shRNA干扰蓝斑核去甲肾上腺素转运体(NET)表达对大鼠抑郁样行为的改善作用

shRNA targeting norepinephrine transporter in the locus coeruleus attenuates depression-like behavior of rats

目的:探讨利用慢病毒在蓝斑核(locus coeruleus,LC)敲除去甲肾上腺素转运体(norepinephrine transporter,NET)对大鼠抑郁样行为的影响。方法:构建NET-shRNA慢病毒载体及阴性对照序列,感染PC12细胞后通过Q-PCR和Western Blot方法测定NET mRNA和蛋白水平;建立慢性不可预见性温和应激大鼠抑郁症模型,双侧蓝斑核注射NET-shRNA;3周后采用强迫游泳实验、糖水偏好实验、旷场实验检测大鼠抑郁样行为;Western Blot检测大鼠NET水平;高效液相色谱法(HPLC)检测大鼠去甲肾上腺素(norepinephrine,NE)水平。结果:Q-PCR和Western Blot检测结果显示:shRNA-1146沉默效率最高(P<0.01);与抑郁症模型组相比,蓝斑核注射NET-shRNA组大鼠强迫游泳不动时间减少、糖水偏好率增加、旷场实验得分增加(P<0.01),蓝斑核NET表达下降(P<0.01)、NE水平升高(P<0.05)。结论:NET-shRNA慢病毒注射蓝斑核能下调NET的表达,改善大鼠抑郁样行为,为抑郁症的基因治疗提供了实验依据。

Objective： To investigate the effect of lentivirus-mediated shRNA targeting norepinephrine transporter （NET） in locus coeruleus on depression-like behavior of rats. Methods： Recombinant lentiviral vector expressing shRNA against NET and nonsense shRNA as negative control were produced, and their efficiency in knocking down of NET in PC12 cells were assessed by Q-PCR and Western Blot analysis. Furthermore, shRNA were injected into the rat locus coeruleus bilaterally to investigate whether it can prevent the depressive-like behavior induced by chronic unpredictable mild stress （CUMS）. 3 weeks later, rats were tested for depressive-like behavior in the forced swimming test, the sucrose preference test and the open field test; the protein expressions of NET were measured by Western Blot; the levels of NE were measured by high performance liquid chromatography （HPLC）. Results： Q-PCR and Western Blot showed that the ShRNA-1146 has the best interference efficiency targeting on NET （ P 〈 0.01 ） ; Compared with the depression model group, the immobility time in the forced swimming test decreased significantly （P 〈 0.01 ） , the sucrose preference in the sucrose preference test and the total scores in the open field test increased significantly （P 〈0.01 ） in the group treated with shRNA in locus coeruleus; compared with the depression model group, NET levels decreased significantly （P 〈 0.01 ） and NE levels increased significantly （ P 〈 0.05 ） in the group treated with shRNA in locus coeruleus. Conclusion ： Lentivirus-mediated shRNA targeting NET in locus coeruleus downregulated NET and led to a significant reduction in depressive-like behavior of rats, which provide evidience for further translational studies.