Construction Of Pires-gfp-hpenk And Its Expression Of Intrathecal Injection

ObjectiveGene therapy of pain is considered as an effective and safe treatment,and it has become the treatment of hot spots. And its key aspect is to choose a valid target gene and the vector. Enkephalin (enkephalin, ENK) is a kind of endogenous opioid peptides. Compared with other opiates, the much superiority of its analgesic has been confirmed. Thus, the human preproenkephalin gene (human preproenkephalin gene, hPENK), as the controlling gene of enkephalin expression has become a relatively ideal selection of gene therapy for chronic pain. It is a commonly research tools that virus vector carrying PENK express ENK by transfecting cells or injecting directly, but its clinical application is limited because of the virus self-replication and human anti-viral immune response. To further study about the PENK and the ENK controlled and synthesised by PENK, we constructed the recombinant plasmid pIRES-GFP-hPENK,which was highly efficient expression, low toxicity and immunogenicity.And we studied it by two aspects.In vitro experiments , we transfered the recombinant plasmid into NIH3T3 cells,and admixtured the supernatant of the NIH3T3 cells and the neuronal cells ,and observed the change in ion channel k of the neuronal cells. In vivo experiments,we injected the recombinant plasmid into the subarachnoid space in rats to observe the expression of location and timeliness and its secretions enk. This experiment will provide a more adequate experimental data for genetic research and application of analgesic.Methods1. Construction of pIRES-GFP-hPENK eukaryotic expression plasmid The target gene in the recombinant plasmid T-hPENK was amplified by PCR.And then the target gene was connected with the corresponding pair of large fragments of pIRES-GFP vector digested. Then the new plasmid was transfected into E.coli JM109 strain with Amp screening. The positive colonies was selected to extract plasmid and to accredit by double digestion.2. In vitro experimentsThe recombinant plasmid pIRES-EGFP-hPENK transfected the natural NIH3T3 cells.Then the supernatant of the stable positive cloned cells was added to the original generation of neurons synchronized culture.And the changes of ion channels of K were detected by patch-clamp technique.3.In vivo experimentsThe recombinant plasmid pIRES-EGFP-hPENK was injected into the subarachnoid space in rats to observe the space-time effects experiments and the role of their products after transfection in vivo. Group A (to observe the space-time effects ): a total of 35 male SD rats. The rats in experimental group (n = 21) were divided into seven sub-groups (24 hours, 1,2,3,4,5,6 weeks groups ,3 rats each group) .The rats in control group(n = 14) were received no treatment. Group B(to observe the role of their products): a total of 24 male SD rats.The experimental group was divided into four sub-groups (1,2,3,4 week group,3 rats each group), and the control group(n = 12) was not treated. The experimental group was injected 100μl plasmid into the subarachnoid space in rats by the respectively correspronding plasmid(density:30μg/100ml). Immunohistochemical technique was used to observe the expression of ENK in the spinal cord dorsal horn of rats.Results1. Eukaryotic expression plasmid pIRES-EGFP-hPENK was successfully constructed and transfected into NIH3T3 cells. And the sequences of its clones were fully consistent with GeneBank rereported sequences.2. The new eukaryotic expression plasmid transfected the NIH3T3 cells ,and they secreted ENK.Patch-clamp results show that ENK caused the efflux of k in neuronal cell to augment conspicuously.3. The recombinant plasmid pIRES-GFP-hPENK injected in the subarachnoid space.Green fluorescence was observed after 24 hours,and it reached the expression peak between 7 and 14 days ,decreased significantly after 4 weeks and disappeared at the sixth week. Compared with the experimental group ,the control group was not found the green fluorescence. Experimental group was observed the spinal cord injury and the animal behavior changes.4. Immunohistochemistry showed that the expression of ENK in experimental group at each time point was significantly higher than the control group(P< 0.01).The expression amount of ENK was the highest at the second week in two groups(P<0.01), and it began to decrease significantly at the fourth week(P<0.01).Conclusions1. The product of natural cells transfected by PIRES-GFP-hPENK can enhance the outflow of k ions in the surface of neuronal cells.2. The pIRES-GFP-hPENK can be directly transfected into the brain meninges and spinal nerve membrane by intrathecal injection.The expression of ENK controlled by hPENK can be observed as long as 4 weeks.