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BACKGROUND: It has been found that xenogenic extracellular matrix (ECM) may cause a strong inflammatory response in humans during clinical application of decellularized porcine heart valve (synergraft valves). An early inflammatory reaction severely weakens matrix structure of valve wall, leading to structural rupture and decay of grafts. From Synergraft's event, the decellularized porcine heart valves still had immunogenicity, especially for pediatric patients. The mechanisms by which the ECM triggers this immune process need to be further evaluated. OBJECTIVE: To find the difference of gene sequence between human and porcine ECM and to identify the ECM immunogenicity based on bioinformatics. DESIGN, TIME AND SETTING: A contrast study between human and porcine ECM based on type IV collagen was performed at the Laboratory of Cardiothoracic Surgery, Changhai Hospital, the Second Military Medical University of Chinese PLA from June 2008 to February May.MATERIALS: The fresh porcine heart valves were obtained from Shanghai Wufengshangshi Slaughter House. Decellularized porcine aortic valves, hybridoma cells, and monoclonal antibodies were provided by our laboratory. METHODS: Similar region and conservative site of gene sequence among human, porcine, and rat were compared so as to look for common similar region, site, and sequence difference and investigate the segment which caused common and different gene sequence. Type IV collagen monoclonal antibody was used to evaluate the persistence of ECM of decellularized porcine heart valve following immunohistochemical staining. MAIN OUTCOME MEASURES: Type IV collagen gene sequence; efficacy of self-made antibody using immunohistochemistry; effect of self-made antibody on type IV collage of decellularized porcine heart valve. RESULTS: The differential gene serial in type IV collagen protein was found out by bioinformatics method. Monoclonal antibodies were successfully produced by human-mouse hybridoma technique. Residual porcine ECM was observed on decellularized porcine heart valve. CONCLUSION: Residual porcine ECM was observed on decellularized porcine heart valve and had immunogenicity.
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OBJECTIVE: To observe the effects of Xuefu Zhuyu Capsule (XFZYC), a compound traditional Chinese herbal medicine, on endothelin-1 (ET-1) release in myocardium and vascular endothelium and nitric oxide (NO)/nitric oxide synthase (NOS) system of swines after acute myocardial infarction (AMI) and reperfusion, and to explore the action mechanisms of XFZYC in improving the endothelium function. METHODS: Forty-five Yorkshire swines were randomized into 3 groups: sham-operated group, untreated group and XFZYC-treated group. A Yorkshire swine model of reperfusion in AMI was established by ligation of left anterior descending coronary artery for 90 min followed by 2 h relaxation. The content of serum ET-1 and NO was measured by radioimmunoassay before and after AMI and after reperfusion, respectively. Twenty-four hours after operation, all Yorkshire swines underwent diagnostic coronary angiography to delineate coronary arteries. The expressions of ET-1 and endothelial nitric oxide synthase (eNOS) in myocardial tissue of ischemic area were quantified with Western blotting. Microvessel density of the implanting sites was assessed by using HE staining. RESULTS: Compared with the untreated group, the levels of serum ET-1 after AMI and reperfusion were significantly decreased in XFZYC-treated group (P<0.01), while the NO levels after AMI and reperfusion in XFZYC-treated group were significantly increased (P<0.01). There was no significant difference in diagnostic coronary angiography between XFZYC-treated group and untreated group (P=0.253). Western blotting showed that the level of ET-1 in ischemic area in XFZYC-treated group was lower than that in the untreated group (P<0.01), while the eNOS protein expression in XFZYC-treated group was higher than that in the untreated group (P<0.01). The results of HE staining and microvessel density analysis of the implanting sites all showed that the degree of telangiectasis was reduced, the cardiac muscle damage was improved, and the density of capillaries was increased obviously in XFZYC-treated group as compared with the untreated group. CONCLUSION: The endothelium injury may be one of the important mechanisms for no-reflow phenomenon. XFZYC may reduce the no-reflow by protecting endothelium cells.
