超极化激活环化核苷酸门控通道2基因转染后293T细胞中目的基因的表达

背景：研究证实超极化激活环化核苷酸门控通道电流在调控心脏的自发搏动中起着非常重要的作用。目的：观察超极化激活环化核苷酸门控通道2基因重组质粒转染后293T细胞中目的基因在核酸和蛋白质水平的表达。方法：采用脂质体转染试剂Lipofectamine2000将含全长超极化激活环化核苷酸门控通道2基因的质粒CMV-hHCN2-3xHA-IRES-EGFP转染至293T细胞中。结果与结论：用脂质体转染试剂Lipofectamine2000成功地将质粒CMV-hHCN2-3xHA-IRES-EGFP转染至293T细胞中。反转录PCR定性地检测到在100-250bp处可见高度特异DNA的条带,与携带超极化激活环化核苷酸门控通道2基因的质粒扩增出的片段位置相同。经过RT-PCR、Western-blot方法检测提示超极化激活环化核苷酸门控通道2基因在mRNA和蛋白水平都有高表达。表明质粒CMV-hHCN2-3xHA-IRES-EGFP成功转染后的293T细胞中超极化激活环化核苷酸门控通道2基因可以在核酸和蛋白水平表达。     

促红细胞生成素抑制急性梗死心肌炎症因子的表达

背景：研究表明,炎症细胞因子可影响心肌梗死后的预后,在心脏重构的进程中起重要作用。同时促红细胞生成素的促红细胞生成之外的非造血效应也被广泛证实：促红细胞生成素可通过与靶细胞膜表面的促红细胞生成素受体结合而减少炎症反应,从而减少心肌缺血后的再灌注损伤。 目的：观察重组人促红细胞生成素对大鼠急性心肌梗死后心脏重构中炎症因子表达的影响。 方法：通过结扎左冠状动脉前降支建立SD大鼠急性心肌梗死模型,分为5组,假手术组注射生理盐水,手术对照组造模后注射生理盐水,SB203580组造模后注射p38 MAPK的高选择性阻断剂SB203580,促红细胞生成素组造模后注射促红细胞生成素注射液,促红细胞生成素+SB203580组造模后注射促红细胞生成素+ SB203580混合液,分别在造模前、造模后1 d、1周、2周及4周进行尾静脉采血,酶联免疫吸附法检测血清中白细胞介素1β、白细胞介素6、肿瘤坏死因子α水平变化。 结果与结论：造模前各组大鼠血清白细胞介素1β、白细胞介素6、肿瘤坏死因子α检测值差异均无显著性意义(P > 0.05)。假手术组各时段白细胞介素1β、白细胞介素6、肿瘤坏死因子α检测值差异无显著性意义(P > 0.05),其余4组不同时段各因子检测值呈现造模后1 d最高,造模后4周回降的趋势(P < 0.05)。造模后手术对照组血清各因子检测值较其他组升高明显,而假手术组血清各因子检测值均低于其他4组(P < 0.05);使用药物进行干预的3组中,促红细胞生成素+SB203580组各因子检测值较低(P < 0.05),而促红细胞生成素组与SB203580组各因子检测值差异无显著性意义(P > 0.05)。提示重组人促红细胞生成素抑制了大鼠急性心肌梗死后心脏重构中炎症因子白细胞介素1β、白细胞介素6、肿瘤坏死因子α的表达,重组人促红细胞生成素抑制炎症因子表达的机制可能与转化生长因子β1-TAK1-p38 MAPK信号路径相关。     

超极化激活环化核苷酸门控通道2基因转染后293T细胞中目的基因的表达

背景：研究证实超极化激活环化核苷酸门控通道电流在调控心脏的自发搏动中起着非常重要的作用。 目的：观察超极化激活环化核苷酸门控通道2基因重组质粒转染后293T细胞中目的基因在核酸和蛋白质水平的表达。 方法：采用脂质体转染试剂Lipofectamine2000将含全长超极化激活环化核苷酸门控通道2基因的质粒CMV-hHCN2-3xHA-IRES-EGFP转染至293T细胞中。 结果与结论：用脂质体转染试剂Lipofectamine2000成功地将质粒CMV-hHCN2-3xHA-IRES-EGFP转染至293T细胞中。反转录PCR定性地检测到在100-250 bp处可见高度特异DNA的条带,与携带超极化激活环化核苷酸门控通道2基因的质粒扩增出的片段位置相同。经过RT-PCR、Western-blot方法检测提示超极化激活环化核苷酸门控通道2基因在mRNA和蛋白水平都有高表达。表明质粒CMV-hHCN2-3xHA-IRES-EGFP成功转染后的293T细胞中超极化激活环化核苷酸门控通道2基因可以在核酸和蛋白水平表达。     

