Objective To study the role and mechanism of sphingosine-l-phosphate (S1P)/sphingosine-1-phosphate receptor 1 (S1P1) signal pathway during post conditioning of hypertrophic cardiomyocytes.Methods Neonatal rat cardiomyocytes were isolated and cultured,then stimulated by norepinephrine (NE) to induce cardiomyocytes hypertrophy.Using tri-gas incubator to create hypoxia and reoxygenation enviroment to mimic ischemia-reperfusion and postconditioning.Hypertrophic cardiomyoctyes were divided into five groups according to the presence or absence of various drugs and postconditiong and relevant signal pathways changes were detected:(1) IPost group (hypoxia + postconditioning);(2) IPost + S1P group (cells were pretreated with S1P (1 μmol/L) for 2 h before IPost);(3) IPost + W-146 + S1P group (cells in IPost + W-146 + S1P group were pretreated with S1P1 inhibitor W-146 (0.4 μmol/L) for 20 min);(4) IPost + PD98059 + S1P group (cells in IPost + S1P group were pretreated with MAPK antagonist PD98059 (125 μmo[/L) for 20 min);(5) IPost + LY-294002 + S1P group (cells in IPost + S1P group were pretreated with PI3K antagonist LY294002 (0.1 μmol/L) for 20 min).Apoptosis was detected by flow cytometry and protein expression of relevant signal pathways were detected by Western blot.Results (1) Apoptosis rate was significantly increased in hypoxia/reoxygenation (27.90 ± 4.49) ％ group compared with normal control group (7.97 ± 2.18) ％,which could be significantly reduced in IPost group (15.90 ± 1.77) ％ (all P ＜ 0.05).(2) Apoptosis rate and caspase-3 expression were both significantly lower in IPost + S1P and IPost + S1P + LY-294002 groups than in IPost and IPost + S1P + W-146 and IPost + S1P + PD98059 group (all P ＜ 0.05).(3)p-ERK1/2 expression was significantly higher in IPost + S1P and IPost + S1P + LY-294002 group than in IPost and IPost + SIP + W-146 group and IPost + S1P + PD98059 group (all P ＜ 0.05) while p-Akt expression was similar among IPost,IPost + S1P + W-146 and IPost + S1P +PD98059 groups.p-ERK1/2 and p-Akt levels in IPost + S1P + W-146 group and IPost + S1P + PD98059 were similar as in IPost group.Conclusions S1P can play protective role on NE induced cardiomyocytes hypertrophy during post conditioning through downregulating caspase-3 expression and reducing apoptosis rate via targeting S1P1 and activating ERK1/2 signal pathway.

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.

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: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: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.

BACKGROUND:The formation of atherosclerotic lesions in apolipoprotein E knockout mice is similar to that of human systemic atherosclerosis, and apolipoprotein E knockout mice are ideal animals for current establishment of atherosclerosis models.
OBJECTIVE:To research the pathological process of atherosclerosis in apolipoprotein E knockout mice aged different weeks, and to explore the effect of different diets on the occurrence and development of atherosclerosis in apolipoprotein E knockout mice.
METHODS:Male apolipoprotein E knockout mice aged 8 weeks old were randomly divided into two groups, and fed with high fat diet and normal diet, respectively, for 8, 12, 16, 20, and 24 weeks.
RESULTS AND CONCLUSION:Serological detection revealed that serum total cholesterol, triglycerides and low density lipoprotein levels were significantly higher in different weeks of mice of high fat diet group than in the normal diet group (P<0.05), in a time-dependent manner. Gross and frozen oil red O staining showed that atherosclerotic plaque area of lumen was significantly larger in the high fat diet group than in the normal diet group (P<0.05), in a time-dependent manner. At this time, significant differences in plaque area of lumen at each week were detected between both groups (P<0.05). Apparent lipid plaque was visible in aorta at 16 weeks of high fat diet in mice. Results demonstrated that apolipoprotein E knockout mice of atherosclerosis were successful y established. The formation of lipid streaks and fiber hyperplasia was faster in high fat diet group than in the normal diet group.

