Effection Of FGF-2 On The Pathogenesis Of SOD1-G93A Transgenic Mice

Objective: Amyotrophic lateral sclerosis(ALS) is a fatal neurodegenerative disease. It’s the most common type of motor neuron disease(MND), characterized by involving of upper and lower motor neurons, affecting cell in ventral horn, motor neurons in cerebellum, etc. and cause degeneration and death of neurons[1]. The clinical manifestations include progressive muscle atrophy and weakness. 60% of ALS patients die in 3 years after the onset, about 10% could survive over 8 years[2]. Almost 5-10% of ALS are familial ALS(f ALS)[3], others are sporadic ALS(s ALS) with no family history. Approximately 10-20 % of f ALS and 4 % of s ALS are linked to mutations in the gene encoding for the ubiquitous copper/mnc superoxide dismutase(SOD1)[4,5].The pathogenesis of ALS is still unknown. Many hypothesis, such as oxidative stress, glutamate toxicity, mitochondrial damage, abnormal folding and aggregation of proteins, autoimmune mechanism, neurotrophic factors deficiency, were proposed with past investigations[6,7].Fibroblast growth factor-2(FGF-2) is a of fibroblast grouth factor, which plays a regulatory role in the replication and differentiation of cells[8,9], widely effects in the nervous system, and expressed in a variety of cell types, including neurons and glial cells[8,10]. FGF-2 level elevated after brain injury and regulated neuroprotetion in many kinds of neuronal cells[9]. There has been experiment confirmed that double mouse mutants transgenic for the human SOD1 mutation and lacking the endogenous FGF-2 gene showed a significant delay in disease onset and less impaired motor performance in comparison to mutant SOD1 mice with normal FGF-2 levels[11].On this basis, we utilized classical animal model of ALS, SOD1G93 A, to discover the differentiation of FGF-2 in motor cortex, cerebellum, lumbar spinal cord and muscle of onset and end stage. With western bloting and RT-PCR technology, we explore the changes of FGF-2 in the tissues and discuss the role and significance in the pathogenesis of the disease.Methods: 1 Breeding of transgenic miceAll animals in this study were kept at constant temperature and humidity in sterile conditions(Speificpathogenfree, SPF) environment. All animals were fed the Sterilized rodents feed. feed.In Order to maintain the transgenic mutated SOD1 gene in B6 SJL. Tg N(SODlG93A)1Gur, hemizygous B6SJLSODlG93A+/- males were mated with B6SJLFI/J female. The offspring genetically identified determine whether human mutant SOD1G93 A gene was successfully inherited. 2 Experimental groupsThe animals were divided into positive group and control group. Both groups include two stages: onset stage(90 days), and end stage. Each group contained at least 3 female SOD1G93 A transgenic mice and 3 of their littermates, and they were used in western blot. 2. The animals were divided into positive group and control group. All the groups include four stages: 30 days, 60 days, 120 days and end stage. Each group contained at least 3 female SOD1G93 A transgenic mice and 3 of their littermates, and they were used in RT-PCR. 3.1 SamplingAfter anesthesia with 10% chloral hydrate, target tissues were preserved in the fixative after transcardially perfusion with phosphate-buffered saline(PBS), followed by 4% paraformaldehyde in PBS, or dissected freshly, along with other needed tissues, put into 1.5ml centrifuge tubes, frozen in liquid nitrogen, and then stored in-80 refrigerator.℃ 3.2 Western blottingThe spinal cord prepared using a total protein extraction kit following the manufacturer’s instruction and quantified using the BCA method. Eighty micrograms of protein from each sample was run on 10% or 12% SDS-PAGE gels, and blotted onto PVDF membranes. After blocking in 5% skim milk for 1 hour, primary antibodies including rabbit anti-ACTIN(1:500), rabbit goat anti-SOD1(1:500) rabbit anti-FGF-2(1:500) was. added respectively and the membranes were incubated overnight at 4℃. Next day, the membranes were rinsed with TPBS for 3 times, 5 minutes every time. Then add anti-rabbit or anti-mouse fluorescence-labeled. secondary antibody(1:10000) for 1 hour at room temperature, then the membranes were rinsed for 3 times with TPBS, and 1 time with PBS, 5 minutes each times. The.bands of interest were detected using an Odyssey Infrared Imaging System( LI-COR, Lincoln, NE). Band intensity was quantified, normalized by the ACTIN or GAPDH band, and statistically analysed. 3.3 Fluorescence quantitative.PCRAfter anesthesia, extract the lumbar spinal cord of the mice immediately, freeze with liquid nitrogen and store at-80℃. RNA was extracted, after reverse transcription and gene amplification, m RNA levels of target genes in different groups were detected with Stratagene Mx3005 P. 3.4 Statistical analysisResults were expressed as means±S.D.( x ±s). Statistical analyses were performed using one-way ANOVA with SPSS 13.0.4 statistical software. Differences were considered significantat at P <0.05.Results:1 Identification oftransgenic micePCR electrophoresis of geneome DNA from offspring mice demonstrates: the band located between 300-400bp(324 bp) is PCR product of IL-2; the bond located between 200-300bp(236 bp) is PCR product of human SOD1 gene..2 The expression of target proteinThe expression of FGF-2 in muscle increased in mice of experimental group along with the progress of the disease(P <0.05), while the level of FGF-2 in the other tissues was not changed obviously.3 results of real-time PCRWe examined the m RNA level of the FGF-2 genes in the brain cortex, cerebellum, lumbar spinal cord and muscle of the transgenic mice at different stages. Compared to their littermates, SOD1G93 A transgenic mice showed no significant changes in the m RNA levels of FGF-2 in all stages.Conclusion:In this study, we found that FGF-2 level of SOD1G93 A transgenic mice was up-regulated in muscle(P <0.05), while the level of FGF-2 in the other tissues was not changed obviously; The m RNA level of FGF-2 was not obviously changed in the tissues of Amyotrophic Lateral Sclerosis SOD1G93 A transgenic mice compared to the littermate. The results suggest that FGF-2 might play a role in the pathegenesis of ALS, and it worth to be studied further.