| This study includes three parts. First, we study the relationship between coagulation factor VII activity( CFVIIa) and intracranial progressive hemorrhagic injury( PHI) in isolated blunt traumatic brain injury( TBI) patients. Then, we explore the effect of coagulation factor VII( CFVII) gene promoter methylation on gene expression and PHI in TBI patients. Each part of the study is summarized as follows:Part 1: Activity of coagulation factor VII in patients with isolated blunt traumatic brain injury: association with intracranial progressive hemorrhagic injuryObjective: To study the relationship between CFVIIa and PHI in TBI patients.Methods: One hundred and twelve isolated TBI patients with moderate or severe injury were recruited between August 2010 and June 2014. Blood samples were collected at admission and then measured the parameters of international normalized ratio( INR), activated partial thromboplastin time( a PTT), D-dimer, platelet count( PLT), fibrinogen( FIB) and CFVIIa. Every 24 hours follow up of patients head computed tomography( CT). PHI was defined as when follow up CT noted any increase in size or number of hemorrhagic lesions.Results: CFVIIa levels and PLT in patients who developed PHI were significantly lower than those without PHI( 77.79% ± 22.82% VS 100.32%±30.39%, 126.95 ± 48.46 VS 148.13 ± 53.41, P<0.05). The plasma levels of a PTT, INR, D-dimer and FIB in patients with PHI were higher than those without PHI, but there were no significant difference between two groups( P>0.05). Stepwise logistic regression analysis identified decreased CFVIIa( OR=1.06, 95% CI=1.03-1.10, P<0.001) and PLT( OR=1.01, 95%CI=1.00-1.02, P=0.009) as predisposing risk factors independently associated with the presence of PHI.Conclusions: The levels of plasma CFVIIa and PLT in PHI group were significantly lower than non-PHI group. Decreased CFVIIa and PLT are predisposing risk factors independently associated with PHI.Part2: The promoter methylation of coagulation factor VII regulating gene expressionObjective: To investigate a possible epigenetic regulation of the CFVII gene at the promoter region.Methods: Gene synthesis two fragments, one containing 781 bp of the promoter( positions-781 to position-1) and one containing 1000 bp of the promoter( from position-1000 to position-1). This two fragments were cloned at PGL3-basic vector, then we got two recombinant plasmids: PGL3-F7UM-781--1 and PGL3-F7UM-1000--1. After enzyme digestion and sequencing validate, we used Sss I( Cp G) methylase do methylation modification on recombinant plasmid, PGL3-Basic and PGL3-Control. This experiment is divided into nine groups: Negative control, PGL3-Basic( UM), PGL3-Basic( M), PGL3-Control( UM), PGL3-Control( M), PGL3-F7UM-781--1, PGL3-F7M-781--1, PGL3-F7UM-1000--1 and PGL3-F7M-1000--1. All the constructs were transiently transfected into the tumoral hepatic cell line Hep G2, and luciferase activity was examined.Results: The luciferase expression levels in methylated plasmid groups were significantly lower than non-methylated plasmid groups. PGL3-F7M-781--1 set of luciferase expression was down about 110 times from PGL3-F7UM-781--1 group( P<0.001); PGL3-F7M-1000--1 set of luciferase expression was down about 244 times from PGL3-F7UM-1000--1 group( P<0.001). There were no significant difference between two non-methylation recombinant plasmids( P>0.05). However, there were significant difference between two methylation recombinant plasmids( P<0.001).Conclusions:The promoter methylation of CFVII regulating gene expression, the hypermethylation resulting in lower gene expression. Located in the promoter region-781 bp to-1000 bp the Cp G methylation have more significant influence on CFVII gene expression.Part3: The promoter methylation of coagulation factor VII in patients with isolated blunt traumatic brain injury: association with coagulation factor VII activity and intracranial progressive hemorrhagic injuryObjective: To study the effect of the promoter methylation in CFVII on CFVIIa and PHI in TBI patients.Methods: Blood was collected and analyzed with a PTT, INR, PLT, FIB and CFVIIa. Genomic DNA was isolated from patient blood, Bisulfite treatment was carried out following the manufacturer’s instructions. Design and Synthesis of primers, PCR amplification of gene fragments. The promoter methylation in CFVII was analysed using Epi TYPER. This experiment adopts two methods of grouping: CFVIIa≥90% group and CFVIIa<90% group, PHI group and non-PHI group.Results: The results showed significant difference in Cp G site 3 methylation levels between CFVIIa≥90% group and CFVIIa<90% group( 0.83±0.05 VS 0.85±0.03, P<0.05), CFVIIa≥90% group Cp G site 3 methylation levels were lower than CFVIIa<90% group, while there were no significant difference in other Cp G sites between this two groups( P>0.05). All of the Cp G sites methylation levels in the group with the PHI or non-PHI have no significant differences( P>0.05).Conclusions: The promoter methylation of CFVII affect plasma CFVIIa concentrations, the higher methylation resulting in lower CFVIIa. There was no relationship between CFVII promote methylation and PHI in TBI patients. |
