Background Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. It can trigger inflammatory cascades which are primarily mediated via nuclear factor-κB (NF-κB). The NF-κB transcription factor family includes several subunits (p50, p52, p65, c-Rel, and Rel B) that respond to myocardial ischemia. It has been proved that persistent myocyte NF-κB p65 activation in heart failure exacerbates cardiac remodeling. Mechods A recombinant adeno-associated virus serotype 9 carrying enhanced green fluorescent protein and anti-NF-κB p65 ribozyme (AAV9-R65-CMV-eGFP) was constructed. The cells were assessed by MTT assay, Annexin V–propidium iodide dual staining to study apoptosis. The expression of P65 and P50 were assessed by Western blot to investigate the under-lying molecular mechanisms. Results After stimulation with H2O2 for 6 h, H9c2 cells viability decreased significantly, a large fraction of cells underwent apoptosis. We observed a rescue of H9c2 cells from H2O2-induced apoptosis in pretreatment with AAV9-R65-CMV-eGFP. Moreover, AAV9-R65-CMV-eGFP decreased H2O2-induced P65 expression. Conclusions AAV9-R65-CMV-eGFP protects H9c2 cells from oxidative stress induced apoptosis through down-regulation of P65 expression. These observations indicate that AAV9-R65-CMV-eGFP has the potential to exert cardioprotective effects against oxidative stress, which might be of great importance to clinical efficacy for cardiovascular disease.

BACKGROUND:Recombinant adeno-associated virus serotype 9 has a high affinity in myocardial tissue, and the expression of recombinant adeno-associated virus serotype 9-enhanced green fluorescent protein (rAAV9-eGFP) in the aorta of atherosclerosis mice is not clear. OBJECTIVE:To explore the optimal time point of rAAV9-eGFP expression in the aorta of atherosclerosis mice. METHODS:Atherosclerosis model was established with high-fat diet in 30 ApoE-/-mice for 16 weeks. Among them, 25 mice were injected with 5.0×1011 vg (virus genomes) rAAV9-eGFP through the tail vein, while the remaining 5 mice were injected with saline, serving as the control group. The virus-transfected mice were kil ed at 14, 21, 28, 35 and 60 days after transfection, and aortic tissue was harvested. The expression of enhanced green fluorescent protein was detected with laser scanning confocal microscope. Western blot assays were used to detect the expression of enhanced green fluorescent protein in aorta. The expression of enhanced green fluorescent protein in vivo was observed and the optimal expression time point was determined. RESULTS AND CONCLUSION:rAAV9-eGFP effectively transfected the aorta of atherosclerosis mice, enhanced green fluorescent protein was expressed in aortic tissue, and the expression intensity increased gradual y with the increasing transfection time. The highest expression level was found at 35 days after transfection and then maintained stable at 60 days. There were significant differences at different time points after transfection (P<0.001). These data indicate that rAAV9-eGFP can be effectively expressed in the aorta of atherosclerosis ApoE-/-mice and rAAV9-eGFP can be regarded as the optimal vector in the treatment of atherosclerosis.

BACKGROUND:A lot of work has been carried out on the development of the primary cultured rat myocardial cel s at home and abroad. The primary culture technology of rat myocardial cel s becomes more mature, but myocardial cel s from neonatal mice are not easy to be obtained under the same experimental conditions. The mouse genome has more similarities with the human genome, which has a higher research value. OBJECTIVE:To improve the primary culture method of neonatal mouse myocardial cel s, and to obtain myocardial cel s with high purity, vitality and original structure and function. METHODS:The mouse cardiac tissues were treated using an enzyme digestion method to isolate isolated single myocardial cel s:first, the cardiac tissues were digested using trypsin, and then col agenous fibers were treated with col agenase to isolate single myocardial cel s. The concentration and action time of trypsin and type II col agenase were adjusted, and the pH values of reagents and temperature of each step were strictly control ed. RESULTS AND CONCLUSION:At 24 hours after inoculation, the myocardial cel s began to be adherent;at 48 hours, independent pulsation of myocardial cel s could be observed;at 72 hours, myocardial cel s were cross-linked;and at 96 hours, myocardial cel s formed cel clusters and presented with consistent beating. The survival rate and purity of myocardial cel s were both over 95%. This modified method could successful y culture myocardial cel s with high purity and viablility from neonatal mice, and the structure and function of myocardial cel s could be retained. Therefore, it is a feasible culture method.

