, t = 0.160, P = 0.23; （1.84 ± 0.30 vs 1.52 ± 0.37） mmol/L, t = 0.330,P = 0.503]. Conclusion The data indicate that hydrodynamics- mediated RNAi for Mfn2 gene can effectively inhibit the expression of target gene in mice liver in 72 and 120 hours after shRNA administration, and the inhibition of hepatic Mfn2 can induce glycometabolic and fat metabolic disorder.