Objective To investigate the effects of different concentrations of norepinephrine (NE) on proliferation and apoptosis of cultured cardiac fibroblasts (CFBs) from neonatal mice and to elucidate related mechanisms.Methods CFBs of Sprague-Dawley (SD) rats were isolated and cultured and divided into normal control group and different concentration of NE intervention groups (0.1,1,10,50,and 100 μmol/L).Water soluble tetrazolium-1 (WST-1) assay was carried out to detect the viability of CFBs.Morphology of apoptosis cells was evaluated by fluorescence microscope with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.The expressions of collagen Ⅰ,collagen Ⅲ,prooncogene c-myc in CFBs were detected by reverse transcription-polymerase chain reaction (RT-PCR).The phospho-mitogen activated protein kinase (p-p38MAPK) and caspase3 protein levels were examined by Western blot.Results Proliferation was significantly increased in 1 μmol/L and 10 μmol/L groups compared with the normal control group (1.05 ± 0.05 and 1.09 ± 0.02 vs.1.00 ± 0.03,all P ＜ 0.05).CFBs apoptosis was significantly enhanced in 50 Pμmol/L and 100 μmol/L groups ((22.69 ± 2.18)％ and (36.40 ± 6.80) ％ vs.(4.50 ± 1.08) ％,all P ＜ 0.05).Expression of Collagen Ⅰ peaked in 10 μmol/L group,expression of collagen Ⅲ and c-myc increased dose-dependently in proportion to increasing NE concentrations (all P ＜ 0.05 vs.control group).The expression of p-p38MAPK and caspase3 was also significantly upregulated in a dose-dependent manner in NE groups (all P ＜ 0.05 vs.control group).Conclusions Low concentration NE induces CFBs proliferation and high concentration NE promotes CFBs apoptosis.p38MAPK phosphorylation may be a major mediator of NE-induced effects on CFBs.

OBJECTIVETo analyze the relationship between different levels of fasting blood glucose over 35-year old and carotid intima-media thickness (IMT) in Han, Uygur and Kazak adult population from Xinjiang Uygur Autonomous Region.

METHODSFrom October 2007 to April 2010, the present study was performed in 13 935 inhabitants among Han, Uygur and Kazak adult population of aged 35 years old and over by multi-stage stratified cluster random sampling principles from 7 regions in Xinjiang Uygur Autonomous and we excluded the IMT over 0.9 millimeter, long-term out and the floating population. All subjects were measured fasting blood glucose and IMT values of carotid artery. The subjects were divided into three groups according to different fasting blood glucose levels: normal, impaired fasting glucose (IFG) and diabetes mellitus (DM) and we used the analysis of variance to compare the differences among groups of IMT. Multiple linear regression model was used to explore factors of carotid IMT.

RESULTSThe IMT of males of Han, Uygur and Kazak were (0.81 ± 0.29), (0.71 ± 0.27) and (0.79 ± 0.21) mm respectively, the differences were significant (F = 88.50, P < 0.05) . The IMT in DM group ((0.82 ± 0.29) mm) was significantly higher than the normal ((0.77 ± 0.26) mm) and the IFG groups ((0.79 ± 0.27) mm) (F = 7.49, P < 0.05). The IMT of females of Han, Uygur and Kazak were (0.72 ± 0.27), (0.63 ± 0.25) and (0.77 ± 0.22) mm, respectively, the differences were significant (F = 173.93, P < 0.05) . The IMT in DM group ((0.75 ± 0.29) mm) and the IFG groups ((0.74 ± 0.26) mm) were significantly higher than the normal group ((0.70 ± 0.25) mm) (F = 10.46, P < 0.05). Multivariate regression analysis showed that diastolic blood pressure (β = 0.101, P < 0.01) , total cholesterol (β = 0.056, P < 0.05) and fasting blood glucose (β = 0.023, P = 0.009) were independent risk factors of IMT.

CONCLUSIONThe level of fasting blood glucose was an independent influence factor of carotid IMT and had a positive correlation in Han, Uygur and Kazak population of Xinjiang Autonomous Region.

Adult ; Aged ; Asian Continental Ancestry Group ; ethnology ; Blood Glucose ; Blood Pressure ; Carotid Arteries ; Carotid Intima-Media Thickness ; China ; epidemiology ; ethnology ; Diabetes Mellitus ; Fasting ; Female ; Humans ; Male ; Middle Aged ; Prediabetic State ; Risk Factors

BACKGROUND:Cyclin A2 is a key regulator of cellcycle. Location and expression of cyclin A2 in neonatal mouse myocardium is not clear.
OBJECTIVE:To observe the location and expression of cyclin A2 in neonatal mouse cardiomyocytes and its relationship with the exit of cardiomyocytes from cellcycle.
METHODS:Neonatal mice were kil ed to take myocardial tissues at 0, 3, 7, 14 and 28 days after birth. Western blot were used to detect the expression of cyclin A2, proliferating cellnucleus antigen and Phospho-histone H3. Immunohistochemitry detection was used to detect the location of cyclin A2 and expression of proliferation cellnucleus antigen at different time after birth.
RESULTS AND CONCLUSION:Western blot showed the decrease of cyclin A2 after birth til disappeared at day 4 (P=0.001). Cyclin A2 located mainly in the nucleus after birth and exported to the cytoplasm at day 14, and basical y disappeared at day 28. Proliferating cellnucleus antigen showed gradual y decreased tendency after birth. Mitosis specific marker, Phospho-histone H3, exhibited a gradual decrease after birth, which was consistent with cyclin A2 in expression intensity.

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.