Dhepo

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  • Publicado : 11 de diciembre de 2010
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Introduction
Herpes simplex virus is a member of the herpesviridae family. This family consists of 25 know herpes virus which only 8 are know to infected human. Herpes simplex virus has a very large genome that encodes of at least 80 proteins and has 150,000 base pairs and can accommodate 30-40 kilo base paris of foreign genetial material (Latchman, 2000). Is because of this capacitiy of theherpes simplex virus is being use as a vector (Zhu, Song, Stroud, & Parris, 2008) (Latchman, 2000) (Chattopadhay, Mata, & Fink, 2009). In order to use the herpes virus as a vector with out causing the disease, the virus was modified by removing the gene that code of the lyric cycle of the virus ( ICP4 or ICP27) (Latchman, 2000). And other advantages of is it capacities to infected cell and itreproduction capacities including neurons (Latchman, 2000). Will uses the herpes simplex virus as a vector to introduce Erythropoietin (EPO) to diabetic nueropathy model to the dorsal root ganglia (DRG). EPO was originally characterized for its haematopoietic effects, but in recent years has been shown to have important functions in the nervous system. Non-erythropoietic actions of EPO include acritical role in the development and maintenance of neural structure (Juul SE, 1998) (Marti HH, 2000), and protection from or enhanced repair after injury to the nervous system (Campana WM M. R., 2003) (Toth C, 2008)Erythropoietin confers protection against development or improvement of peripheral neuropathies associated with diabetes (Bianchi R, 2004), HIV (Keswani SC, 2004) and ageing (Lauretani F,2008). Study with the transduction of DRG neurons by subcutaneous inoculation of an HSV vector expressing EPO can prevent the progression of diabetic neuropathy in a mouse model (Chattopadhay, Mata, & Fink, 2009). While in vitro studies with vascular endothelial cells, exposed to elevated glucose, have elucidated a strong cytoprotective effect of EPO. Administration of EPO can significantlyimprove endothelial cell survival in a very low dose (Chong ZZ, 2007).
Methods
Vector DHEPO was constructed by insertion of a cassette consisting of the human cytomegalovirus into the immediate early promoter (HCMV IEp) and the human EPO coding sequence into the UL41 locus of a nonreplicating HSV vector defective in expression of the immediate early genes ICP4, ICP27 and UL41. Control vector DHZ.5 wasconstructed by insertion of a cassette consisting of the human cytomegalovirus into the immediate early promoter (HCMV IEp and the reporter gene lacZ
After the creation of the DHEPO vector, mice were use as model of study. Fifty Swiss Webster mice weighing 25-30 g were use following compliance with approved institutional animal care and use protocols. Thirty mice were rendered diabetic byinjection of ST3 beta-galactoside alpha-2,3-sialytransferase 4 (STZ) (100 mg/kg twice in 48 h; Sigma-Aldrich, St Louis, MO, USA). Two weeks after STZ blood glucose was 29_0.72 mmol/l for the twenty diabetic animals and 8.3_0.5 mmol/l ten control mice. Two weeks after STZ administration, subsets of 15 diabetic mice received 10 ml containing 1x〖10〗^7 plaque forming units (pfu) of HSV-based therapeuticvector DHEPO or control vector DHZ.5 subcutaneously into each footpad. Four weeks after the vector inoculation and 6 weeks after STZ-induction, the neuropathy o the mice was measure by electrophysiological and behavioral parameters. The nerves condition was measure on the left hind foot of all mice using the Nicolet Viking II. In order to acquire exact results, the mice were anaesthetized withketamine/xylazine (80/10 mg/kg i.p.). The body will be secured and the foot will be at angle of 40-45o from his body axis. An recording elector rod was place in the gastrocnemius muscle and the stimulant rod was place in the knee. A reference rod will be place in the first digit and the ground rod was place in the tail (Finkel & Bookman, 2001). The hotplate test was use to determent the respond of...
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