ABSTRACT
<p><b>BACKGROUND</b>NR2B containing N-methyl-D-aspartate (NMDA) receptor plays an important role in the facilitation and maintenance of neuropathic pain. The discrete distribution of NR2B subunit in the central nervous system (CNS) may support reduced side effects of agents that act selectively at this site. Therefore, we investigated the hypothesis that a humoral autoimmune response targeting the NR2B subunit of NMDA receptor relieves pain like behaviours produced by peripheral injury.</p><p><b>METHODS</b>Rats were immunized subcutaneously with NR2B-Keyhole Limpet Hemocyanin (NR2B-KLH) three times at two-week intervals. NR2B specific IgG titres in sera and cerebrospinal fluid were determined by indirect ELISA. Seven days after the third immunization, 2 of the 3 terminal branches of the sciatic nerve (tibial and common peroneal nerves) were tightly ligated. Behavioural testing was carried out on every other day after surgery, until 7 days after surgery. The lumbar spinal cord (L4-6) was removed on day 7 after ligation. The expression of NR2B protein in the lumbar spinal cord was determined using Western blotting.</p><p><b>RESULTS</b>After the second vaccination, NR2B specific IgG in sera was detected to be > 0.5 microg/ml in six of nine rats. After the third vaccination, all the immunized rats had > 2.2 microg/ml. Titres of NR2B specific IgG in sera peaked 42 days post initial immunization and persisted for over 70 days. No NR2B specific IgG was detected in sera from PBS or KLH group. The behavioural thresholds in NR2B group were significantly higher than those in PBS and KLH groups on day 7 after ligation. NR2B specific IgG in CSF in NR2B group could not be detected on day 1 before spinal dissection; but could be detected on day 7 after surgery. The expression of NR2B protein in group NR2B was significantly lower than in PBS and KLH groups on day 7 after surgery.</p><p><b>CONCLUSION</b>The NR2B peptide could be used as a vaccine against neuropathic pain, which could be associated with reduction of NR2B protein in the lumbar spinal cord.</p>
Subject(s)
Animals , Female , Rats , Adjuvants, Immunologic , Blotting, Western , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Hemocyanins , Allergy and Immunology , Immunoglobulin G , Allergy and Immunology , Neuralgia , Allergy and Immunology , Pain Measurement , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate , Allergy and Immunology , Metabolism , Recombinant Fusion Proteins , Allergy and Immunology , Spinal Cord , Metabolism , Time Factors , Vaccines , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To provide a sound cell source for further ex-vivo gene therapy for chronic pain, we attempt to develop an immortalized rat astrocyte cell line that expresses enkephalin regulated by doxycycline.</p><p><b>METHODS</b>Retrovirus infection method was employed to develop an immortalized rat astrocyte cell line that could express enkephalin regulated by doxycycline. The hPPE gene expression level of immoralized astroyte cells (IAC)/ hPPE was detected by RT-PCR, indirect immunofluorescence staining and radioimmunoassay.</p><p><b>RESULTS</b>IAC carrying Tet-on system transfected with preproenkephalin gene could secrete enkephalin that was regulated by doxycycline in a dose-dependent manner and hPPE gene activation could be repeated in on-off-on cycles through administration or removal of doxycycline.</p><p><b>CONCLUSION</b>An immortalized rat astrocyte cell line that secrete enkephalin under the control of doxycycline is established successfully, which provides a research basis for transgenic cell transplantation for analgesia.</p>
Subject(s)
Animals , Rats , Anti-Bacterial Agents , Pharmacology , Astrocytes , Cell Line, Transformed , Chronic Disease , Doxycycline , Pharmacology , Enkephalins , Genetics , Metabolism , Genetic Therapy , Methods , Genetic Vectors , Neurotransmitter Agents , Genetics , Metabolism , Pain Management , Protein Precursors , Genetics , Metabolism , Retroviridae , GeneticsABSTRACT
Objective To construct an immortalized rat astrocyte strain expressing enkephalin which can be regulated by doxycycline.Methods pRevTet-On and recombinant plasmid pRevTRE/hPPE were transfected into packaging cell PT67 respectively by standard lipofectamine methods.The supernatant containing RevTet-On virus was used to infect immortalized rat astrocyte strain(IAST)and RevTRE/hPPE virus was used to infect the IAST/Tet-On cell.Immortalized rat astrocyte strain that stably expressed Tet-regulated enkephalin was established. hPPE gene expression level of IAST/Tet-On/hPPE cell strain was detected by real time-PCR,immuno- cytochemistry and radio-immunoassay.Results Real time-PCR,immuno-cytochemistry and radio-immunoassay confirmed the level of enkephalin expression was regulated by doxycycline in a dose-dependent manner.Conclusion An immortalized rat astrocyte strain which secrets enkephalin as controlled by doxycycline has been constructed successfully.