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AIM:To evaluate the effects of atorvastatin on nitric oxide(NO),endothelin-1(ET-1)and myocardial no-reflow in a rabbit model of acute myocardial infarction and reperfusion(AMI/R). METHODS:Twenty-four rabbits were randomized into 3 groups:8 in AMI/R group,8 in atorvastatin-treated group(5 mg·kg~(-1)·d~(-1))and 8 in sham-operated group. Animals in the former two groups were subjected to 60 min of coronary occlusion followed by 120 min of reperfusion. Data on haemodynamics were collected. NO in blood sample,and in normal,and in infarcted reflow and no-reflow myocardium were evaluated respectively by nitrate reductase method. The levels of ET-1 in blood sample,and in normal,infarcted reflow and no-reflow myocardium were determined by radioimmunoassay. RESULTS:(1)Compared to the baselines,the heart rate(HR),systolic blood pressure(SBP),diastolic blood pressure(DBP),left ventricular systolic pressure(LVSP),maximal rate of increase and decline in left ventricular pressure(±dp/dt_(max))and cardiac output(CO)in AMI/R and atorvastatin-treated groups were significantly declined,whereas left ventricular end-diastolic pressure(LVEDP)was increased after 60 min of coronary occlusion and 120 min of reperfusion(P<0.05 or P<0.01). However,in atorvastatin-treated group,LVSP,LVEDP,±dp/dt_(max) and CO at the time point of 120 min of reperfusion recovered more significantly than those at the time point of 60 min of coronary occlusion(P<0.01),which was more significant than those in AMI/R group(P<0.05 or P<0.01). Compared to AMI/R group,the SBP and DBP were significantly heigher in atorvastatin-treated group(P<0.01).(2)In atorvastatin-treated group,the levels of ET-1 in blood sample were significantly lower than those in AMI/R group(P<0.01),and the levels of NO were significantly higher(P<0.01). Moreover,the levels of NO or ET-1 in infarcted reflow myocardium were significantly lower than that in AMI/R group(P<0.05 or P<0.01).(3)Atorvastatin could ameliorate myocardial function. CONCLUSION:Atorvastatin is effective in increasing NO and reducing ET-1 in blood plasma and local myocardium,and in protection of endothelial cells. Atorvastatin also has a beneficial effect on improving left ventricular function during acute myocardial infarction and reperfusion in rabbits.
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<p><b>OBJECTIVE</b>To establish a reliable approach to primary culture and identification of sinoatrial node (SAN) cells.</p><p><b>METHODS</b>The SAN cells were cultured from SAN tissue removed from neonatal Wistar rats and purified with differential attachment and 5'-bromodeoxyuridine (BrdU) treatment. The obtained cells were morphologically observed with inverted microscopy and transmission electron microscopy. Its action potential was recorded using electrophysiological methods.</p><p><b>RESULTS</b>Three distinctly different cells were observed in the cultured SAN cells: spindle, triangle and irregular. Of these, the spindle cells comprised the greatest proportion, with their shape, structure and electrophysiological characteristics consistent with those of the pacemaker cells of SAN. The triangle cells were similar in features to the similarly shaped myocytes located in the atrial myocardium.</p><p><b>CONCLUSIONS</b>The culture method of differential attachment combined with BrdU treatment is a reliable approach to growing SAN cells. Of the cells cultured from SAN, the spindle cells appear to function as pacemaker cells.</p>
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Animals , Female , Male , Rats , Animals, Newborn , Cells, Cultured , Microscopy, Electron , Rats, Wistar , Sinoatrial Node , Cell Biology , PhysiologyABSTRACT
Objective:To construct the recombinant adenovirus of AT2R by using the method of homogenous recombination in bacteria. Methods:AT2R gene was get from the vector of PUHD-AT2R by PCR ,and subcloned into shuttle vector of pAdTrack-CMV, forming transfer vector of pAdTrack-CMV/AT2R.Then it was linearized with PmeⅠ and transformed into Adeasier-1 cell. The DNA of identified recombinant plasmid was digested with PacⅠ and transfected to 293cells to package adenovirus. The PCR technique was used to detect target gene . The titre of the Ad-AT2R was measured with the aid of GFP expression. Results:PCR test indicated each the recombinant adenovirus contained the insert of AT2R. The titre of purified recombinant adenovirus was 1.5?10 12pfu/ml. Conclusion:The method of homologous recombination in bacteria is more convenient and efficient compared with that in cell .The prepared Ad-AT2R paves a sound foundation for further study.
