Expression Changes Of Insulin-like Growth Factor Binding Protein 2 And 4 In The Lumbar Enlargement Of SOD1-G93A Transgenic Mice

Objective: Amyotrophic Lateral Sclerosis (ALS) is a chronic and fatal degenerative disease of central nervous system, characterized by selective loss of cortex, brainstem and spinal cord anterior horn motor neurons, which is the most common type of motor neuron disease【1】, it was predicted as "the cancer of nervous system". Patients are usually middle-aged after 40, and especially the male, invasion is a slow and progressive procedure, and muscle weakness, atrophy occur, tendon reflex may be active, and even pathological reflex can be checked out, about 3 to 5 years after the onset symptoms appear, the majority of ALS patients may die of respiratory failure.According to their clinical features ALS were divided into familial amyotrophic lateral sclerosis and sporadic amyotrophic lateral sclerosis. Pathogenesis of SALS is unclear up to date which accounts for more than 90%, may be mainly correlated to the following aspects: 1. Glutamate excitotoxicity; 2.Ca2+ overload; 3. Toxic effects of free radicals; 4. Immunological mechanism; 5. Disfunction of mitochondrial; 6. Neurofilament hyperphosphorylation; 7. Deficiency of neurotrophic factors.Insulin-like growth factor, as a neurotrophic factor, may not only play an important role on promoting neuron survival and inhibiting apoptosis, but also may have a role in the promotion of neuronal regeneration. Insulin-like growth factor binding proteins as differential carriers could combine together with these factors and have vital effect in the regulation of insulin-like growth factor conveying, distribution, and half-life and biological activity. The expression alloeosis of these binding proteins is likely to affect its normal biological regulating role, but up to now there are few studies on the expression diversity of these proteins on amyotrophic lateral sclerosis. This experimental study was designed to test the expression of insulin-like growth factor binding protein 2 and 4 in the lumbar enlargement of SOD1-G93A transgenic mice, the model of amyotrophic lateral sclerosis, and compared the expression changes before and after disease onset.Method:1 The groups of experimental animal 36 SOD1-G93A transgenic mice were divided into non-transgenic littermate control group, pre-symptomatic group, clinical onset group (using law of snowG93A physically clinical score = 2.0 points definited as clinical onset) and end-stage group (using law of snowG93A physically clinical score = 5.0 points definited as endstage), each group with 9.2 Specimens of each animal group obtained To obtain the non-transgenic littermate control group, pre-symptomatic group, clinical onset group (using law of snowG93A physically clinical score = 2.0 points definited as clinical onset), and end-stage group (using law of snowG93A physically clinical score = 5.0 points definited as endstage) in different periods, the animals were quickly taken out of eyeball for bloodletting and then immediately decapitated and removed the spinal cord from the vertebral canal, quickly and carefully stripped off spinal pia mater, spinal ganglia and the spinal nerve roots, lumbar enlargement was selected immediately and put into liquid nitrogen and then stored at -80℃conditions.3 The detection of IGFBP 2 and 4 Western blot assay used to detect spinal cord in each group, according to the manufacturing company instruction stringent of the tissue protein extraction kit to extract tissue protein of lumbar spinal cord, then using BCA Protein Assay Kit to test the protein concentration of each specimen. 50μg of protein samples were run for electrophoresis in sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) gel, after that transfer the proteins to PVDF membrane, 5% skimmed milk blocking the specific antigens, then anti-mouse IGFBP-2 monoclonal antibody and anti-rabbit IGFBP-4 polyclonal antibody were added, put on rocking bed 4℃overnight。The next day wash the membrane for three times with TPBS first, each time 10 minutes, then IRDye ? 800-conjugated goat anti-rabbit secondary antibody and IRDye ? 700-conjugated goat anti-mouse secondary antibody at room temperature on rocking bed protecting from light, TPBS and PBS are used to wash the membrane, the bands of interest were detected using an Odyssey Infrared Imaging System.Results: Insulin-like growth factor binding protein-2 of SOD1-G93A transgenic mice spinal cord tissue of end-stage group and clinical onset group relatively density values (0.28667±0.13, and 0.44667±0.16) compared with the non-transgenic littermate control group and pre-symptomatic group (0.16000±0.04, and 0.13667±0.05) significantly increased, with statistical significance, while the insulin-like growth factor binding protein-4 expression did not change significantly among the four groups.Conclusion : In the SOD1-G93A transgenic mice, the model of amyotrophic lateral sclerosis, insulin-like growth factor binding protein-2 expression levels of lumbar spinal cord tissues of the end-stage group and clinical onset group increased than the non-transgenic littermate control group and pre-symptomatic group, the increase may be related to the relevance of occurrence and progress of this disease. The insulin-like growth factor binding protein-4 expression was not significantly different among the four groups and it still can not explain the relationship with this disease.