目的制备针对胚胎小鼠心肌L型钙通道Cav1.3编码的α1D亚基胞内末端的多克隆抗体。方法通过基因重组从小鼠胚胎心脏中获得2种原核表达质粒pGEX-4T-α1DN/α1DC,在大肠杆菌中经异丙基-β-D硫代半乳糖苷(IPTG)诱导表达谷胱甘肽S转移酶(GST...目的制备针对胚胎小鼠心肌L型钙通道Cav1.3编码的α1D亚基胞内末端的多克隆抗体。方法通过基因重组从小鼠胚胎心脏中获得2种原核表达质粒pGEX-4T-α1DN/α1DC,在大肠杆菌中经异丙基-β-D硫代半乳糖苷(IPTG)诱导表达谷胱甘肽S转移酶(GST)融合蛋白,纯化得到α1D基因N端和C端产物GST-α1DN/α1DC,用此抗原经弗式佐剂、弗式不完全佐剂乳化后免疫新西兰家兔,收集的免疫血清经protein G Agarose亲和纯化后获得IgG型抗α1D蛋白的多克隆抗体。对这2种抗体采用Westernblot法进行特异性检测,ELISA法测定进行效价分析。结果成功构建α1D胞内片段的原核表达载体,表达纯化了GST融合蛋白GST-α1DC/α1DN,制备2种IgG型多克隆抗体,经ELISA检验证实二者效价均较高,滴度在1:256000时仍有信号,其中GST-α1DN的效价更高。用Westernblot法检测发现,2种抗体都均在相对分子质量240000处得到单一的蛋白印迹条带,特异性良好,能识别小鼠心肌细胞中α1D蛋白。结论用重组融合蛋白GST-α1D作为抗原制备出了具有高效价、强特异性的α1D抗体,为探讨α1D蛋白在小鼠胚胎心脏发育过程中分布的时空特征及研究胚胎发育过程中的钙离子相关信号调控活动奠定了基础。展开更多
Background Studies on human, rat and chicken embryos have demonstrated that during the period of outflow tract septation, retraction of the distal myocardial margin of the outflow tract from the junction with aortic ...Background Studies on human, rat and chicken embryos have demonstrated that during the period of outflow tract septation, retraction of the distal myocardial margin of the outflow tract from the junction with aortic sac to the level of semilunar valves leads to the shortening of the myocardial tract. However, the mechanism is not clear. So we investigated the mechanism of outflow tract shortening and remodeling and the spatio-temporal distribution pattern of α-SMA positive cells in the outflow tract cushion during septation of the outflow tract in the embryonic mouse heart Methods Serial sections of mouse embryos from embryonic day 9 (ED 9) to embryonic day 16 (ED 16) were stained with monoclonal antibodies against α-SCA, α-SMA, or desmin, while apoptosis was assessed using the terminal deoxyribonucleotidy transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay Results Between ED 11 and ED 12, the cardiomyocytes in the distal portion of the outflow tract were observed losing their myocardial phenotype without going into apoptosis, suggesting that trans-differentiation of cardiomyocytes into the cell components of the free walls of the intrapericardial ascending aorta and pulmonary trunk The accumulation of α-SMA positive cells in the cardiac jelly began on ED 10 and participated in the ridge fusion and septation of the outflow tract Fusion of the distal ridges resulted in the formation of the facing walls of the intrapericardial ascending aorta and pulmonary trunk Fusion of the proximal ridges was accompanied by the accumulation of α-SMA positive cells into a characteristic central whorl, in which cell apoptosis could be observed Subsequent myocardialization resulted in the formation of the partition between the subaortic and subpulmonary vestibules Conclusions The shortening of the embryonic heart outflow tract in mice may result not from apoptosis, but from the trans-differentiation of cells with cardiomyocyte phenotype in the distal portion of the outflow tract into the cel展开更多
Background： Outflow tract （OFT） septation defects are a common cause of congenital heart disease. Numerous studies have focused on the septation mechanism of the OFT, but have reported inconsistent conclusions. Thi...Background： Outflow tract （OFT） septation defects are a common cause of congenital heart disease. Numerous studies have focused on the septation mechanism of the OFT, but have reported inconsistent conclusions. This study, therefore, aimed to investigate the septation of the aortic sac and the OFT in the early embryonic human heart. Methods： Serial sections of 27 human embryonic hearts from Carnegie stage （CS） 10 to CS19 were immunohistochemically stained with antibodies against α-smooth muscle actin （α-SMA） and myosin heavy chain. Results： At CS10-CS11, the OFT wail was an exclusively myocardial structure that was continuous with the aortic sac at the margin of the pericardial cavity. From CS13 onward, the OFT was divided into nonmyocardial and myocardial portions. The cushion formed gradually, and its distal border with the OFT myocardium was consistently maintained. The aortic sac between the fourth and sixth aortic arch arteries was degenerated. At CS16, the α-SMA-positive aortopulmonary septum formed and fused with the two OFT cushions, thus septating the nonmyocardial portion of the OFT into two arteries. At this stage, the cushions were not fused. At CS19, the bilateral cushions were fused to septate the myocardial portion of the OFT. Conclusions： Data suggest that the OFT cushion is formed before the aortopulmonary septum is formed. Thus, the OFT cushion is not derived from the aortopuhnonary septum. In addition, the nonmyocardial part of the OFT is septated into the aorta and pulmonary trunk by the aortopulmonary septum, while the main part of the cushion fuses and septates the myocardial portion of the OFT.展开更多
