The Construction And The Analgetic Effect Of Recombinant Lentiviral-vector With Human Interleukin-10 Gene

Neuropathic pain is a refractory chronic pain syndrome. Despite many decades of drug development, effective therapies for neuropathic pain remain elusive. Some recent researches consider that spinal cord glia and glial pro-inflammatory cytokines are intimate relevant to the neuropathic pain. Glial activation and its downstream consequences probably will be important targetes of therapy for neuropathic pain. Interleukin-10(IL-10) is a powerful anti- inflammatory cytokines, can suppress many kinds of cells from producing and releasing pro-inflammatory cytokines. Some researches prove that IL-10 has a notable therapeutic efficacy on neuropathic pain of rats (caused by such as spinal injury, chronic constriction injury, intrathecal administration gp 120, etc). Studies indicate that the transduction of analgetic relative gene into central nervous system can show efficient analgetic effect. The replication-defective virus vector has the characters of high efficiency, safeness and stableness in theory, has a satisfied utilizing prospect. Lentiviral vector, as a kind of special retrovirus, originated from the human immunodeficiency virus (HIV), whose greatest character is it can mediate the efficient delivery, integration and stable expression of transgenes in nondividing cells as well as dividing cells, and it can express in vivo very safely for a long time. Thus, we plan to construct recombinant lentivirus vector (Lentivector, LV) which contains human Interleukin-10 (hlL- 10), to observe IL- 10 overexpression after infection both in vitro and in vivo, and its efficiency of controlling neuropathic pain.ObjectiveTo construct the recombinant Lentiviral-vector which contain human interleukin-10 gene(LV/hlL-10). To research activated astrocytes' immunological cell-liked effect and LV/hlL-10's intervention efficiency. To evaluate the analgetic effect of LV/hlL-10 on chronic constriction injured (CCI)pain of rats' sciatic nerve. To investigate whether HMGB 1 contribute to the maintenance of pain facilitation.Methods 1. First compund suitable primer, then amplify hIL-10 gene from pCYIL-10 plasmid and recombinate hIL-10 gene into Lentivector pWPXL-GFP, take the recombinant plasmid pWPXL-hIL-10-GFP,envelope plasmid pMD2.G and packaging plasmid psPAX2 cotransfection in the 293T cells, to pack out lentivirus particle that has the ability of duplicated-deficiency, then undertake virus titer determination.2. Cultivate DI TNC1 (astrocytes cell come from cerebral cortex tissue of rat) and lay into the bottles separately, add Lipopolysaccharide (LPS) of different density (0,250,500,750,1000ng/mL) to DI TNC1, then to determine the delivery of earlier period inflammatory factor TNF-α,IL-1βat 8h, add 500ng/ml LPS to affect on DI TNC1, determining the expression of mRNA and the delivery of protein from earlier period inflammatory factor TNF-α,IL-1βand advanced stage inflammatory factor HMGB1 mRNA in different time cells; transient transfection DI TNC1 with LV/IL-10,LV-GFP, then detect mRNA expression of IL-10; determin mRNA expression and protein delivery of earlier period inflammatory factor TNF-α,IL-1βand advanced stage inflammatory factor HMGB1 caused by LPS after transfection in different time cells.3. 135 sheer breed pathogen-free adult male Sprague-Dawley rats, divided into 9 arrays at random: CCI models 4 arrays (C0,C1,C2,C3), sham rats 4 arrays (S0,S1,S2,S3) and a normal contrast array (N), each respectively intrathecal injection LV/hIL- 10(C1,S1),LV-GFP(C2,S2),isotonic Nachloride (C3,S3) and control (no implanted catheters and no administration, C0,S0), to observe each array's alteration of algesic ethology on day 3,7,14,21,28 after CCI and sham surgery, and the expression of mRNA and protein of IL-10,IL- 1β,TNF-α,HMGB1 in their spinal cord,pallium,seahorse on day 3,7,14 after surgery or administration. After finish measuring pain threshold of no administration CCI array,sham operated array and normal control array on 7d, pick out 3 rats from each array, to detect the expression of GFAP,NF-kB in the rats' spinal cord,pallium,seahorse by immunohistochemical method.Results1. Obtain the IL-10 genic fragment contain a popular site for cloning PmeI from pCYIL-10 plasmid by PCR, recombinate IL-10 gene into pWPXL-GFP, electrophoresis result of enzymetomy appraisement demonstrate that pWPXL-hIL-10 plasmid has a insert part of about 530bp, is in line with IL-10cDNA (534bp). Result of sequencing demonstrate that the series of insert part in pWPXL-hIL-10 plasmid coincidence with hIL-10 gene order. Obtain high titer (2x10¹⁰) and highly purified lentiviral particles.2. Different density LPS activated on DI TNC1cell 8h later, we found LPS of 500ng/ml will make the most notable effection on the delivery of earlier period inflammatory factor TNF-α,IL-1β, after LPS of 500ng/ml affect on the astrocytes(DI TNC1), the mRNA expression of earlier period inflammatory factor TNF-α,IL-1βwill be the most notable at 2h, while the mRNA expression of HMGB1 will be the most notable at 12h, protein expression will reach the peak at 18h, and still maintain in a fairly high level at 24h. HMGB1 protein delivery in the supernatant of medium will be the most notable at 18h, conspicuous IL-10 overexpression will arise at 4h after LV/IL-10 transfection DI TNClcell, IL-10 overexpression will make a notable depressant effect on the mRNA expression and protein delivery of earlier period inflammatory factor TNF-α,IL-1βand advanced stage inflammatory factor HMGB1 of DI TNC1 cell caused by LPS.3. CCI rats show pain facilitation on 3d, most obviously on about 1 week, conspicuous mechanical allodynia (mirror image pain) can be seen in the contralateral hind paw of CCI rats on 7d after surgery. The expression of GFAP,NF-kB in their spinal cord,pallium,seahorse show significant on 7d, expression of TNF-α,IL-1β,IL-10 in spinal cord of lumbar segments show most obviously on 3d,7d after surgery, almost no expression on 14d; the expression of HMGB1 begin to reinforce on 7d, maintain in the fairly high lever on 14d; among the tissues of spinal cord,pallium,seahorse, the expression of indexes measured in this experiment in the spinal cord is of the most significant. Intrathecal injection LV/hIL-10 will manifest relieve mechanical allodynia and thermal hyperalgesia in the CCI array, expression of IL-10 will reach the peak after LV/hIL-10 injection for 3 days, especially in the spinal cord, while the expression of TNF-α,IL-1βin the spinal cord down-regulate mostly, the expression of HMGB1 in the spinal cord down-regulate mostly on the day 7,14, difference is very significant in contrast to the control group.Conclusions1. Succeed in constructing the lentivirus vector: LV/hIL-10, which contains gene of human IL-10.2. Activated astrocytes by LPS have the ability of immunological cell, who can release inflammatory cytokines TNF-α,IL-1βand HMGB1 (the secretion of HMGB1 appear later and maintain longer), LV/hIL-10 can restrain its secretory action.3. Confirmed that the activated astrocytes are critical to the creation and maintenance of chronic neuropathic pain facilitation through the release of inflammatory cytokines, intrathecal injection LV-h-IL10 can efficiently reverse hyperalgesia of CCI rats, which is relevant to the expression ofTNF-β,IL-1β,HMGB1 of the spinal cord.4. HMGB1 may be contribute to the development and maintenance of chronic neuropathic pain, propably HMGB1 will be a new target of treatment of chronic pain.