siRNA沉默PPARγ基因对大鼠心肌细胞缺血再灌注致胰岛素抵抗现象的影响

目的观察小干扰RNA（siRNA）沉默过氧化物酶体增殖物激活受体基因（PPARγ）的表达对成年大鼠缺血再灌注损伤（IRI）心肌细胞胰岛素抵抗的影响。方法培养成年大鼠心肌细胞，构建沉默大鼠心肌细胞PPARγ基因特异siRNA，以脂质体介导转染离体培养的大鼠心肌细胞。制备心肌细胞IRI模型。采用RT-PCR检测PPARγ和葡萄糖转运蛋白4（GLUT-4） mRNA表达，Western blot检测PPARγ蛋白表达；同位素示踪法检测细胞葡萄糖（Glu）摄取率变化。结果 IRI模型组心肌细胞PPARγ mRNA及蛋白表达较空白组不同程度增加（P均<0.05）；siRNA-PPARγ组，PPARγ mRNA及蛋白表达量较空载体组和IRI模型组明显下降（P<0.01），空载体组PPARγ mRNA及蛋白表达接近IRI模型组水平；GLUT-4 mRNA表达量空白组各时间点无明显差别，IRI模型组0 min表达量明显降低，之后随着再灌注时间增加逐渐恢复至空白组水平，空载体组GLUT-4 mRNA表达与IRI模型组无明显差别；沉默PPARγ后，细胞GLUT-4 mRNA表达较IRI模型组和空载体组减低更明显（P<0.05），恢复较IRI模型组更慢。在胰岛素刺激下，与IRI模型组比较，siRNA-PPARγ组各时间点Glu摄取率均降低（P<0.05），0 min下降49.78%，15 min、1 h 、2 h分别降低38.94%、18.61%、11.54%，6 h开始恢复至IRI模型组水平。空载体组与模型组比较Glu摄取率无明显差异。结论沉默PPARγ的表达，可加重IRI心肌细胞胰岛素抵抗，其可能的机制是PPARγ基因沉默导致的GLUT-4 mRNA表达下降或转位障碍。

英文摘要:

Objective To observe the influenece of siRNA-mediated PPARγ gene knockdown on insulin resistance induced by myocardial ischemia-reperfusion in adult rats. Method The targeting PPARγ siRNA was synthesized. The myocardial cells of adult rats were isolated and cultured. They were divided into four groups: IRI group, siRNA-PPARγ group, empty group and blank control group. Two groups of rat cardiac cells were transfected with PPARγ-targeting siRNA (siRNA-PPARγ group), or empty small interfering RNA (NC group), respectively. Real-time quantitive PCR was performed to detect the mRNA levels of PPARγ and GLUT-4. PPARγ protein expression level was determined with Western blot test. The uptake rate of glucose was determined by the isotope tracer method. Result The PPARγ mRNA and protein expression of IRI group were significantly higher than those in blank control group (P<0.05). The PPARγ mRNA and protein expression of siRNA-PPARγ group were significantly less than those in blank control and IRI group (P<0.01). There was no significant difference in the PPARγ mRNA and protein expression between the blank group and IRI group. The mRNA expression of GLUT-4 in blank control was no significant difference at each time point. The mRNA expression of GLUT-4 in IRI group was significantly less at 0 min, but increased gradually over the following time point. Finally, The mRNA expression of GLUT-4 in IRI group restored the same level as blank control. There was no significant difference in the GLUT-4 mRNA expression between the empty group and IRI group. The GLUT-4 mRNA expression in siRNA-PPARγ group was significantly less than that in IRI group or NC group (P<0.05), and recovered more slowly than IRI group. After given insulin, The uptake rate of glucose in siRNA-PPARγ group was significantly less at each time point compared with those in IRI group (P<0.05), declined by 49.78%, 38.94%, 18.61%, 11.54% at 0 min, 15 min, 1 h,2 h, respectively. At 6 h time point, the uptake rate of glucose in siRNA-PPARγ group reached the same level as IRI group. There was no significant difference was observed in the uptake rate of glucose between the empty group and IRI group. Conclusion The siRNA-mediated PPARγ gene knockdown may enhance the myocardial insulin resistance. The molecular mechanisms that trigger myocardial cell insulin resistance might because the silence of PPARγ expression decreasing the expression of GLUT-4 and decline its transportation from cytoplasm to membrane.

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