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The red cell deformability (RCD) has been taken as a diagnostic aid in recent years. The dilute red cell suspension passes through the silicon microch annels under constant suction pressure. Such three parameters of RCD are measure d as IF (filtration index), and The results show that the above-mentioned parameters can reflect the changes of cell rheological characteristics from dif ferent aspects. With the observation of the flow passage, the rehological charac teristics can be evaluated accurately and comprehensively.
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Objective Overexpressing gax gene in vascular smooth muscle cells(VSMC) mediated by adenovirus transfection.Methods Replication defective adenovirus type 5 (Ad5) vector encoding the rat gax gene (AdCMV gax) was constructed via site specific recombination and amplified in 293 cells. The resulting adenovirus was purified for a high viral titer. The expression of gax mRNA and protein was evaluated by a series of methods such as reverse transcription polymerase chain reaction (RT PCR), flow cytometry and immunocytochemical technique after gax gene was transferred to VSMCs. Results When AdCMV gax was transfected to VSMCs, the expression of gax protein increased significantly in VSMCs, and reached 80% in 24 hours. High expression of gax protein could keep for over 5 days. Before AdCMV gax was transfected to VSMCs, gax mRNA and protein could be down regulated by platelet derived growth factor (PDGF) BB. After AdCMV gax was transfected to VSMCs, the level of gax expression was significantly higher than that before transfection, and no correlation with the stimulation of PDGF BB.Conclusions gax gene can be transfected to VSMC by AdCMV gax and translated to protein, which is advantageous to exploring the effects of gax gene on biological behaviour of VSMC.
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Objective: To construct recombinant adenovirus vector of Antisense BTEB2 cDNA and study the effect of antisense BTEB2 on proliferation of vascular smooth muscle cells and neointimal hyperplasia.Methods:BTEB2 cDNA was prepared by RT PCR technique and was subcloned reversedly into shuttle plasmid pDC315 to constructed recombinant shuttle plasmid pDC315AS BTEB2.Then the recombinant shuttle plasmid and adenovirus genomic plasmid pBHGlox?E1,3Cre were cotransfected into 293 cell to obtain recombinant adenovirus. The PCR technique was used to detect target gene fragment and adenovirus genomic characteristic fragment. After the recombinant adenovirus infected vascular smooth muscle cells, antisense RNA expression of BTEB2 was detected by RT PCR. Results:There was recombinant adenovirus containing BTEB2 cDNA in the lysate of cotransfected 293 cells.The recombinant adenovirus infected 293 cells and replicated in 293 cells. The expression of BTEB2 antisense RNA was very obvious in vascular smooth muscle cells after infected by recombinant adenovirus. Conclusion:The construction of recombinant adenovirus vector of Antisense BTEB2 has been achieved, and Antisense RNA of BTEB2 can express in vascular smooth muscle cells. This study has paved the way for furthur research.
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Objective To evaluate the immediate and short term therapeutic efficacy of percutaneous transluminal septal myocardial ablation (PTSMA) in patients with hypertrophic obstructive cardiomyopathy (HOCM) and to investigate the prevention of the related complications. Methods PTSMA was conducted in 3 patients with HOCM refractory to medication. The immediate effect was evaluated by pressure monitor, but the follow up effect by observation of the clinical symptoms and ultrasonic cardiogram. Results The left ventricular outflow tract gradients triggered by ventricular premature beats decreased from 143 mmHg (ranging from 70 to 180 mmHg) to 53 mmHg (ranging from 30 to 80 mmHg). Complete atrioventricular block (Ⅲ?AVB) with atrioventricular node rhythm was found in 1 case, and complete right branch bundle block (CRBBB) in another one. Follow up of the 3 cases at 6 months after operation revealed remarkable improvement of the patients' cardiac function (NYHA), disappearance of angina pectoris, and obvious attenuation of ventricular septal hypertrophy and SAM (systolic anterior motion of mitral) phenomenon. Conclusion PTSMA is an effective method with satisfactory short term effect for the patients with HOCM refractory to medication.
