中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2010年
10期
778-782
,共5页
血糖%甘油三酯类%RNA干扰%线粒体融合素基因-2
血糖%甘油三酯類%RNA榦擾%線粒體融閤素基因-2
혈당%감유삼지류%RNA간우%선립체융합소기인-2
Blood sugar%Triglycerides%RNA interference,%Mitofusin-2 gene
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.
目的 觀察流體力學介導的RNA榦擾(RNAi)對小鼠肝髒線粒體融閤素基因-2(Mfn2)、空腹血糖(FBS)和血清甘油三酯(TG)水平的影響.方法 將56隻雄性BALB/c小鼠隨機分為空白對照組(NC組,n=8)、陰性對照組(HK組,n=24)和Mfn2質粒榦擾組(Mfn2組,n=24).HK組小鼠利用流體力學註射75μg陰性對照質粒溶液1.5 ml,Mfn2組小鼠利用流體力學註射75 μg Mfn2 shRNA質粒溶液1.5 ml.應用逆轉錄-聚閤酶鏈反應和Western blot方法分彆測定註射24、72、120 h後,小鼠肝髒Mfn2的mRNA和蛋白質錶達;同時分彆取血測定小鼠FBS和TG水平.多組間比較採用單因素方差分析,兩兩比較採用Scheffe's t檢驗.結果 質粒註射72、120 h後,Mfn2組小鼠肝組織Mfn2 mRNA相對錶達量分彆為1.00±0.03和1.01±0.053,較HK組(分彆為1.14±0.07和1.18±0.07)明顯下降(f值分彆為4.027和4.234,P值均＜0.01); Mfn2蛋白分彆為7.81±0.80和8.05±0.15,較HK組(分彆為8.01±0.08和8.56±0.01)也明顯下降(f值分彆為2.941和4.883,P值均＜0.05).註射24 h後,Mfn2組小鼠FBS低于HK組[(2.65±0.70)mmol/L比(5.28±0.82)mmol/L,t=6.879,P＜0.01],TG高于HK組[(1.96±0.32)mmol/L比(1.12±0.16)mmol/L,t=-6.711,P＜0.01)],HK組與NC組之間FBS和TG差異無統計學意義(F值分彆為1.412和2.711,P值均＞0.05);註射72、12h後,Mfn2組小鼠FBS高于HK組(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組,但差異無統計學意義[(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)].結論 流體力學介導的RNAi在榦擾72、120h後可有效抑製肝髒目的基因的錶達,抑製Mfn2錶達可導緻小鼠葡萄糖和脂肪代謝異常.
목적 관찰류체역학개도적RNA간우(RNAi)대소서간장선립체융합소기인-2(Mfn2)、공복혈당(FBS)화혈청감유삼지(TG)수평적영향.방법 장56지웅성BALB/c소서수궤분위공백대조조(NC조,n=8)、음성대조조(HK조,n=24)화Mfn2질립간우조(Mfn2조,n=24).HK조소서이용류체역학주사75μg음성대조질립용액1.5 ml,Mfn2조소서이용류체역학주사75 μg Mfn2 shRNA질립용액1.5 ml.응용역전록-취합매련반응화Western blot방법분별측정주사24、72、120 h후,소서간장Mfn2적mRNA화단백질표체;동시분별취혈측정소서FBS화TG수평.다조간비교채용단인소방차분석,량량비교채용Scheffe's t검험.결과 질립주사72、120 h후,Mfn2조소서간조직Mfn2 mRNA상대표체량분별위1.00±0.03화1.01±0.053,교HK조(분별위1.14±0.07화1.18±0.07)명현하강(f치분별위4.027화4.234,P치균＜0.01); Mfn2단백분별위7.81±0.80화8.05±0.15,교HK조(분별위8.01±0.08화8.56±0.01)야명현하강(f치분별위2.941화4.883,P치균＜0.05).주사24 h후,Mfn2조소서FBS저우HK조[(2.65±0.70)mmol/L비(5.28±0.82)mmol/L,t=6.879,P＜0.01],TG고우HK조[(1.96±0.32)mmol/L비(1.12±0.16)mmol/L,t=-6.711,P＜0.01)],HK조여NC조지간FBS화TG차이무통계학의의(F치분별위1.412화2.711,P치균＞0.05);주사72、12h후,Mfn2조소서FBS고우HK조(7.23±0.82)mmol/L비(5.18±0.69)mmol/L,t=2.050,P＜0.01;(7.00±0.67)mmol/L비(6.05±0.76)mmol/L,t=3.57,P＜0.05)],혈청TG고우HK조,단차이무통계학의의[(1.53±0.27)mmol/L비(1.37±0.18)mmol/L,t=0.160,P＞0.05;(1.84±0.30)mmol/L비(1.52±0.37)mmol/L,t=0.330,P＞0.05)].결론 류체역학개도적RNAi재간우72、120h후가유효억제간장목적기인적표체,억제Mfn2표체가도치소서포도당화지방대사이상.
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.
