The Application Of SiRNA In Treatment Of Neuropathic Pain By Inhibiting The Expression Of DREAM Gene

Pain is an unpleasant experience that involves the conscious awareness of noxious sensations, hurting and aversive feelings associated with actual or potential tissue damage. This wide definition recognizes that the experience of pain is modulated by a complex set of emotional, environmental and psychophysiological variables. It is not only a clinical syndrome, but actual a disease. Everyone maybe suffer from pain in all their life. Although pain does not lead to death directly, chronic pain syndromes make some people feeling too painful to live, even suicide. The treatment options for pain are mainly depended on opiate compounds and non-steroidal anti-inflammatory drug (NSAIDs). Even though chronic neuropathic and inflammatory pain can be relieved to some extent with opiate compounds, these agonists of the endogenous opiate receptors which involveμ,δandκopiate receptors also affect cognition, perception, mood, respiration, and gastro-intestinal function, and clinical use ofμagonists leads to tolerance, withdrawal, and addiction. The treatment of chronic neuropathic pain is more difficult than other acute or chronic pain syndromes. Because it is not sensitive to opiate compounds and the efficiency of the tricyclic antidepressants or carbamazepine are also limited.DREAM (Downstream regulatory element antagonist modulator) was identified in vitro studies as a putative transcriptional repressor for the prodynorphin gene, which contains a consensus DNA sequence, or downstream regulatory element (DRE), required for direct association with DREAM. The DRE is important for the active repression of prodynorphin transcription. Because DREAM serves as a "transcriptional swath" for repressing and derepressing endogenous modulators of pain processing, it is regarded that inhibiting the expression of DREAM may active the endogenous dynorphin/κopiate receptors to result in antinociception.With the further studies of RNAi in the last few years, more about its mechanisms has been elucidated. It works by the small interference RNA's specificity silence of the homology genes. The siRNAs combine with corresponding mRNA and made it degradation, and then the translation of mRNA is terminated. Moreover, as a technique to inhibit gene expression, RNAi is becoming more and more perfect in the studies of functional genomics. Because of its high efficiency and specificity, it also provides a powerful tool for studying the functions of genes and gene therapy. Now, some researchers supply the RNAi technique in gene therapy of pain successfully. Their results indicated that intrathecal administration of siP2X3 displayed markedly reduced pain behaviors in models of chronic neuropathic pain with no side-effects. In a word, the RNAi technique has opened up new possibilities for studying the mechanisms of acute and chronic pain and designing new and more selective drugs. The objective of this study is to investigate the gene therapy of neuropathic pain by application of RNA interference by inhibiting the expression of DREAM gene. Our study includes three parts. The first one is to confirm the efficient position of RNAi of DREAM gene and observe the result after RNAi. Firstly, we design three siRNA sequences for DREAM gene by software of online web, and then, we construct three plasmids of siRNA, which are based on pRNAT vector. Lastly, we transfer the plasmids into neuron cells (C6 cells) instantly by liposome and detect the contents of DREAM mRNA and protein by Realtime-PCR and Western blot analyses respectively. We find two efficient RNAi positions of DREAM. The mRNA levels are down-regulated by 59% and the expression of protein is also down-regulated in some extent. The second one is to construct Lentivectors of siRNA which can work stably in neuron cells with information from the part one. At first, we construct the plasmids on the base of Lentivirous vectors, pKCSHR-Puro/GFP, and then, both of the pKCSHR-Puro/GFP-DREAM and Lentivector package plasmids mix are transferred into the 293T cells together. We detected the concentration of the virus after transferring 48 hours. In conclusion, we construct pKCSHR-Puro/GFP-DREAM successfully and obtain higher concentration lentivirus which can express siDREAM stably in neuron cells. The concentration of the lentivirus is 1×10~8 ifu/ml. The last one in our research is to test the efficiency of RNAi of DREAM in mice. We expect the test in mice can help the application in human body in the future. We assessed the pain behavior and detected contents of DREAM mRNA and protein by Realtime PCR and Western Blot analysis respectively in the model of neuropathic pain (chronic constriction of the sciatic nerve, CCI) that mimics peripheral nerve injury in humans. Intrathecal administration of pKCSHR-Puro/GFP-DREAM after CCI can reduce pain behaviors markedly, and the levels of DREAM Myrna is down-regulated by 55% and the expression of protein of DREAM is also decreased in same extents.At last, we come to some conclusions: 1. we designed siRNA sequence of DREAM and constructed the plasmids of siRNA which were transferred into neuron cells effectively. The efficiency of transferring was 85%. 2. We confirmed the effective siRNAs sequence of DREAM successfully. The effective siRNAs of DREAM gene can down-regulated mRNA level of DREAM gene and depress the express of DREAM protein. 3. We constructed and packaged the pKCSHR-Puro/GFP-DREAM successfully and obtained higher concentration of lentivirus. 4. We established neuropathic pain model by constricting of sciatic nerves. 5. The in vivo test indicated that intrathecal administration of pKCSHR-Puro/GFP-DREAM can attenuate pain behavior and down-regulate DREAM mRNA and protein of DREAM in neuropathic pain model in mice.