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Objective To evaluate the therapeutic efficacy and safety of transcatheter closure of atrial septal defect (ASD) and patent ductus arteriosus (PDA) using Amplatzer occluder. Methods Routine cardiac catheterization and angiography were performed in 50 patients (23 male, 27 female, age ranging from 3 to 64 years old), including 19 cases of ASD and 31 cases of PDA under local or general anesthesia. After balloon sizing of the ASD, the optimal Amplazter septal occluder (ASO) was transmitted into the left atrial, and the left and right disks were released in turn. The Amplatzer occluder was completely released after transthoracic echocardiography confirmed that there was no residual shunts or new onset mitral valve regurgitation. The Amplatzer duct occluder (ADO) size was selected according to the narrowest point of PDA measured by angiography, and the occluder was released after the repeated angiography showed no residual shunts. Results ① The mean diameter of the ASD measured by balloon was 13-31 (23?6) mm and the diameter of ASO was (17-40) mm. The immediate closure rate was 100%. ② Angiography confirmed that closure of the ductus using ADO was achieved in 30 patients, and closure of the large size (12 mm) was achieved in 1 case of PDA patient using ASO (17 mm). No complications were encountered. Conclusion Transcatheter closure of ASD and PDA using Amplatzer device, with the advantages of simple operation, confirmative occlusion efficacy, minimal invasiveness, wide indications, and less complications, has a bright future of clinical application.
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<p><b>OBJECTIVE</b>To determine the biotic effects of angiotensin II (Ang II) on the migration of rat smooth muscle cells (VSMCs) and investigate the mechanisms involved in the development of vascular injury.</p><p><b>METHODS</b>VSMCs isolated from aortic media of Wistar rats and cultured by the modified explant method were adopted. In the presence and absence of Ang II, the expression of Ang II receptor (ATR) and reorganization of the actin cytoskeleton and focal adhesion of VSMCs were studied by an immunocytochemistry technique and fluorocytochemistry technique. Migration assays were performed with a modified Boyden's chamber. The effects of AT(1)R antagonist (CV-11974), AT(2)R antagonist (PD123319) on the aforementioned target were studied.</p><p><b>RESULTS</b>VSMCs migration was stimulated by adding Ang II. The dynamic reorganization of actin cytoskeleton and focal adhesions may be an important mechanism by which Ang II facilitates VSMCs motility. The expression of AT(1)R in VSMCs could be upregulated initially after treatment with Ang II, then decreased gradually. The expression of AT(1)R was downregulated by AT(1)R antagonists. The effect of Ang II on VSMCs migration was mediated by AT(1)R, while AT(2)R had no significant effect.</p><p><b>CONCLUSIONS</b>The dynamic reorganization of focal adhesions and the actin cytoskeleton is required for Ang II-induced VSMCs migration. This effect is mediated by AT(1)R.</p>
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Animals , Female , Male , Rats , Actins , Metabolism , Angiotensin II , Pharmacology , Angiotensin Receptor Antagonists , Benzimidazoles , Pharmacology , Cell Movement , Cells, Cultured , Cytoskeleton , Dose-Response Relationship, Drug , Focal Adhesions , Metabolism , Imidazoles , Pharmacology , Immunohistochemistry , Muscle, Smooth, Vascular , Cell Biology , Metabolism , Pyridines , Pharmacology , Rats, Wistar , Receptor, Angiotensin, Type 1 , Receptors, Angiotensin , Metabolism , Tetrazoles , PharmacologyABSTRACT