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Objective To develop a novel method for treating complete heart block by autotransplantation of simus node node cells to right ventricular anterior wall.Methods Twenty healthy mongrel dogs were involved in the present study.The dogs were randomly assigned to transplant group or control group(n=10).The sinus node (SN)was harvested and isolated in vitro after an electronic pacemaker was implanted and complete heart bolck was introduced.The SN cells from dogs of transplant group were injected to autogenic right ventricular wall.Commensurable culture medium was implanted to ghe same position of dogs in control group.Two weeks later,detailed electropohysiological study was performed.For investigating the variation of the rhythm,epinephrine was administrated through femoral vein to dogs of transplant group.Results Most of isolated SN cells from dogs were thin-spindle shape,and cell activity was fine.The SNs by VG stained displayed typical structural feature.2 weeks after cell autotransplantation,higher heart rates were achieved from transplant group than that in control group(P＜0.05).This rhythm was stable in 4 weeks and became faster remarkably after administration of eninephrine(P＜0.05).Conclusion SN cell of dogs tutorgrfted into right ventricular anterior wall can form new pacemaker site in ventrcle and improve ventricular rate of complete heart bolck.This pacemaker site can also be regulated by epinephrine.
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Objective To observe the inhibition of vascular endotheilal growth factor (VEGF) gene transcribition and translation with antisense VEGF165 adenovirus vector transfection. Method To transfect the synovial cells with antisense VEGF165 adenovirus and detect the gene translation level with northern-blot method. Result The Northern-blot showed that: the transcription of VEGF gene were inhibited markedly after the antisense VEGF165 gene transfection for 3 days and the inhibition efficiency was obivious in 4th day. In the 5th day, the VEGF translation signal was hardly detected. Conclusion The antisense VEGF165 adenovirus gene transfection can inhibite not only the transcription but the translation of VEGF gene in synovial cells. Thus it can inhibit the secretion of VEGF in synovial cells with high efficiency.
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Objective To construct the replication-deficient recombinant adenovirus coding ARP2 gene before the studies of gene transfection to ischemic myocardium.Methods From Dec.2004 to Oct.2005,in the Department of Cardiology,Changhai Hospital of the Second Military Medical Univesity,the ARP2-pAxCAwt was constructed by inserting the cDNA of interest into the SwaI site of pAxCAwt.Results The right direction of the insert was confirmed by restriction.Conclusion The direction of the insert must be confirmed by restriction analysis,and the DNA package protein is much helpful during the transfection of the recombinant cosmid to E.coli.
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AIM: To generate the angiopoietin-1 recombinant adenovirus for the further studies about adenoviral mediated angiopoietin-1 gene transfer in ischemic myocardium and its effect on the development of neoangiogenesis.METHODS: Angiopoietin-1-PAxCAwt cosmid DNA and adenoviral DNA-TPC were cotransfected to 293 cells by the use of calcium phosphate precipitation method. All recombinant adenoviruses were propagated and titrated in 293 cells, purified by cesium chloride density purification, dialyzed, and stored at-70 ℃. RESULTS: The result shows that the direction of the insert in the cosmid is correct and the replication-deficient angiopoietin-1 recombinant adenovirus was generated efficiently by COS/TPC homologous recombination, with the titers of 5.6?10 11 pfu/L. The viral stocks were demonstrated to be free of replication-competent wild type adenoviruses. The virus stocks in high titer were harvested in our experiment. CONCLUSION: The COS/TPC is an efficient method to prepare recombinant adenovirus and the angiopoietin-1 recombinant adenovirus could be further used in gene therapy.