BACKGROUND:Platelet-derived growth factor-B (PDGF-B) is an effective pro-angiogenic growth factor, and adeno-associated virus type 9 (rAAV9) has a strong cardiomyocyte targeting affinity, which is an ideal vehicle for ischemic heart disease gene therapy.
OBJECTIVE:To explore the PDGF-B gene transfection of in vitro neonatal rat myocardial cells mediated by rAAV9 against ischemia and hypoxia-induced cardiomyocytes apoptosis.
METHODRat neonatal myocardial cells were isolated and cultured, and then transfected by rAAV9-PDGF-B and empty virus, rAAV9 with enhance green fluorescent protein (eGFP), under multiplicity of infection (MOI) of 105, 106 and 107, respectively. We observed the expression of eGFP under fluorescence microscopy every day, and used flow cytometry to measure transfection efficiency of vector rAAV9. Western blot and immunofluorescence were used to evaluate protein expression of PDGF-B. Myocardial ischemia and hypoxia injury model was established in vitro on the 5th day of transfection of rAAV9-eGFP and rAAV9-PDGF-B with 107 MOI. The number of myocardial apoptosis was measured by TUNEL assay. Western blot was employed to detect the protein expression of Bax and Caspase-3 which were related apoptosis, and the effect and its possible mechanism of PDGF-B gene overexpression against myocardial apoptosis were explored.
RESULTS AND CONCLUSION:rAAV9 vector can efficiently transfect neonatal rat myocardial cells. eGFP and PDGF-B protein expressed in myocardial cells correctly and efficiently, and the expression intensity increased gradual y with the increasing of time course and MOI. The expression became stable on the 5th day, and the transfection efficiency showed significant difference among these groups (P<0.01). Myocardial apoptosis rate was significantly reduced in the rAAV9-PDGF-B group than the rAAV9-eGFP group (P<0.05), and protein levels of Bax and Caspase-3 in the rAAV9-PDGF-B group were significantly lower than those of the rAAV9-eGFP group (P<0.05). These data indicate that overexpression of PDGF-B gene can effectively reduce ischemia and hypoxia-induced myocardial apoptosis, and the possible mechanism might be by inhibiting Bax and Caspase-3 protein expression, which can provide evidence of rAAV9-PDGF-B vector in the gene therapy of ischemic heart diseases.

Objective To investigate the association between acute myocardial infarction (AMI) and the (GT)n repeat sequence polymorphism in promoter region of heme oxygenase-1 (HO-1) , and to study the influence of serum bilirubin on AMI as well for HO-1 as a rate-limiting enzyme of bilirubin production in patients from Uighur national minority. Method Totally 287 patients with AMI evidenced by coronary arteriography admitted from January 2006 to June 2008 were eligible for being studied, and another 190 healthy subjects without anomaly in coronary arteriography, and with normal findings in physical examination and in variety of biochemical assays were enrolled as controls. Serum total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDL-C), serum bilirubin were detected. Polymerase chain reaction-nondenaturing polyacrylamide gel electrophoresis was used to detect HO-1 promoter (GT)n repeat polymorphism, and at the same time the serum bilirubin was determined. The group representation of samples was tested with HardyWeinberg balance test. Differences in distributions of genotypes and alleles between AMI patients and control subjects were analyzed using Chi-square test. Comprehensive evaluation of the factors associated with myocardial infarction using multi-factor Logistic regression analysis. P < 0.05 was considered as significantly different. Results Body mass index, triglyceride, high density lipoprotein cholesterol and the proportion with hypertension in myocardial infarction group was significantly higher than those in control group ( P < 0.01) . The X~2 values of HO-1genotype distribution in the myocardial infarction group and the control group were 2.09 and 0.05, respectively (P > 0.05), consist with the results of Hardy-Weinberg balance test. The HO-1 genotype was classified into three groups, L/L, L/S and S/S. The L/L genotype frequency (35.5%) and L-allele frequency (57.8%) in AMI group and in control group showed statistically significant differences, respectively (X~2 = 11.65, P = 0.001; X~2= 11.32, P = 0.003). The bilirubin level of L/L genotype significantly decreased compared with that of S/S, L/S genotype ( P all < 0. 001) . Logistic regression analysis showed that body mass index, high blood pressure,triglycerides, blood bilirubin and HO-1 gene polymorphism are risk factors of myocardial infarction. Conclusions To the Xinjiang Uighur ethics, HO-1 promoter ( GT) n repeat polymorphism and the occurrence of myocardial infarction are relevant. People with L allele genotype have lower serum bilirubin and higher risk of myocardial infarction.