Objective To construct the recombinant adenovirus of Angiotensin Ⅱ type 2 receptor (AT2R), and study the effect of AT2R on prevention of neointimal hyperplasia in rat carotid arteries after balloon angioplasty. Methods The recombinant adenovirus of AT2R with green fluorescent protein was constructed by using the method of homogenous recombination in bacteria. AT2R gene was transduced into rat carotid arteries with pAdCMV/AT2R after the reproduction of rat carotid balloon injury restenosis model. The expression and the transfection efficiency of AT2R gene were evaluated by RT-PCR, immunofluorescence staining, and confocal microscope. The intimal/medial area ratio was measured by digital analysis system. Results The titre of purified recombinant adenovirus was 1.5?10 12pfu/ml, and it was proved by PCR. pAdCMV/AT2R transfection efficiency in carotid artery was about 40% at day14, and localized to the neointima, as well as to the media and the adventitia. pAdCMV/AT2R delivered into injured rat carotid arteries significantly up-regulated AT2RmRNA and protein expression in neointima from day 7 to 21 after injury. Compared with pAd-GFP transfection group, pAdCMV/AT2R transfection reduced I/M ratio significantly on day 21(0.78?0.06 vs. 1.36?0.21 respectively, P
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Objective To study the value of imaging characters in diagnosing mediastinal tumors.Methods X-ray and CT findings of pathologically proved mediastinal tumors in 58 cases were analysed,the findings enabling qualitative diagnosis were recommended. Results There were 40 cases of anterior mediastinal tumors including intrathoracic thyroid tumors [n=32,5 tyroid carcinomas and 1 thyroid cyst ,thymomas (n=3) and teratomas (n=5, 3 cases ruptured)]. All the other 18 cases in posterior mediastinum were neurogenic tumors.Conclusion Based on the combination of the mediastinal compartment knowledge, X-ray and CT findings of the tumors and the clinical information, the qualitative diagnosis of mediastinal tumors will be characterized correctly.
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Objective:To analysis the appearances and to evaluate the diagnostic value of X-ray CT and MR imaging in osteoid osteoma.Methods:22 cases 19 females and 3 males of osteoid osteoma proved by surgical pathology and their X-ray,CT and MRI were reviewed.The ability of X-ray,CT and MRI to demonstrate the nidus and surrounding reaction were analyzed.Results:The nidus were appeared as round or oval shape and the diameter was less than 2 cm.It was fomd in 17 cases of X-ray film，22 CT and 20 of MRI .There were different degree of bone sclerosis,periosteal reaction and soft tissues or bone marrow edema around the nidus.The diagnostic accuracy was 77.3% for X-ray，100% for CT and 90.9% for MRI.Conclusion:Most of osteoid osteoma have the typical appearances and is no difficult to make diagnosis.CT scan is the most accuracy method to demonstrate the nidus.It is possible to make misdiagnosis only with X-ray or MRI for the case that the hidus not be demonstodted.
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Objective To constructed the cell model transferred angiotensin Ⅱ (AngⅡ ) type 2 receptor (AT2R) in vascular smooth muscle cells(VSMC) Method The VSMCs, isolated from the aorta of rat, were cultured by routine method. Recombinant adenoviral vector, AdCMV-AT2R, containing rat AT2 receptor gene was constructed by homologous recombination, and then it was used to transfer AT2 receptor gene to VSMC in vitro. The rate of AT2R expression in VSMC was analysed by flow cytometry. The expression of mRNA, protein were detected by RT-PCR, Western blot and immunohistochemistry respectively. The angiotensin Ⅱ (AngⅡ) type 1 receptor (A T1R) was determined as well. Result The expression rate of AT2R in VSMC was increased signific antly after transferred by AdCMV-AT2R with time, and the peak value detected by flow cytometry was about 89.51% at 48 hours. RT-PCR, Western blot and immunohistochemistry showed that the expression of AT2R mRNA and protein were increased obviously in transferred VSMC. There were no significantly change of AT1R expression during AT2R expression. Conclusion Our study indicates that AdCMV-AT2R did generate high level expression of AT2 receptor and its expression did not affect AT1R exp ression in cultured VSMC. The VSMCs transferred AT2R gene may be used as a good model to study the effect of AT2R on their biological action such as proliferation, migration and apoptosis.
