Fibrinogen-like protein 2 expression correlates with microthrombosis in rats with type 2 diabetic nephropathy

Fibrinogen-like protein 2 (fgl2),a novel prothrombinase,is involved in microthrombosis.We examined fgl2 expression in the glomerular and tubulointerstitial capillaries and its correlation with microthromsis in rats with streptozocin-induced type 2 diabetic nephropathy.Our RT-PCR and immunoblotting analysis showed that fgl2 mRNA and protein levels were increased in microvascular endothelial cells of the glomeruli and renal interstitia at week 19 and became significantly elevated with the development of diabetic nephropathy (P < 0.01).Fgl2 was not or only weakly expressed in the renal tissues of normal rats.Furthermore,a direct significant correlation (r=0.543,P < 0.01) was found between fgl2 expression and microthrombotic capillaries in the renal tissues.Enzyme linked immunosorbent assays (ELISA) additionally showed that circulating TNF-α levels in rats with type 2 diabetes were significantly elevated and closely correlated with fgl2 expression (r=0.871,P < 0.01).Our results suggest that fgl2 may activate renal microthrombosis,thus contributing to glomerular hypertension and renal ischemia.

Effectiveness and feasibility of transradial approaches for primary percutaneous coronary intervention in patients with acute myocardial infarction

Objective: To evaluate the efficacy and feasibility of the transradial approach for primary percutaneous coronary intervention (PCI) in patients with acute myocardial infarction(AMI). Methods: 195 patients with acute myocardial infarction were randomly divided into two groups according to the different PCI operation pathways. 105 cases were assigned to the transfemoral artery group and 90 cases to the transradial artery group. We analyzed the data from the two groups, including the achievement ratio of paracentesis, cannulation time, the time from local anesthesia to the first time balloon inflation, the time of the total procedure, achievement ratio of PCI, incidence rate of vascular complications, total duration of hospitalization, and the six-month follow-up results in both groups. Results: Our results showed that the achievement ratio of arteriopuncture, cannulation time and the time from local anesthesia to the first time balloon inflation in the transradial and transfemoral groups were 98.9% vs. 100%, 3.15 ± 1.56 min vs. 2.86 ± 0.97 min, and 18.56 ± 4.37 min vs. 17.75 ± 3.21 min, respectively. These differences between the two groups were not statistically significant. The total operating time was 29.75 ± 4.38 min for the transradial group and 27.89 ± 3.95 min(P < 0.05) for the transfemoral group. The operation achievement ratio in the transradial group was 96.7%, and 96.2% in the transfemoral group. The incidence of puncture point complications was 2.2% in the transradial group and 11.4% in the transfemoral group, and this difference was significant. The duration of hospitalization was 10.56 ± 2.85 days for the transradial group and 13.78 ± 3.15 days(P < 0.05) for the transfemoral group. At the six-month follow-up, the rate of survival without cardiac event was 86.1% vs. 86.4% respectively in the transradial and transfemoral groups(P > 0.05). Conclusion: The transradial approach was as effective as the transfemoral approach, and there were fewer puncture point complications as well as a shorter span of hospitalization in the transradial group. PCI via the transradial approach is safe, effective and feasible in patients with AMI.

Effects of sodium ferulate on the ultrarapid delayed rectifier K^+ current in human atrial myocytes

Objective:To study the effects of sodium ferulate on the ultrarapid delayed rectifier K+ current(IKur) in human atrial myocytes. Methods:Human atrial myocytes were isolated by enzyme dispersion method. IKur in human atrial myocytes were recorded by using the whole cell patch clamp. The changes of IKur were compared in the absence and the presence of sodium ferulate. Results:There was no effect of 0.4 g/L sodium ferulate on I-V relation of IKur. However, 0.4 g/L sodium ferulate inhibited IKur to some degrees at each test pulse. The current densities of IKur at +60 mV decreased from 4.997 ± 0.35 PA/PF to 3.331 ± 0.26 PA/PF(n = 6, P < 0.05). The inhibitory effect was concentration-dependent. IC50 was(0.41±0.03)g/L and the Hill coefficient was 0.95±0.05. Conclusion:Sodium ferulate as a potassium channel blocker can inhibit IKur in human atrial myocytes effectively.

