SiRNA干扰慢性炎性痛大鼠患侧背根神经节MrgC表达及PKCε磷酸化的研究
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国家自然科学基金资助项目(81202755);浙江省医药卫生科技计划(2015KYA172)。


SiRNA interference on expression of MrgC and phosphorylation of PKCε in ipsilateral dorsal root ganglion of rats with chronic inflammatory pain
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    摘要:

    目的 观察鞘内注射小干扰RNA(small interference RNA, siRNA)对完全弗氏佐剂(complete freund's adjuvant, CFA)诱导的慢性炎性痛大鼠患侧背根神经节(dorsal root ganglion, DRG)中MrgC(Mas-related G protein- coupled receptor C, MrgC)mRNA与蛋白表达的干扰作用,并观察该作用对大鼠患足痛阈及患侧DRG PKCε丝氨酸729点位磷酸化 (phosphorylation of PKCε Ser729, p-PKCε Ser729)水平的影响。 方法 健康雄性SD(Sprague-Dawley,SD)大鼠16只,随机分为对照siRNA组,MrgC siRNA组,每组8只。大鼠脊髓鞘内插管成功后,两组大鼠给予相应药物鞘内注射4 d,1次/d,5 μg/d/只。给药第4 d,大鼠右后足底注射CFA 0.1 mL建立慢性炎性痛模型,此后隔日注射药物,直至给药第11 d处死。分别于鞘内置管前、给药前、给药4 d(CFA造模0 h)、给药5 d(CFA造模24 h)、给药11 d(CFA造模7 d)5个时点检测大鼠患足机械缩腿阈(Paw withdrawal thresholds, PWTs)的变化。荧光定量PCR法检测患侧DRG MrgC的mRNA的表达,免疫荧光法检测患侧DRG MrgC表达量及p-PKCε Ser729的含量。 结果 与给药4 d比较,给药5 d两组大鼠的PWTs均有显著的下降(P<0.01);给药前后各时点,两组大鼠之间PWTs没有明显差异。观察给药11d时大鼠患侧L4-L6 DRG MrgC mRNA的表达,与对照siRNA组比较,MrgC siRNA组各神经节MrgC mRNA的表达均明显下降(P<0.01);观察大鼠给药11d时大鼠患侧L4-L6 DRG MrgC与p-PKCε Ser729的表达,与对照siRNA组比较,MrgC siRNA组患侧DRG的MrgC阳性细胞率明显减少(P<0.01),而p-PKCε Ser729的阳性细胞率显著上升(P<0.05)。 结论 MrgC siRNA片段可有效干扰CFA慢性炎性痛大鼠患侧L4-L6 DRG MrgC mRNA与MrgC的表达,对MrgC的干扰作用能显著上调PKCε Ser729磷酸化的水平,但不影响大鼠患足机械缩腿阈。

    Abstract:

    Objective To observe the Interference effects of siRNA(small interference RNA) intrathecal injection on the expression of mRNA and protein of MrgC, on PWTs (paw withdrawal thresholds) and the phosphorylation of PKCε Ser729 in ipsilateral DRG(dorsal root ganglion) of rats with chronic inflammatory pain induced by CFA(complete freund's adjuvant). Methods 16 health adult male SD (Sprague-Dawley) rats were randomly divided into 2 groups: control siRNA group and MrgC siRNA group, 8 rats in each group. After success of intrathecal catheterization, corresponding siRNA was injected in rats for 4d, once a day, 5μg/d per rat. The model of chronic inflammatory pain was established by CFA (0.1ml per rat) subcutaneously injected into the right hind paw at 4th day post-administration, then two groups were administrated corresponding siRNA on alternate day and executed at the 11th day post-administration. The PWTs were measured at 5 time points of pre-intrathecal catheterization, pre-administration, 4th day post-administration(0h post-CFA injection), 5th day post-administration(24h post-CFA injection), 11th day post-administration(7 d post-CFA injection). The expression of MrgC mRNA in ipsilateral DRG was detected by fluorogenic quantitative PCR, and the expression of MrgC and the phosphorylation of PKCε Ser729 in ipsilateral DRG was detected by immumofluorescence method. Result Compared with 4th day post-administration, PWTs of both two groups at 5th day post-administration decreased significantly (P<0.01). While there was no significant difference of PWTs between two groups at every detective time point. Compared with control siRNA group, the expression of MrgC mRNA and the rate of MrgC positive cells in MrgC siRNA group both decreased significantly (P<0.01,P<0.05), whereas the rate of p-PKCε Ser729 positive cells increased obviously (P<0.05) at 11th day post-administration. Conclusion MrgC siRNA can effectively interfere the expression of mRNA and protein of MrgC in L4-L6 ipsilateral DRGs of rats with chronic inflammatory pain induced by CFA, and the siRNA interference on MrgC can obviously up-regulate the phosphorylation of PKCε Ser729, while it has no significant effect on PWTs of rats.

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林小溪,方芳,方剑乔,刘盈君. SiRNA干扰慢性炎性痛大鼠患侧背根神经节MrgC表达及PKCε磷酸化的研究[J].中国比较医学杂志,2015,25(7):39~45.

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  • 最后修改日期:2015-07-02
  • 在线发布日期: 2015-07-28
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