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Objective To study the proliferation and migration of vascular smooth muscle cell (VSMC) after transferred angiotensin Ⅱ (AngⅡ) type 2 receptor (AT2R) gene. Methods The recombinant adenoviral vector, AdCMV-AT2R, containing rat AT2 receptor gene was constructed by homologous recombination, and transfered to rat VSMC in vitro. The expression of AT2R mR NA was detected by RT-PCR. The rate of expression and the change of cell cycle in VSMC were analysed by flow cytometry. These assays including cell devision index. Incorporation of bromodeoxyuridine(BrdU) and 3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenyltetrazolium bromide( MTT) were used to determine the proliferation of VSMC. The modified Boyden's chamber method was used to test the migration of VSMC. The r e-organization of F-actin in VSMC was analysed by confocal microscopy. Results RT-PCR showed that the expression of AT2R mRNA increased substantially in transferred VSMC, and the peak value of expression rate is about 89.51% at 48 hours. When the expression of AT2R is at peak value, the ratio of S, G2 and M periods was reduced from 31.7% to 13.9%(P<0.05). The OD values of MTT and BrdU incorporation were reduced by 61.4% and 51.6% respectively(P<0.0(1). the number of VSMC migration was reduced by 62.2%(P<0.05) and the expression of F-actin was also decreased signific antly. Conclusion AdCMV-AT2R induced high level expression of AT2 receptor in cultured rat VSMC and its expression can significantly inhibit the proliferation and migration of rats VSMC in vitro.
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Objective To investigate the role of angiotensin Ⅱ receptors in metabolism of m yocardial collagen. Methods The collagen volume fraction(CVF), perivascular circumferential area(PVCA), hydroproline concentration(HC), ratio o f collagen type Ⅰto type Ⅲ(Ⅰ/Ⅲ),angiotensin Ⅱ(AngⅡ) content and the maximal binding capaci ty (Bmax) of ATRs were studied. The methods of radioimmunoassay(RIA),biochemistry assay, and pathological examin ation were used. Results Ventricular myocardial CVF, PVCA, HC, Ⅰ/Ⅲ, AngⅡ and B max of ATRs in WKY rats were significantly higher than those in sham -operation(SO)(P<0.05 or P<0.01),suggesting the collagen was abnormal ly accumulated in rats heart muscles. AngⅡ was positivily correlated with HC(r1=0.9045, P<0.01). After treament of Irbesartan, All above parameter were reduced significantly(P<0.05 or P<0.01), abnormally accumulated collagen was disap peared. While after treament of CGP42112A,an ATR2 antagonist, Bmax of ATRs was markedly decreased(P<0 .01), but CVF, PVCA, HC,Ⅰ/Ⅲ, AngⅡ showed little changes(P >0.05). Conclusion Obvious hyperplasia of myocardial collagens and remode ling of collagen network occurred after pressure overload. Local produced AngⅡ involves in the process,it's effects is mainly mediated by angiotensin Ⅱ 1 type receptor(ATR1)
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Objective To study the effect on the proliferation of vascular smooth muscle cells(VSMC) after transferring angiotensin Ⅱ(AngⅡ) type 2 receptor(AT2R) gene.Methods The recombinant adenoviral vector AdcCMV-AT2R, containing rat AT2 receptor gene which was constracted by homologous recombination, was used to transfer AT2 receptor gene to rat VSMC in vitro. The expression of AT2R mRNA was detected by RT-PCR. The rate of expression and the change of cell cycle in VSMC were analysed by flow cytometry. Cell devision index, incorporation of bromodeoxyuridine(BrdU)and 3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenytetrazolium bromide(MTT) were used to determine the proliferation of VSMC, respectively.Results RT-PCR showed that the expression of AT2R mRNA increased obviously in transferred VSMC, and the peak value of expression rate was about 89.51% at 48 hours. When the expression of AT2R was at peak value, the ratio of S and G2-M periods was reduced from 31.7% to 13.9%(P<0.05) and the index of cell division from 37.4% to 9.6%(P<0.01). The OD values of MTT and BrdU incorporation were reduced by 61.4% and 51.6% respectively(P<0.01).Conclusion Our study indicates that AdCMV-AT2R can generate high level expression of AT2 receptor in cultured rat VSMC and its expression can significantly inhibit the proliferation of rats VSMC in vitro. It may be a new selection for the gene therapy of restenosis after angioplasty.