AIM: To explore the changes in extracellular regulated protein kinase (ERK1/2) in the hypertrophic myocardium induced by pressure overload at the different time courses and to determine the molecular mechanism in the myocardium from hypertrophy to heart failure. METHODS: C57/BL mice, aged 12 week old, were subjected to sham-operation (SH) or transversing aortic constriction (TAC) to establish left ventricular hypertrophy. Echocardiographic assessments, hemodynamic determination, organ weight measurement, morphological and histological examination were performed at 1, 4, 8, 12 and 16 weeks after surgery. Meanwhile mRNA levels of atrial natriuretic peptide (ANP), α-myosin heavy chain (α-MHC), bcl-2 and bax were measured by RT-PCR, and ERK1/2 levels were detected by Western blotting. The animals in SH group were performed the same tests then sacrificed at 16 weeks. RESULTS: (1) Compared to SH group, LVESd, LVEDd, Awsth, Awdth, Pwsth and Pwdth progressively increased after TAC. Meanwhile, ejection fraction (EF%) significantly decreased at 16th week (P<0.05). LVSP, dp/dt_(max) and dp/dt_(min) in TAC group were progressively increased after 4 weeks. From 8-12 weeks these parameters maintained stable and then sharply decreased at 16th week (all P<0.05). However, LVEDP was statistically increased at 8th week. These echocardiographic and hemodynamic changes indicated a development of LVH and eventually progressing towards to heart failure. (2) Histologically, cardiac collagen measured by percentage of Sirius red positive stained area and apoptosis index showed progressive increases from 4 to 16 weeks. (3) Compared to SH group, mRNA levels of ANP was time-dependently increased while α-MHC and Bcl-2 were time-dependently decreased. The ratio of Bcl-2 /Bax was decreased. Phosphorylation of ERK1/2 was increased at 4th week, then decreased with age of TAC (all P<0.05). CONCLUSION: Pressure-overload induced by TAC results in a development of LVH from early concentric hypertrophy to late eccentric hypertrophy, and eventually toward cardiac dysfunction or heart failure. Those changes are associated with increase in cell size and cardiac fibrosis. ERK1/2 signaling pathway may involve in the regulation of myocardial cell apoptosis in hypertrophic and failure heart.

Objective To investigate the association between matrix metalloproteinase-9 (MMP-9) gene polymorphism (-1562C ＞ T/R279Q) and acute coronary syndrome (ACS) in Uygur nationality of Xinjiang Autonomous Region of China. Methods A total of 352 patients with ACS including 213 patients with unstable angina pectoris and 139 patients with acute myocardial infarction evidenced by using coronary arteriography and 421 control subjects were recruited in this study. The MMP-9-1562C ＞ T and R279Q genotypes were detemined by using PCR-RFLP method. The relationship between the polymorphism in the MMP-9 gene and the severity of coronary arterial stenosis was analyzed. All polymorphisms were determined for confimation with Hardy-Weinberg expectations in both groups separately. Differences in distributions of genotypes and alleles between two groups were analyzed with x2 test. The association between the MMP-9 polymorphisms and the risk of ACS was estimated by odds ratio(Ors) and their 95% confidence intervals (CIs), and the comprehensive evaluation of the factors associated with ACS was determined by using multifactor logistic regression. P ＜ 0. 05 was considered to be statistically significant. Results The genotype frequencies for CT + TT genotypes and T allele were 25.9 and14.5 percent in ACS subjects and 15.7 and 8.4 percent in control subjects, respectively. The genotype frequencies were different significantly between the two groups (x2 = 12.26,P ＜ 0.01;x2 = 14.15,P ＜ 0.01, respectively). No relationship between R279Q polymorphism and ACS was found in this study ( P ＞ 0.05). The multifactor logistic regression analysis showed that the T allele carrier (CT + TT) significantly increased the risk of ACS compared with the CC genotype ( OR = 1.791,95 % CI: 1. 088 - 2.951, P = 0.022) after adjustment for tradition risk factors. The frequencies for CT + TT and CC genotypes of the -1562C ＞ T polymorphism were not statistically different among ACS patients with one, two and three or more significantly diseased vessels ( x2 = 1.15, P = 0.56). Conclusions The findings suggest that the polymorphism in MMP-9 gene promoter (-1562C ＞ T) is associated with the susceptibility to the ACS. The T allele might be an independent risk factor for the ACS. But the -1562C ＞ T polymorphism may not be useful as a predictor of the severity of coronary arterial stenosis. The R279Q polymorphism of MMP-9 gene was not significantly associated with ACS in this studied population.

