A Preliminary Study On The Downregulation Of Nogo-A Expression Of Oligodendrocytes In Vitro With Antisense Oligodeoxynucleotides

Background and Purpose: Optic nerve injury is a common ocular injury. In craniocerebral injury, the incidence of optic nerve injury is 0.3% to 5.2%. About 40-50% patiens will lose sight after injury, there is scarcely effective method for their management. Peripheral nerve injury can regeneration after injury, while central nervous can(t. The failor of regeneration of optic nerve injury is due to the poor regenerative ability of its neuron named retinal ganglion cells, nerve growth inhibitors exist in its micro- environment and the lack of neurotrophic factors. Schwab reported that the neurite growth inhibitors NI-35/250 expresed by oligodendrocytes had the strong inhibitory effects on growing neurites of CNS. A nerve growth inhibitor gene named Nogo was found in 2000. Nogo gene has three transcriptional mRNA: Nogo-A, Nogo-B, and Nogo-C. Nogo-A mRNA encodes protein NI-250, only expresses in oligodendrocytes. Many investigations proved that using monoclonal antibodie IN-1 of NI-35/250 can neutralize its activity and improve the regeneration of CNS, while articles of utilizing antisense technique to repress or block the expression of Nogo were not found. The purpose of this study is: to detect Nogo-A mRNA expression in oligodendrocytes, to study the effect of antisense oligonucleotides (SODN) on Nogo-A mRNA expression in oligodendrocytes and to screen safe and effective SODN concentration used for in vivo research.Method: The research was divided into three parts: (1) Oligodendrocytes were obtained by inoculating the optic nerve of newborn 2 days rats and adjusting medium; the cells were identified with GC antibody immunocytochemicalstain. (2) Nogo-A mRNA expression was detected by probe labeled by digoxin with In situ hybridization immunocytochemical stain. (3) The uptake of SODN labeled by 6-FAM in oligodendrocytes was studied under fluorescent invert microscope; in order to observed the effects of Nogo-A SODN on cultured cells, the expriments was divided into five groups: 2μM,5μM,10μM Nogo-A SODN groups,random sequence groups and control groups. RT-PCR was adopted to study the effects of SODN on the expression of Nogo-A in oligodendrocytes.Result: (1) 3 days after inoculation, a few of round or fusiform shape cells migrated from optic nerve tissue; About 11 days later, The coverlips were completely covered by the cells; The cells identified with GC antibody immunocytochemical stain were positive cells. (2) The plasm of oligodendrocytes showed buffy positive reaction with Nogo-A probe in situ hybridization immunocytochemical stain. (3) Under fluorescent invert microscope, over 80 per cent of cells showed green fluorescence; the result of RT-PCR study showed that Nogo-A SODN could significantly and specificly inhibit the expression of Nogo-A after 24 hours (P<0.01). Random sequence has no effect on Nogo-A expresson. Nogo-A SODN has no obvious effect on survival of the cells when the concentration below 10μM.Conclusion: (1) Oligodendrocytes cultured by the method were purified cells and can meet the demands of further reaseach. (2) Oligodendricytes express Nogo-A mRNA. (3) Oligodendrocytes can directly intake Antisense Oligonucleotides. (4) Nogo-A SODN can effectively and specifically inhibit the expression of Nogo-A (P<0.01). Nogo-A SODN has no obvious effect on survival of the cells when the concentration below 10μM.