Inhibitory effect and mechanism of chuanxiongzine on multiplication of VSMC

在脉管的光滑的肌肉房间(VSMC ) 上学习 chuanxiongzine 的禁止的效果的目的增长并且探索它的分子的生物学基础。我们选择了 VSMC 的方法从是的老鼠主动脉 thoracalis 的有教养的 4-8 产生研究目标。对象被划分成四个组(I) 控制组，(II ) chuanxiongzine (50 渭 g /ml ) 组，() chuanxiongzine (100 渭 g /ml ) 组并且(lv ) chuanxiongzine (200 渭 g /ml ) 组。VSMC 增长上的 chuanxiongzine 的禁止的效果被房间数， M'IT 和 WTdR 加入试金调查。以便照亮禁止 VSMC 增长的 chuanxiongzine 的分子的生物学机制，增殖的房间的表示原子抗原(PCNA ) 和 C-myc 被检测。结果 Chuanxiongzine 能在一个剂量依赖者和时间依赖者举止显著地禁止 VSMC 的增长，与控制组相比(P < 0.05 ) 。PCNA 和 c-myc 的表示显然被禁止并且与 chuanxiongzine 的集中相关(P < 0.05 ) 。结论 Chuanxiongzine 可以在 VSMC 增长过程起一个可观的作用。在一个剂量依赖者和时间依赖者举止的 chuanxiongzine 的禁止的效果能被认识到经由在调整 PCNA 和 c-myc 的表示下面。在这研究，关于中国药的大理论臀部，被用来对待动脉粥样硬化，作为) ，被获得了。

Constructing retroviral vector carrying green fluorescent protein(GFP)and investigating the expression of GFP in primary rat myoblast

Objective:To construct green fluorescent protein(GFP)retroviral vector(pLgXSN),and to investigate the expression of GFP in primary rat myoblast.Methods:GFP cDNA was subcloned into the plasmid pLgXSN,and the recombinant vector was transfected into packaging cell PT67.G418 was used to select positive colony.Myoblasts were infected by a high-titer viral supernatant.The recombinant retroviral plasmid vector was identified by restriction endonuclease analysis and DNA sequence analysis.Confocal microscopy and flow cytometry were used to detect the expression of GFP.Results:The GFP cDNA sequence was identical to that of GenBank.Recombinant retroviral plasmid vector pLgGFPSN was constructed successfully.The titer of the packaged recombinant retrovirus was 1 × 106 cfu/ml.Bright green fluorescence of the transfected cells was observed under confocal microscope 48 h after transfection.The transfection rate was 33%.The effective expression of GFP in myoblast infected by recombinant retrovirus lasted for 6 weeks.Conclusion:GFP gene could be effectively and stably expressed in myoblast,which suggests that GFP could act as a marker for studies on myoblast.

Cardiovascular Immunology Research in Wuhan Union Hospital:Over the Past 25 years

Cardiovascular immunology research in Wuhan Union Hospital began in 1991.Anti-heart antibodies in dilated cardiomyopathy and acute viral myocarditis began to be reported from 1993.It was found that a new autoantibody against L-type calcium channel results in ventricular tachycardia and sudden death in patients with dilated cardiomyopathy.Through the Intervention Study of Diltiazem in Dilated Cardiomyopathy,diltiazem was verifi ed to reduce mortality and the chronic heart failure hospitalization rate signifi cantly in patients with dilated cardiomyopathy.The autoantibodies against angiotensin II receptor type 1 andα1-adrenoceptor were associated with the increased recurrence of and death from stroke in hypertensive patients.Through many clinical and experimental studies,the functional imbalance of T-cell subsets was suggested to mediate myocardial injury and chronic heart failure,which provided a new theoretical basis for immunoregulation therapy for heart failure.The first antihypertensive polypeptide vaccine(ATRQβ-001)was invented.In addition to these achievements,there will be more research on cardiovascular immunology in Wuhan Union Hospital in the future.