BACKGROUND:Studies have shown that fibrin glue can promote the survival of myoblast grafts, reduce infarct size and induce neovascularization of infarct zone. OBJECTIVE:To understand the condition of revascularization of infarcted heart muscle using endothelial progenitor cells combined with degradable fibrin glue materials. METHODS:A total of 27 Sprague-Dawley rats were randomized into three groups, 9 rats in each group:non-myocardial infarction group, immediate transplantation group and 1-week post-infarction transplantation group. Then, these three groups were sub-grouped into two groups, respectively:endothelial progenitor cells+fibrin glue group (experimental group) and fibrin glue group (control group). At 3 and 8 weeks after transplantation, the rats were sacrificed in each group. The revascularization and function of infracted heart muscle were observed by microscope, immunohistochemistry and echocardiography. RESULTS AND CONCLUSION:Under the microscope, there were some lax connective tissues between the heart and chest in the experimental groups, but no difference existed between the experimental and control groups. The heart structure was normal relatively and difficult to be distinguished between the experimental and control groups histological y and immunological y, and there was no angeioma, vascular malformation and tumor. The number of revascularization of heart muscle showed no difference between experimental and control groups as wel as between different experimental groups. Additional y, there was no significant difference in cardiac function between experimental and control groups. Although there are no positive results of endothelial progenitor cells, we wil modify and improve the strategy and believe that the celldelivery system is of benefit and efficacy.

Objective To optimize primary cultures techniques of isolating neonatal rat cardiomyocytes and to com?pare three different methods of extractingβ3-adrenergic receptor(β3-AR)membrane protein from cultured neonatal rat car?diomyocytes. Methods TypeⅡcollagen and differential velocity adhesion were used to collect primary cardiomyocytes. To?tal protein method, ultracentrifugation method, extract kit method were used to isolate cardiomyocytesβ3-AR membrane pro?teins. The BCA method was applied for protein quantification. Relative content ofβ3-AR membrane protein and GADPH in the sample were examined by western blot. Results Optimizing culture and isolation skills can produce a great quantity of cardiomyocytes in high concentration.The kit method acquired a higher level of protein concentration(8.26±0.29)g/L than to?tal protein method(5.12±0.47)g/L does than ultracentrifugation method(3.20±0.37)g/L does all of which were with signifi?cant difference(P < 0.05). The concentration of β3-AR membrane protein was higher if obtained by kit method(0.22 ± 0.05)than ultracentrifugation method(0.09 ± 0.03)than total protein method (0.01 ± 0.01) with significant difference(P <0.05). Conclusion optimizing methodology can obtain abundant myocardial cells in high concentraion. The kit method of isolating primary culturedβ3-AR membrane proteins result in improved concentration and specificity of membrane protein.

BACKGROUND:Macrophage migration inhibitory factor (MIF) is a multi-potent cytokine that makes considerable contribution to the regulation of inflammatory response and immune response in the body. MIF rs1007888 is associated with various inflammatory diseases, but the correlation between rs1007888 and coronary heart disease in the Kazakhs of China has been rarely explored. OBJECTIVE:To investigate the relationship between rs1007888 gene polymorphisms in MIF gene and coronary heart disease in the Kazakhs from Xinjiang Uygur Autonomous Region, China. METHODS:A total of 230 Kazakh patients with coronary heart disease evidenced by coronary arteriography between December 2012 and July 2014 were recruited, and another 478 Kazak controls were free from coronary artery disease with normal angiograms. Real-time fluorescence quantitative PCR assay was used to detect the rs1007888 polymorphisms of MIF gene. Alele and genotype distributions of the rs1007888 polymorphism were compared between patients and controls. RESULTS AND CONCLUSION:Distribution of genotypes in the two groups appeared to be in Hardy-Weinberg equilibrium (P> 0.05). The alele frequencies and genotypes of MIF-rs1007888 showed no significant difference between the two groups (P > 0.05). Therefore, the genetic variation of rs1007888 in MIF gene is not associated with coronary heart disease in the Kazakhs of China.