本文应用不同浓度的硒(0.50、0.75、1.00μg/ml)和锌(0.10、0.50、1.00μg/ml)分别加入心肌细胞培养液中,观察两者对~3H-TdR 对 DNA 参入率的影响.实验结果显示,加入 Se 0.50μg/ml 及 Zn 0.50～1.00μg/ml,能增加~3H-TdR 的参入率。
文摘目的制备针对胚胎小鼠心肌L型钙通道Cav1.3编码的α1D亚基胞内末端的多克隆抗体。方法通过基因重组从小鼠胚胎心脏中获得2种原核表达质粒pGEX-4T-α1DN/α1DC,在大肠杆菌中经异丙基-β-D硫代半乳糖苷(IPTG)诱导表达谷胱甘肽S转移酶(GST)融合蛋白,纯化得到α1D基因N端和C端产物GST-α1DN/α1DC,用此抗原经弗式佐剂、弗式不完全佐剂乳化后免疫新西兰家兔,收集的免疫血清经protein G Agarose亲和纯化后获得IgG型抗α1D蛋白的多克隆抗体。对这2种抗体采用Westernblot法进行特异性检测,ELISA法测定进行效价分析。结果成功构建α1D胞内片段的原核表达载体,表达纯化了GST融合蛋白GST-α1DC/α1DN,制备2种IgG型多克隆抗体,经ELISA检验证实二者效价均较高,滴度在1:256000时仍有信号,其中GST-α1DN的效价更高。用Westernblot法检测发现,2种抗体都均在相对分子质量240000处得到单一的蛋白印迹条带,特异性良好,能识别小鼠心肌细胞中α1D蛋白。结论用重组融合蛋白GST-α1D作为抗原制备出了具有高效价、强特异性的α1D抗体,为探讨α1D蛋白在小鼠胚胎心脏发育过程中分布的时空特征及研究胚胎发育过程中的钙离子相关信号调控活动奠定了基础。
基金ThisworkwassupportedbytheScientificResearchFoundationforReturnedOverseasChineseScholars,StateEducationMinistryandEducationMinistryofShanxiProvince (No 9845 )
文摘Background Studies on human, rat and chicken embryos have demonstrated that during the period of outflow tract septation, retraction of the distal myocardial margin of the outflow tract from the junction with aortic sac to the level of semilunar valves leads to the shortening of the myocardial tract. However, the mechanism is not clear. So we investigated the mechanism of outflow tract shortening and remodeling and the spatio-temporal distribution pattern of α-SMA positive cells in the outflow tract cushion during septation of the outflow tract in the embryonic mouse heart Methods Serial sections of mouse embryos from embryonic day 9 (ED 9) to embryonic day 16 (ED 16) were stained with monoclonal antibodies against α-SCA, α-SMA, or desmin, while apoptosis was assessed using the terminal deoxyribonucleotidy transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay Results Between ED 11 and ED 12, the cardiomyocytes in the distal portion of the outflow tract were observed losing their myocardial phenotype without going into apoptosis, suggesting that trans-differentiation of cardiomyocytes into the cell components of the free walls of the intrapericardial ascending aorta and pulmonary trunk The accumulation of α-SMA positive cells in the cardiac jelly began on ED 10 and participated in the ridge fusion and septation of the outflow tract Fusion of the distal ridges resulted in the formation of the facing walls of the intrapericardial ascending aorta and pulmonary trunk Fusion of the proximal ridges was accompanied by the accumulation of α-SMA positive cells into a characteristic central whorl, in which cell apoptosis could be observed Subsequent myocardialization resulted in the formation of the partition between the subaortic and subpulmonary vestibules Conclusions The shortening of the embryonic heart outflow tract in mice may result not from apoptosis, but from the trans-differentiation of cells with cardiomyocyte phenotype in the distal portion of the outflow tract into the cel
文摘Background： Outflow tract （OFT） septation defects are a common cause of congenital heart disease. Numerous studies have focused on the septation mechanism of the OFT, but have reported inconsistent conclusions. This study, therefore, aimed to investigate the septation of the aortic sac and the OFT in the early embryonic human heart. Methods： Serial sections of 27 human embryonic hearts from Carnegie stage （CS） 10 to CS19 were immunohistochemically stained with antibodies against α-smooth muscle actin （α-SMA） and myosin heavy chain. Results： At CS10-CS11, the OFT wail was an exclusively myocardial structure that was continuous with the aortic sac at the margin of the pericardial cavity. From CS13 onward, the OFT was divided into nonmyocardial and myocardial portions. The cushion formed gradually, and its distal border with the OFT myocardium was consistently maintained. The aortic sac between the fourth and sixth aortic arch arteries was degenerated. At CS16, the α-SMA-positive aortopulmonary septum formed and fused with the two OFT cushions, thus septating the nonmyocardial portion of the OFT into two arteries. At this stage, the cushions were not fused. At CS19, the bilateral cushions were fused to septate the myocardial portion of the OFT. Conclusions： Data suggest that the OFT cushion is formed before the aortopulmonary septum is formed. Thus, the OFT cushion is not derived from the aortopuhnonary septum. In addition, the nonmyocardial part of the OFT is septated into the aorta and pulmonary trunk by the aortopulmonary septum, while the main part of the cushion fuses and septates the myocardial portion of the OFT.
文摘本文应用不同浓度的硒(0.50、0.75、1.00μg/ml)和锌(0.10、0.50、1.00μg/ml)分别加入心肌细胞培养液中,观察两者对~3H-TdR 对 DNA 参入率的影响.实验结果显示,加入 Se 0.50μg/ml 及 Zn 0.50～1.00μg/ml,能增加~3H-TdR 的参入率。