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Objective To study the transfection efficiency, protein expression, and effect of adenovirus-mediated transfection on microvascular endothelial cells transfected by angiopoietin-related protein 2(Ad. ARP2)gene. Methods Mice coronary microvascular endothelial cells(CMECs) were isolated, cultured and transferred by Ad-ARP2. The transfection efficiency and cellular toxicity of adenovirus vector to CMECs were detected by immunofluorescence staining. Expression of Ad. ARP2 in CMECs and the secreted materials in culture medium were measured by Western blot and ELISA and compared among groups of Ad. ARP2, Ad. null, and PBS control. Vascular endothelial cells were incubated with conditional medium containing secreted ARP2, and effects on cells sprouting were observed in matrigel. Results Adenovirus-transfected CMECs showed a very high efficiency. When multiplicity of infection (MOD was 200, the transfection efficiency was 93. 5% ,and no harmful effect on CMECs growth was found. When CMECs were transfected with Ad. ARP2, there was a high ARP2 expression, which was significantly different from that with Ad. Null or PBS. The conditional medium containing ARP2 had an excellent ability to stimulate sprout of CMECs which phenomenon could not be seen in the control groups. Conclusions Adenovirus vector can be transferred into CMECs efficiently and safely. Ad. ARP2 gene transfection allows a high transient expression, and the expression products can stimulate the sprout of microvascular endothelial cells in vitro very well.
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Objective To investigate the evaluation of multimodality MR imaging on the transplantation of autologous marrow stromal cells transfected ex vivo by vascular endothelial growth factor(VEGF)gene in a porcine chronic ischemic heart disease model. Methods Pigs of chronic ischemic heart disease model were randomly divided into two groups: treatment with marrow stromal cells transfected ex vivo by VEGF(Group I,n=9) and treatment with adenovirus served as control(Group Ⅱ,n=7).Four weeks after therapy,ejection fraction,ischemic segment and infracted segment were detected by multimodality MR imaging.The percentage of infarction area,CM-DiI labeled cells on fluorescence microscopy,vessel density were detected on specimen. Results In Group Ⅰ,in comparison with Group Ⅱ,the ejection fraction was significantly improved,the ischemic and infarction segments decreased(P
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Objective:To construct a recombinant adenovirus containing cytosine deaminase( CD ) gene and thymidine kinase( TK ) fusion gene for the gene therapy research of malignant tumors. Methods: A recombinant cosmid containing CD and TK fusion gene was constructed, and then mixed with DNA TPC and co transfected to the 293 cells by calcium phosphate coprecipitation. Results: The results of restriction and PCR showed the insertion was right and the recombinant adenovirus generated contained the CD and TK fusion gene without replication competent adenovirus. Conclusion: The recombinant adenovirus generated is E1 and E3 deleted and contains double suicide gene needed, which can be further studied for gene therapy.
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Vascular endothelial growth factor (VEGF)165 cDNA were inserted into the expression plasmid pcDNA3.1 site in an antisense orientation to produce antise VEGF165 expression vector.Then electrophoresis and sequencing were carried out,the antisense vector were verified and could be used for related study of antisense gene expression.
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Gap junction, a special membrane structure communicating many kinds of cells, is composed of connexin.Changes of connexin levels and distribution can lead to changes of conduction velocity of electric couple and increase of reentrant arrhythmia, finally to atrial fibrillation. This article elucidates the relationship between cardiac gap junction protein connexin and atrial fibrillation.
