5 µl of RT reaction of each
cDNA were processed for PCR. Ten μL from each PCR reaction product were separated on a 2% agarose gel then stained with ethidium bromide. The appearance of specific bands (Bax 516 bp, β-actin 540 and FasL 345 bp) was evaluated under ultraviolet light and photographed. Photos were scanned and quantification of each band was carried out using GeneTools version 4 (Syngene, Cambridge, UK). Each quantified data point was related to its individual β-actin. Soluble Fas protein was measured using a commercially available #Fostamatinib solubility dmso keyword# sandwich enzyme-linked immunosorbent assay (ELISA) (29). DNA Fragmentation Assay: This is done according to the method of Ioannou and Chen 1996 (30). Separation of both fragmented and total DNA is carried out using DNA separating kit (Takara, Japan). DNA fragments were gradient separated from the intact DNA using polyethelene glycol (5% in Ethyl ether) and then quantified spectrophotometrically using Hoechst 33258 (0.2 µg/ml) as a chromophore. ELISA Bcl-2: The amounts of Bcl2 in circulating Inhibitors,research,lifescience,medical lymphocytes were determined by a sandwich enzyme linked immunosorbent assay (ELISA) purchased from Cliniulab, using
two anti-human BCL2 monoclonal murine antibodies Inhibitors,research,lifescience,medical (31). Plasma analysis of the cytokine TNF-α was performed using ELISA R & D Kits (32), for the growth factor VEGF using the ACCUCYTE Human VEGF immunoassay kit (33) and for bFGF using human bFGF immunosorbant assay (ELISA) Quantitin kit (34). Statistical analysis Each experimental condition was performed and expressed Inhibitors,research,lifescience,medical as mean ± SD. Comparisons were made by Student’s t-test (two-tailed for independent samples). Results Percentage of DNA fragmentation
per total DNA in plasma showed a significant increase in DMD patients compared to controls (mean = 0.38% ± 0.12 vs. 0.2% ± 0.15, p < 0.001) as shown in Figure Figure44. Figure 4 Markers of degeneration: Percentage of plasma DNA fragmentation Inhibitors,research,lifescience,medical per total DNA, FasL mRNA, Bax mRNA and Fas protein in DMD patients compared to controls. Fas protein in plasma showed a significant increase in DMD patients compared to controls (mean 9.9 ± 2.8 vs. 2 ± 0.1, p < 0.001) (Fig. (Fig.44). Histone demethylase FasL mRNA relative expression (Fig. (Fig.1)1) related to β-actin mRNA expression (Fig. (Fig.2)2) in circulating lymphocytes showed a significant increase in DMD patients compared to controls (mean 0.47 ± .09 vs. 0.24 ± .04, p < 0.001) (Fig. (Fig.44). Figure 1 FasL mRNA expression in DMD patients compared to controls. Figure 2 β-actin mRNA expression in DMD patients compared to controls. There is an inverse relationship between Bax and Bcl-2 gene expression. Bax mRNA relative expression (Fig. (Fig.3)3) in circulating lymphocytes related to β-actin mRNA expression (Fig. (Fig.2)2) showed a significant increase among DMD patients compared to controls (mean 0.19 ± 0.07 vs. 0.05 ± 0.01, p < 0.001) (Fig. (Fig.4).4).