Construction Of GRIN2B ShRNA And Its Gene Silencing Effects Of NR2B In Mouse

Objective:1. To design and construct short hairpin RNA(shRNA) eukaryotic expression plasmids targeting the gene of N-methyl-D-aspartate(NMDA) receptor NR2B subunit(GRIN2B shRNA) and identified.2. To investigate the intrathecal injection of polyethylenimine (PEI)-mediated shRNA in interfering the expression of NR2B gene and explore the feasibility of chronic pain treatment by using GRIN2B shRNA to block the expression of NR2B gene.Methods:1. According to NMDA receptor NR2B subunit mRNA coding sequence, the specific RNA interference(RNAi) fragments targeting the gene of NR2B subunit were designed and synthesized, which were cloned into pYr-1.1plasmid vector, and the shRNA eukaryotic expression vector pYr-1.1-GRIN2B-shRNA targeting the gene of NR2B subunit was constructed,and then identified by restriction analysis and sequencing test.2. The60male C57BL/6mice were equally and randomly assigned to four groups:control group (no treatment; n=15), normal saline(NS) group (intrathecal injection of10μL NS; n=15), scrambled shRNA(Scr) group (intrathecal injection of10μL scrambled shRNA; n=15), PEI-shRNA group (intrathecal injection of10μL PEI-GRIN2B shRNA complex; n=15).Pain behavior was observed after the dorsal surface of the mice injected with50μL of1%formalin solution on the7th day after the intrathecal injection, and L4-6spinal cords were separated for PCR, western blotting or immunohistochemistry to analyze the location and expression levels of the NR2B.Results:1. The results of restriction analysis and sequencing test showed that NMDA receptor NR2B subunit shRNA was successfully inserted into the plasmid vector pYr-1.1.2. The observation of the pain behavior:The nociceptive response of the mice in I phase (the acute phase) was indistinguishable among all groups(p>0.05); while in II phase (the tonic phase) the nociceptive response of PEI-GRIN2B shRNA group was decreased significantly as compared to the three other groups (p<0.05). There was no significant difference between control group, NS group and Scr group (p>0.05).3. The results of NR2B mRNA and protein expression in the spinal dorsal horn:The NR2B mRNA and protein levels were significantly decreased in PEI-GRIN2B shRNA group as compared to the three other groups (p<0.05), while there were no significant differences among control, NS and Scr groups.4. The results of immunohistochemistry:the NR2B subunit was mainly located at the superficial dorsal horn in L4-L5spinal cord. There were no significant differences among control, NS and Scr groups (p>0.05), while the NR2B expression levels decreased significantly in the PEI-GRIN2B shRNA group as compared to the three other groups(p<0.05).Conclusion:1. The pYr-1.1-GRIN2B-shRNA plasmid was constructed successfully, which lay the foundation for the NR2B as an analgesic target in gene therapy.2. The intrathecal delivered PEI-GRIN2B shRNA can significantly inhibit NR2B expression in the spinal dorsal horn.3. PEI-mediated GRIN2B shRNA could relieve formalin-induced nociception in tonic phase. This furnished further evidence that NR2B plays an important role in maintaining and central sensitization of pain, which provides new methods and ideas for the treatment of pain.