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AIM: To investigate the mechanism of vascular smooth muscle cells (VSMC) apoptosis after balloon injury. METHODS: VSMCs apoptosis, Bax protein and Bcl-2 protein was measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and immunohistochemical technique after balloon injury.RESULTS: VSMCs apoptosis occurred in vascular media at 3 days after balloon injury and reached a peak in media and intima where apoptosis was mainly present at 7 days, then decreased. At 28 days after balloon injury, only a few apoptosis cells were in intima. Irbesartan significantly increased VSMCs apoptosis ( P
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AIM: To study the effects of ischemia/reperfusion (I/R) on primary cultured sinoatrial node (SAN) cells and the influence of pinacidil (a K_(ATP) channel activator). METHODS: The SAN cells were isolated from newborn rats and purified. The 48 h cultured cells were cultivated in following mediums: simulated reperfusion solution as normal control, simulated ischemia/reperfusion solution (I/R), Pinacidil+I/R (P+I/R), 5-HD+P+I/R and 5-HD+I/R. Spontaneous action potentials were recorded by ruptured-patch whole-cell technique in current clamp ((I=0)) and the maximum diastolic potential (MDP), upstroke velocity (UV), action potential overshoot (APO), interbeat interval (IBI) and action potential durations at 50% repolarization (APD_(50)) were measured. RESULTS: Compared with control group, simulated ischemia/reperfusion shorten APD_(50), reduced UV, MDP and APO. Exposed to pinacidil, MDP of cells in I/R groups was hyperpolarized; IBI, UV and APO were increased; APD_(50) was shorten. 5-HD couldn't block the effects of pinacdil on APD_(50), IBI and MDP, but reversed its actions on increasing UV and APO. CONCLUSIONS: Pinacidil made changes of AP in I/R group by opening different K_(ATP) channels of SAN cells. The role of this changes on protection in SAN cells during ischemia/reperfusion requires further investigation. [
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Objective To investigate the mechanism of affecting smooth muscle cells apoptosis after balloon injury to vessel Methods The percentage of vascular smooth muscle cells (VSMC) apoptosis and the expression of angiotensin Ⅱ subtype 1 receptor (AT 1R) was measured by terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labeling (TUNEL) and immunohistochemical technique after balloon injury Results Compared with sham's, the expression of AT 1R protein in vascular media was significantly increased at 3 days after balloon injury ( P
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Objective To study the change of the expression of cellular growth factor receptor and its effect on the proliferation and migration of vascular smooth muscle cells (VSMCs) under electrical field. Methods 1) A electrophoresis apparatus was designed for applying the direct current electric fields to the cultured VSMCs, which were separated from the aorta of rats. Interval photographs were used to analyse the direction and distance of VSMCs migration. Cellular growth factors such as platelet derived growth factor receptor (PDGFR), angiotensin II type 1 receptor (AT1R) and its type 2 receptor(AT2R) were studied by immunohistochemistry or immunofluorescence to show the mechanism relation to the migration of VSMCs. Results Under the direct current electric field, the expression of PDGFR was increased obviously and distributed asymmetricly on the membrane of VSMCs. The receptor mainly focused on the cathode side of cellular membrane in electric field. The AT1R were also enhanced in the cells after exposure to the electric field, but the expression of AT2R was uncharged. The longer exposure time to the 150 mV/mm electric field, the more electrotaxis the cells showed. The distance of cellular migration increased obviously with the time to exposure the electric field comparing with the control group. There was no significant difference in the expression of proliferating cell nuclear antigen (PCNA) between two groups. Conclusion External electric fields applying to the rat VSMCs can change the growth characteristic and enhance directional migration of VSMCs. These effects maybe related to the up-regulated expression and re-distribution of some cellular growth factors such as PDGFR, AT1R and AT2R.