The Effect Of Analgesia And Immunologic Modulation By HSV-I Amplicon Vector Transferring HPPE To Rats CNS

Treatment of chronic pain, particularly of neuropathic etiology, is extremely difficult and resistant to many available pharmacologic therapies. Current analgesic agents may be limited with regard to analgesic efficacy or side effects.Newer and experimental pharmacologic agents may also have significant limitations. By targeting a specific receptor or other specific protein targets, a gene therapy approach to the treatment of pain may provide greater analgesic efficacy without the limitations associated with current pharmacotherapy. In theory this method has the characters of high efficiency, safeness and stableness.Advances in the field of gene therapy, along with significant increases in our understanding of the neurobiology of nociception and knowledge of the fundamental genetic structure of many nociceptive targets, have made gene therapy for the management of pain a conceivable reality. In our study we test the possibility that gene transfer therapy might be further developed as a useful treatment for pain. In our study evaluate the effect antinocicepition and immune function of HPPE gene transferred into rat CNS by Herpes Simplex Virus type I amplicon vectorObjective: Targeted gene delivery using HSV-based vectors offers a means to utilize peptides to produce specific effects in the nervous system.Neurotropic HSV- I amplicon vector was adopted to transfer the Human proenkephalin gene(HPPE) into rat subarachnoid space or PAG neural cells that whose product has strong analgesic effect. The lacZ gene in the vector and the foreign HPPE expression were investigated to demonstrate whether or not this gene therapy transferred by HSV- I may be effective for radiant heat hot pain and formalin pain , and to evaluate the effect of immune function of HPPE gene transferred into rat CNS .Methods: The HPPE gene fragment was cut down from pCMVHPPE plasmid and recovered. Then the ligation of HPPE fragment and HSV-1 amplicon vector pHSVIRES-lacZ(SHZ) were done to construct a recombinant plasmid pHSVIRES-HPPE-lacZ(SHPZ). The HSV-tsk was used to help the recombinant plasmid pachaged and amplified in BHK-21 cells. The amplicon virus titer and the expression of lacZ gene were examined by X-gal staining. The HPPE expression was detected by the method of RT-PCR and radioimmune assay, rats were intrathecally delivered with recombinant plasmid Sffi^ SHZ or NS and were injected periaqueductal gray ( PAG) by stereo-orientation microinjection. 5% formalin 50ul was injected into left hindpaw, pain intensity scoring (PIS) was utilized to assess antinociceptive effect of morphine. To investigate the effect The paw withdrawal latency (PWL) induced by radiant heat was measured weekly for 5 weeks or 6 weeks. Spleens were aseptically removed to obtain splenic cells. T lymphocytefunction was evaluated based on Concanavalin-A(ConA) induced splenocyte proliferation. A modified lactic acid dehydrogenase release assay to assess NK cell activity. And phenotypic expression of cell surface markers of T lymphocyte subsets (CD3+ > CD3+CD4+ -> CD3+CD8+ , CD4+/CD8+) and NK cell(CD161+) in the spleen were analyzed by flow cytometry.Results: The construction of recombinant SHPZ was comfirmed by digestion with restriction enzyme. The BHK-21 cells imfected by amplicon virus had obvirously morphological changes and beta-galactosidase expression. The amplicon virus titers were determined at 8xl05pfu/ml. After in vivo transferred into neurocyte demonstrated strong positive sigals with X-gal immunohistochemical staining. RT-PCR and L-enkephalin radioimmune assay found that the neural cells transferred foreign gene(HPPE) has effective expression.The results showed that the PWL was not affected by intrathecal delivery of vehicle or SHZ compared with the PWL before intrathecal delivery.However, intrathecal delivery of SHPZ showed antinociceptive effects on basal hot nociceptive response.The antinociceptive effect of SHPZ reached peak 3 week after intrathecal delivery and could be maintained for 5 weeks. Meanwhile, PAG delivery of SHPZ also showed antinociceptive effects on basal nociceptive response.The antinociceptive effect of SHPZ reached peak 2 week after intrathecal delivery and couldbe maintained for 6 weeks. Intrathecal delivery of SHPZ showed antinociceptive effects on formalin induced pain for 4 weeks compared with SHZ; PAG delivery of SHPZ showed antinociceptive effects on formalin induced pain for 5 weeks.After Intrathecal or PAG delivery SHPZ. SHZ or NS for 1 week, There were not significantly changes between three groups about spleen indexes. There were significantly changes between SHPZ group and SHZ groups about T lymphocyte proliferation. NK cell activity. CD3+CD4+. CD3+CD8+ . CD4+/CD8+and CD161+. splenocyte proliferation induced by ConA and NK cell activity were significantly suppressed by intrathecal pumping morphine (50ug/h); There were significantly changes between SHPZ. SHZ group and control groups about T lymphocyte proliferation . NK cell activity. CD3+. CD3+CD4+. CD3+CD8+and CD161+,but no effect to CD4+/CD8+.Conclusions: We have constructed a recombinant HSV- I amplicon vetor SHPZ.The amplicon virus which pachaged and amplified with the helper virus HSV-tsk has high infective ability. This amplicon virus can transfer HPPE into rat CNS neural cells and make it express efficiently,suggest SHPZ is satisfactory treatment tools for gene therapy of chronic pain; Intrathecal or PAG delivery SHPZ demonstrated antinociceptive effects on formalin induced pain and basal hot nociceptive response for several weeks;The expression product of HPPEtransferred by HSV- I amplicon vector had no effect on immune function while produce significant antinociceptive effect,but to promote immune function. Our study could facilitate and intensify further research in the field of gene therapy of chronic pain.