The Construction Of Human NGF-Beta Recombinant Adenorivus Vector And Expression And Biological Activity In Astrocyte As Well As In CCI Rats

Objective To construct a recombinant adenovirus vector containing human beta-nerve growth factor gene(h β NGF) by the homologous recombination in bacteria. to prepare investigating the h β NGF gene therapy in chronic pain. Methods NGF gene was digested from pBLAST44-h β NGF v02,subcloned into plasmid of pBluescript II sk(+) and formed plasmid of pBluescript II sk(+)-hNGF β .Then hNGF β gene was digested from plasmid of pBluescript II sk(+)-hNGF β ,subcloned into shuttle plasmid of pShuttle-CMV and formed transfer plasmid of pShuttle-CMV-hNGF β .Adenovirus genomic plasmid of pAdEasy-1 was transformed into BJ5183 bacteria and prepared ultracompletent BJ5183 containing pAdEasy-1. Results The linealinzed pShuttle- CMV-hNGF β was transformed into ultracompletent BJ5183 containing pAdEasy-1 .There were 30% positive recombinant plasmid. PCR test indicated that the recombinant adenovirus plasmid pAdEasy-1-hNGF β contained NGF gene. Conclusion The homologous reconbiation in bacteria is a convenint and efficient method to prepare recombinant adenovirus plasmid pAdEasy-1 -hNGF β .This offers a good gene transfer vector for the gene therapy in pain management.Part IIInfection of astrocyte from dorsal horn with recombinant adenovirus containing human NGF-beta gene and theiridentificationObjiective: The astrocyte was cultured from neogenesis SD Rat spinal cord in vitro, then exploring whether it can act directly as a gene target cell which can be infected by the recombinant adenovirus vector. And cell toxic action and cytoactive were observed to evaluate the expression of exogenous gene after infection Ad-hNGF β gained from Part I . Methods: The astrocytes from neogenesis SD Rat spinal cord were mechanically dissociated, and the DMEM/F-12 medium was adopted to primary culture and passaged the cells, and then identified by GFAP (glial fibrillary acidic protein) immumofluorescence method. The recombinant Ad-hNGFβ was transfected into AST cells at a MOI of 100. Immunohistochemistry staining was applied to determine the expression of exogenous genes after three days. ELISA methods were employed to identify the expression of NGFβ in astrocyte at 3^th day 6^th day 9^th day. The activety of astrocyte to the Ad-hNGFβ was determined by MTT(dimethylthiasol, mithiazoly blue)method. And cell cycle distribution was analyzed using flow cytom. Results The astrocytes from neogenesis SD Rat spinal cord were successfully cultured, which showed positive activity to GFAP. The expression of NGF β were detected by immunohistochemistry staining , there were significant difference between control group and testing group. ELISA methods revealed that the productions of exogenous genes were higher in testing group than control group(P <0.01). The productions of NGF β were gradually decreased with the extension of time, the quantity of the ninth day was lower than that in third day and in sixth day(P<0.01). MTT method revealed that the OD₅₇₀ of testing group was higher than that of control group(P<0.01). Conclusions The dosal horn astrocytes can be infected directly by the recombinant adenovirus containing hNGF-belta gene and the apex time of the expression was about the third day. There was no obvious cell toxic action and it could elevate the cytoactive at a MIO of 100.Part IIIThe effect of Intrathecal injection of Ad-hNGF β on neuropathic pain in rats following chronic constriction injury ofthe sciatic nerveObjective: We investigated the influence of intrathecal injection of Ad-hNGF β on neuropathic pain by chronic constriction injury (CCI) of the sciatic nerve. Methods: Make CCI model rats, then administration of Ad-hNGF β (group I ) and artificial cerebral-spinal fluid ACSF(group II), Ad-hNGF β and ACSF was administered respectively by the intrathecal injection after sciatic nerve constriction injury. Record the responsiveness to mechanical and thermal stimuli before and after Infusion of Ad-hNGF β or ACSF. Determine hNGF β ,Substance P and calcitonin gene-related peptide(CGRP)immunoreactivity. Results: with Ad-hNGF β or ACSF injection, significantly increased responsiveness to mechanical and thermal stimuli and significantly increased pain behavior was observed on postoperative days after sciatic nerve ligation compared with before surgery. With Ad-hNGF β injection, significantly increased hNGF β in the spinal cord, and decreased the responsiveness to thermal stimuli and pain behavior than that of ACSF injection, in this case, SP decreased significantly in the spinal cord, CGRP was not significantly changed in the spinal cord. Conclusion: Intrathecal injection of Ad-hNGF β in CCI rats attenuated the neuropathy-induced warm hyperalgesia and pain behavior. This effect could be related to the increase of hNGFβ and decrease of SP in the spinal cord. We demonstrate that Intrathecal injection of Ad-hNGF β could be a useful therapy for patients suffering from neuropathic pain.