Biomarker Screening Of Brain Tissue And Cerebrospinal Fluid In Patients With Drug-Resistant Epilepsy And Their Possible Clinical Value

PART ONE: EXPRESSION OF N-WASP AND ITS UPSTREAM AND DOWNSTREAM GENE AND PROTEIN EXPRESSION IN BRAIN TISSUE OF PATIENTS WITH DRUG-RESISTANT EPILEPSYObjective To investigate the expression of neuronal Wiskott-Aldrich syndrome protein (N-WASP), and its upstream elements cell division cycle 42 GTP-binding protein (Cdc42), downstream molecules actin-related protein 2/3 (Arp2 / 3) in the brain tissues obtained from patients with drug-resistant epilepsy and discuss their significances in epileptogenesis.Methods cDNA microarray scanning has shown that N-WASP significantly increased in brain tissue of patients with drug-resistant epilepsy. Western blot, immunohistochemistry and immunofluorescence were used to evaluate the expression of N-WASP, immunohistochemistry and immunofluorescence were used to evaluate the expression of its upstream regulator Cdc42. Western blot was used to detect its downstream effector Arp2/3 expression in 40 cases of drug-resistant epilepsy, respectively. Comparison was made with the control group.Results cDNA microarray result showed the Cy5/Cy3 ratio of N-WASP was 3.021. Western blot gray value was 0.9030±0.0492 ( x±s) in drug-resistant epilepsy group and 0.4320±0.0727 ( x±s) in control group (P<0.05). The immunohistochemistry OD value of epilepsy group was 0.384±0.072 ( x±s), and 0.143±0.046 ( x±s) for control group (P <0.05). The data of immunofluorescence analysis complied with the outcome of immunohistochemistry. OD values of Cdc42 in drug-resistant epilepsy group and control group were 0.032±0.016 ( x±s), 0.020±0.005 ( x±s), respectively (P <0.05). Gray value of Arp2 / 3 was 0.7765±0.0230 ( x±s) in case group, compared to 0.4986±0.0823 ( x±s) in control group (P <0.05). These results indicated that N-WASP, Cdc42 and Arp2 / 3 were upregulated in brain tissue of patient with drug-resistant epilepsy.Conclusions Overexpression of N-WASP and its upstream Cdc42, downstream Arp2 / 3 in patients with drug-resistant temporal lobe epilepsy, suggesting that this pathway may play an important role in the mechanisms of drug-resistant epilepsy. PART TWO: BIOMARKER SCREENING OF CEREBROSPINAL FLUID IN PATIENTS WITH DRUG-RESISTANT EPILEPSYObjective To investigate the proteomic changes of cerebrospinal fluid (CSF) in patient with drug-resistant epilepsy in order to discover cerebrospinal fluid biomarkers of epilepsy.Methods Two-dimensional gel electrophoresis followed by liquid chromatography electrospray ionization tandem mass spectrometry were used, these proteins were identified by database searching. Expression of some proteins was validated by western blot.Results Eight protein spots showed significant differential expression (p<0.05): vitamin D-binding protein (DBP) was elevated in the CSF of TLE patients whereas pigment epithelium-derived factor (PEDF), cathepsin D (CTSD), gelsolin, apolipoprotein J (apoJ), Ras protein-specific guanine nucleotide-releasing factor 1 (RASGRF1), Fam3c, and superoxide dismutase 1 (SOD1) were decreased in the CSF of TLE patients. Additional six protein spots presented only in the CSF of epilepsy patients were identified as tetranectin (TN), talin-2, apolipoprotein E (apoE), immunoglobulin lambda light chain (IGL@), immunoglobulin kappa variable light chain 1-5 (IGKV1-5), and procollagen C-endopeptidase enhancer 1 (PCOLCE). Expression of DBP,SOD1and talin-2 was validated by western blot.Conclusions Our results suggest that there exsist disease specific biomarkers of cerebrospinal fluid in patients with intractable epilepsy, which may provide better understanding of the pathophysiologic mechanisms underlying epileptogenesis.