Depressive Effect of the Antisense Oligonucleotides of C-myc and PCNA on the Proliferation of VSMC

Objectives To study the depressive effect of the antisense oligonuceotides (ASODN) of c-myc and proliferating cell nuclear antigen (PCNA) on the proliferation of VSMC. Methods Taking the VSMC obtained from rat aorta thoracalis cultured 4～8 generation as research object. The objects were divided into three groups to carry out control study: control group, PCNA ASODN group and c-myc ASODN group. The ASODNs' working concentration all were 1:50. The depressive effect of ASODN on VSMC proliferation was investigated by cell counting, MTT and 3H-TdR incorporation assay; PCNA and c-myc expression were detected by immunohistochemical method after transferring PCNA and c-myc ASODN into VSMC. Results PCNA and c-myc ASODN could inhibit the proliferation of VSMC significantly, compared with control group (P<0.05). ② Transferring PCNA and c-myc ASODN into VSMC obtained successfully; the corresponding gene was inhibited obviously; compared with control group (P<0.05). Conclusions PCNA and c-myc might play a considerable role in the VSMC proliferation process. The corresponding gene could be depressed successfully after transferring PCNA and c-myc ASODN into VSMC, and then the proliferation of VSMC was slowed down. This study presented a beneficial proposal and theoretical fundament for atherosclerotic treatment.

The altered expression of inflammation-related microRNAs with microRNA-155 expression correlates with Th17 differentiation in patients with acute coronary syndrome

MicroRNAs(miRNAs)are a novel class of small,non-coding RNAs that play a significant role in both inflammatory and cardiovascular diseases.Immune cells,especially T helper(Th)cells,are critical in the development of atherosclerosis and the onset of acute coronary syndrome(ACS).To assess whether inflammation-related miRNAs(such as miR-155,146a,21,125a-5p,125b,31)are involved in the imbalance of Th cell subsets in patients with ACS,we measured the expression of related miRNAs in patients with acute myocardial infarction(AMI),unstable angina(UA),stable angina(SA)and chest pain syndrome(CPS);analyzed the relationship between miRNA expression and the frequency of Th cell subsets;and observed the co-expression of miR-155 and IL-17A in peripheral blood mononuclear cells(PBMCs)of patients with ACS.The results showed that the expression of miR-155 in the PBMCs of patients with ACS was decreased by approximately 60%,while the expression of both miR-21 and miR-146a was increased by approximately twofold.The expression patterns of miRNAs in plasma correlated with those in PBMCs,except for miR-21,which was increased by approximately sixfold in the AMI group and showed no significant difference between the UA group and the CPS group.We also found that the expression of miR-155 inversely correlated with the frequency of Th17 cells(r520.896,P,0.01)and that miR-155 was co-expressed with IL-17A in patients with ACS.In conclusion,our study revealed the expression patterns of inflammation-related miRNAs in patients with ACS and found that miR-155 may be associated with Th17 cell differentiation.

Agonistic AT_1 Receptor Autoantibody Increases in Serum of Patients with Refractory Hypertension and Improves Ca^(2+) Mobilization in Cultured Rat Vascular Smooth Muscle Cells