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Objective:To investigate the survival of rabbits receiving autologous left auricle cardiomyocytes transplantation into the infarcted myocardium and to assess the effect of transplantation on the peripheral blood supply, heart rate, and cardiac function. Methods: Healthy adult rabbits were randomly divided into transplantation group (n=8) and control group (n=8). The myocardial infarction (MI) models were established by ligating the left anterior descending arteries in all rabbits. Four weeks later the left auricles were harvested and the auricle cardiomyocytes were isolated and labelled with DAPI ex vivo. Rabbits in the transplantation group were injected with DAPI-labelled cell suspension into the infarcted areas and those in control group received culture medium. All the rabbits were examined by electrocardiogram (ECG) and 2-D ultrasonic cardiogram (UCG) 4 weeks after transplantation. Specimens were harvested from the transplanted areas and observed histologically. Results: All rabbits survived 4 weeks after transplantation. ECG showed that the heart rate in transplantation group was faster than that in control group (P
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Objective:To modify the pulsatile bioreactor we constructed previously for simulating the high-flow, high-pressure hemodynamics of heart valve in vivo, and to experimentally cultivate the tissue-engineered heart valves (TEHV) in the modified bioreactor. Methods: T-PLS system (NewheartBio Co., Ltd Korea) was used to generate pulsatile flow in the modified bioreactor and we designed a new air-exchange pathway to avoid contamination. The TEHV were made by seeding human bone mesenchymal stem cells (BMSCs) on de-cellularized porcine heart valve. After cultured under static condition for 4 d, the TEHVs were moved to the modified bioreactor and exposed to low-flow (0-600 ml/min) or high-flow(0-4 800 ml/min) pulsatile hydrodynamics for 7d, then the cells on TEHVs were evaluated. Results: After modification, the flow range expanded from (0-1 200) ml/min to (0-6 000) ml/min and the pressure range expanded from (0-40) mmHg to (0-180) mmHg. In culture experiments, 26.3% of the seeded cells remained under low-flow environment and cells were completely lost under the high-flow dynamics. Conclusion: The modified bioreactor can basically simulate the dynamics of heart valve in vivo and can be used in TEHV cultivation research. However, the current TEHV can not tolerate the high-flow pulsatile hydrodynamics.
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Objective: To investigate the angiogenic potentiality of angiogenin in ischemic myocardium and estimate the possibility of its application in biologic treatment. Methods: Recombinant human angiogenin derivative Asp116His was applied in an acute myocardial infarction model in rabbits which were established by direct intramyocardial injection into the border zone of the ischemic myocardium. The animals were sacrificed on the eighth postoperative day and their hearts were harvested and subjected to histologic studies. Results: Myocardium around the injection sites was apparently less ischemia in the experiment group as compared with control groups. A large number of small vessels were observed in the interstitial tissues and even more were revealed in the myocardium on immunohistochemistry studies. Conclusion: The results indicate for the first time that angiogenin or its derivatives may act on ischemic myocardium and stimulate neo angiogenesis, which holds great promise for their future application in biologic revascularization of ischemic myocardium.
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Objective:To evaluate the protective effects of adenovirus-mediated gene transfer of soluble complement receptor type 1(sCR1) on acute myocardium ischemia in mice.Methods: Twenty-seven SD rats were subjected to left anterior descending coronary artery(LAD) occlusion(30 min) and reperfusion.In the treatment group(n=14),a mixture of adenovirus carrying sCR1 and LacZ was injected into the ischemic zone(100 ?l,10~(10)pfu)5 min before reperfusion;in the control group,only adenovirus carrying LacZ was injected(n=13).Echocardiography was performed 2 weeks later,followed immediately by pathologic examination.Results: Echocardiographic results of the treatment group were better than those of the control group((P
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Objective To culture normal adult esophageal epithelial cells and establish a normal esophageal epithelial cell line to provide experimental materials for further studies in vitro.Methods Normal esophageal epithelium samples were obtained in sterile phosphate buffered saline supplemented with antibiotics from the normal part of the esophagus of a patient with esophageal carcinoma.Tissue was cleaned of all connective tissues and muscles and rinsed in phosphate buffered saline 5-6 times.The mucosal layer was minced into small pieces and dissociated into a single cell suspension by 0.25% Dispase and 0.25% trypsin/0.02%EDTA.Cells were grown in keratinocyte serum-free media.The cultured cells were identified through their morphological characteristics and immunohistochemical staining.The proliferative capacity of the cultured cells was also examined.Results The cultured cells showed microscopic features of epithelial cells and were positive in keratin and epithelial membrane antigen staining.Eight days after primary culture,the cells displayed a cobblestone morphology reaching 80%-90% confluency and were passaged successfully with 0.25% trypsin/0.02%EDTA.The cell cycle analysis showed about 77.60% of the cultured cells was in G0/G1 phase and the others in S/G2/M phase.Conclusion\ The culture methods and techniques used in the experiments are convenient and suitable for the primary culture and subculture of normal adult esophageal epithelial cells.