| Coronary heart disease is a serious hazard to human health, from the view of Tranditional Chinese Medicine, coronary heart disease belongs to" obstruction in the chest", "cardiac pain" category, and blood stasis syndrome is the most common type of coronary heart disease. As the emergence of proteomics and its application in the field of medical research, providing us a new perspective and method to look for the molecular basis of blood stasis syndrome.Objective:To establish stable experimental conditions for Two-dimensional gel electrophoresis of serum,and observe the differentially expressed serum proteins between different periods of coronary heart disease and also between coronary heart disease with blood stasis syndrome and healthy people. Finding specificic related proteins to explain the molecular biology essentialism of CHD and blood stasis syndrome, but also for the clinical diagnosis of Coronary Heart Disease and therapy.At the same time,observe the effect of different periods serum on the proliferation of HUVEC.Method:1 Research on serum proteome of CHD and blood stasis syndrome1.1 According to strict inclusion and exclusion criteria to collect 4 groups of patients with CHD each of 8 cases, and 8 CHD patients in the④group are all diagnosed of blood stasis syndrome.In the early morning collected the peripheral venous blood of each people of the 3ml, and then centrifuged to obtain serum samples from the 4 groups.1.2 The total protein concentration of the serum was determinated by Brandford method, and then separated by two-dimensional electrophoresis (2-DE). Immobilized pH gradient (IPG) DryStrips (24 cm, pH 3-10NL) were rehydrated with each serum sample (1 mg of protein)。IEF was carried out using the IPGphor IEF system and then the strips were equilibrated in two steps for 15 min each with gentle shaking. After equilibration, the IPG strips were applied onto 12% gradient SDS-PAGE gels (25.5cm×20.5cm×1mm), the Second-dimensional SDS-PAGE was performed using an Ettan DALTⅡsystem. Proteins were visualized by the Coomassie brilliant blue staining method, and gels were analysised with Imagemaster and PDQuest software after scanning to identify differentially expressed proteins between the two groups. Spots of difference cutted from the gel were subjected to in-gel trypsin digestion,and then detected by the MOLDI-TOF-MS. To identify proteins, information of the spots were searched against protein sequences from swissprot and NCBlnr databases using the MASCOT search program.2 The effect of 4 groups serum on the proliferation of HUVEC2.1 After recovery,the HUVECs were cultured with 10% fetal bovine serum (FBS) culture medium at 37℃,5% CO2 concentration in the culture incubator.2.2 By 3000 cells/well were seeded in 96 well plates, the 4 groups serum were filtrated with 0.22um membrane for sterilization, and then 5% serum of each group was mixed with DMEM culture medium respectively, adding the serum into 96-well plate, each at least 6 holes.2.3 As the culture time was to 24h,48h,72h,96h, each hole cells viabilities were detected by MTT method.Result:1 Research on serum proteome of CHD and blood stasis syndrome1.1 Clear two-dimensional gel maps-of serum proteins were obtained, and 4 groups:①healthy group,②CHD in high-risk population groups,③acute CHD patients and④old groups of patients with CHD,the average protein spots respectively were 153.6±27.57,148.8±23.15,120.8±6.57,146.6±19.99.1.2 Through analysis and comparison,13 different proteins were detected between the 4 groups. Identificated by peptide mass fingerprint(PMF), finally got 6 meaningful proteins, respectively:retinol binding protein 4 (RBP4), apolipoprotein E (ApoE), apolipoprotein A1(ApoA1), CD5 antigen-like protein (CD5L), haptoglobin (Hp),clusterin(CLU)and serum albumin (ALB).1.3 There were 6 proteins:RBP4, ApoE, ApoA1, CD5L, Hp, ALB differentially expressed between coronary heart disease with blood stasis syndrome and healthy people. 2 The effect of 4 groups serum on the proliferation of HUVECWhether in the four groups cultured with or without 5% FBS, the role of 4 groups sera on HUVEC proliferation is similar:the trend of promoting proliferation by①,②,③,④group serum was gradually increased,and the④group had the strongest effectConclusion:1 Established a reproducible and stable experimental conditions for two-dimensional gel electrophoresis of serum,and got a better serum protein expression profiles of CHD and blood stasis syndrome2 The 7 proteins identified in this study:Retinol binding protein 4 (RBP4), apolipoprotein E (ApoE), apolipoprotein A1 (ApoA1), CD5 antigen-like protein (CD5L), haptoglobin(Hp),clusterin(CLU) and serum albumin (ALB),may associated with Coronary Heart Disease, and six of these proteins were involved in blood stasis syndrome.Also, we considered that lipid metabolism and inflammation disorders may relate to the pathological process of the blood stasis syndrome of CHD, but need further verification.3 Four groups of serum on promoting proliferation of HUVEC was gradually increasing,so we think that the serum of patients with CHD may contain some cytokine or protein fractions to promote vascular regeneration when the myocardial ischemia occurs... |
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