Agonistic AT1 receptor autoantibodies (AT1-AAs) have been described in the patients with malignant hypertension or preeclampia. Furthermore, AT1-AAs were highly associated with refractory hypertension. Function of vascular smooth muscle cells (VSMCs) is important in the regulation of blood pressure. We investigated and compared the ability of angiotensin II (Ang II) and AT1-AAs to stimulate the intracellular calcium mobilization and cellular proliferation of rat VSMCs. Twenty-two patients with refractory hypertension, 24 patients with non-refractory hypertension and 37 normotensives were recruited. The serum of each patient was detected for the presence of AT1-AAs by ELISA. Ang II and the AT1-AAs from the sera of patients were used to stimulate rat VSMCs in vitro. AT1-AAs were detected in 10/22, 3/24 and 3/37 of patients with refractory hypertension, non-refractory hypertension and normotensives, respectively. AT1-AAs led the increase intracellular calcium mobilization in a dose-dependent manner and cellular proliferation of VSMCs just as Ang II. Both of these effects caused by AT1-AAs were blocked with losartan or a peptide corresponding to a part of the second extracellular loop of AT1 receptor. Since AT1-AAs exhibited pharmacological activity in rat VSMCs just as Ang II, they might play a role in the elevation of peripheral vascular resistance and in vascular remodeling. And AT1-AAs were suggested to involve in resistance to antihypertensive therapy.

Myosin Specific-T Lymphocytes Mediated Myocardial Inflammation in Adoptive Transferred Rats

Myosin specific-T lymphocytes might mediate myocardial inflammation and remodeling after AMI. Myosin- activated or unactivated T lymphocytes in vitro were transferred into na?ve syngeneic rats, respectively. T lymphocyte infiltration and myocyte apoptosis were explored by the H&E and TUNNEL. Proteins and mRNA levels of cytokines (IL-1β, IL-6 and TNF-α) in myocardium were determined by RT-PCR and immunohisto- chemistry. T lymphocyte infiltration was evidently observed after one week of activated T cell transfer. The expressions of cytokines were elevated markedly one week later. The myocyte apoptosis occurred after T lymphocyte infiltration in myocardium. Our findings suggest that cardiac myosin activated-T lymphocytes may mediate myocardial inflammation and remodeling.

Molecular genetics of Brugada syndrome

Brugada syndrome(BrS)is a life-threatening cardiac rhythm disorder characterized by persistent STsegment elevation in leads V1–V3 and right bundle branch block on electrocardiograms(ECG),and by syncope and sudden death from ventricular tachycardia(VT)and ventricular fibrillation(VF).BrS is responsible for nearly 4%of sudden cardiac deaths and considered to be the most common cause of natural death in males younger than 50 years in some Asian countries.Since the first diseasecausing gene for BrS(the cardiac sodium channel gene SCN5A)was identified in 1998,extensive investigations on both clinical and basic aspects of BrS have occurred rapidly.SCN5A mutations remain the most common cause of BrS;nearly 300 SCN5A mutations have been identified and are responsible for 20%–30%of BrS cases.Commercial genetic testing is available for SCN5A.Recently,seven other disease-causing genes for BrS have been identified and include GPD1L(BrS2),CACNA1C(Cav1.2,BrS3),CACNB2(Cavβ2,BrS4),SCN1B(Navβ1,BrS5),KCNE3(MiRP2,BrS6),SCN3B(Navβ3,BrS7),and HCN4(BrS8).This article will briefly review the progress made over the past decade in our understanding of the clinical,genetic and molecular aspects of BrS.

Preliminary study on preparation of E.coli cell-free system for protein expression

In the new era of‘‘Omics’’,the traditional techniques of protein expression in vivo can not come up with the exponential increase of genetic information.The cellfree protein synthesis system provides a new strategy of protein expression with advantages of rapid,convenient and high-throughput expression.The preparation of cell extracts,the optimization of substrate concentrations and the energy regeneration system are the key factors for the successful construction of cell-free protein expression system.In this work,the cell extract was prepared from RNase I-defective strain E.coli A19.The cell growth phase,the pressure for cell disruption and the storage condition of cell extracts were optimized.Meanwhile,the optimal substrate concentrations and the energy regeneration system were selected.Under the optimized conditions,the green fluorescent protein(GFP)reporter gene was expressed in the E.coli cell-free system with high expression level(Ca.154 mg/mL)which was 29 times higher than the expression level before optimization.