体外培养猪骨髓间充质干细胞hHCN2基因的转染及表达

背景：通过超极化活化的阳离子电流调制心脏自律性成为该领域研究的焦点。 目的：将起搏基因质粒CMV-hHCN2-3xha-IRES-EGFP转染到猪骨髓间充质干细胞,检测其在骨髓间充质干细胞中核酸和蛋白水平是否表达。 方法：采用单纯直接贴壁法或密度梯度离心结合直接贴壁法分离、纯化、增殖获得骨髓间充质干细胞,脂质体2000转染质粒CMV-hHCN2-3xha-IRES-EGFP至骨髓间充质干细胞。 结果与结论：单纯直接贴壁法收集到的细胞增殖速度慢,而密度梯度离心结合直接贴壁法细胞增殖速度较快,原代细胞培养第3代后转染48 h荧光显微镜下可见骨髓间充质干细胞发出荧光,对照组无荧光。细胞转染率达15%-20%。RT-PCR检测及Western blot 印迹检测证明了hHCN2基因在骨髓间充质干细胞有表达。密度梯度离心结合直接贴壁法较单纯直接贴壁法培养骨髓间充质干细胞的培养效率更高。转染目的基因hHCN2的骨髓间充质干细胞蛋白有表达。     

替罗非班治疗急性心肌梗死患者发生血小板减少症的临床分析

目的探讨急性心肌梗死患者应用替罗非班期间发生血小板减少症的临床特点、处理及预后。方法 2005年4月至2010年6月,341例接受替罗非班治疗的急性心肌梗死征患者中,发生5例生血小板减少症,发生率为1.47%。5例患者中,4例为男性,年龄45~78岁。1例为女性,年龄为72岁。回顾性分析5例患者的相关临床情况。结果 5例患者发生血小板减少的时间为接受替罗非班治疗后的血小板减少症2~12 h,血小板最低值在(10~81)×109之间,其中重度1例,极重度1例。停用替罗非班24~144 h后血小板计数值恢复正常。其中3例发生出血并发症;无需要输血小板和输红细胞的病例,未发生再梗死及死亡。结论急性心肌梗死患者应用替罗非班过程中可以发生严重的血小板减少症,虽然发生率相对较低,但在应用过程中仍需要加强监测。     

促红细胞生成素对高糖环境诱导下体外培养心脏成纤维细胞的影响

目的探讨促红细胞生成素(EPO)对高糖环境诱导过程中体外培养的乳鼠心脏成纤维细胞(CF)增殖及其表型转化的影响。方法用乳鼠原代细胞培养CF,在促进CF细胞表型转化的过程中,选用高糖培养基进行诱导,添加EPO进行干预。将同一批CF细胞随机分为3组:对照组(5.5 mmol/L葡萄糖)、高糖组(25 mmol/L葡萄糖)、高糖+EPO组(25 mmol/L葡萄糖,20 k U/L EPO)。采用免疫组织化学法鉴定CF,酶联免疫吸附法检测Ⅰ型、Ⅲ型胶原含量,Western blot检测纤维化指标α-平滑肌肌动蛋白(α-SMA)的表达。结果免疫组织化学染色结果为纤维连接蛋白、波形蛋白阳性,α-肌动蛋白阴性,证实培养的细胞为心脏成纤维细胞。与对照组比较,高糖组Ⅰ型、Ⅲ型胶原表达量都明显增加(P0.01),而高糖+EPO组Ⅰ型、Ⅲ型胶原表达量都显著低于高糖组(P0.01)。高糖组α-SMA表达较对照组明显增高(P0.05),而高糖+EPO组α-SMA表达较高糖组明显降低(P0.05)。结论高糖环境下能有效的诱导CF发生表型转化,而EPO能够逆转在高糖环境作用下诱导发生的心肌纤维化。     

二氮嗪减轻兔心肌缺血再灌注损伤后的心肌细胞凋亡

目的探讨二氮嗪对兔心脏缺血再灌注损伤过程中心肌细胞凋亡和心肌细胞bcl-2和bax基因表达的影响.方法24只兔随机分成假手术组(P组)、缺血再灌注组(IR组)、缺血预适应组(IP组)和二氮嗪组(DP组).阻断和松开左冠脉前降支制作缺血再灌注模型,缺血5 min/再灌注5 min连续3次循环诱导预适应.DP组在缺血前缓慢静脉注射二氮嗪3 mg/kg.再灌注结束后测算左室心肌梗死面积,取缺血区心肌行TUNEL法检测心肌凋亡细胞和免疫组化检测bcl-2和bax蛋白的表达.结果DP组和IP组梗死面积明显小于IR组(P＜0.001).凋亡细胞百分数DP组(34±5)%和IP组(32±6)%较IR组(56±8)%显著减少(P＜0.001).和IR组相比,bcl-2表达在IP组和DP组明显升高(P均＜0.01),bax基因表达在IP组和DP组则明显降低(P均＜0.001).结论二氮嗪能通过调节bcl-2和bax的表达来减轻兔心肌缺血再灌注损伤后的心肌细胞凋亡.     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.     

Erythropoietin inhibits angiotensin Ⅱ induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway

Objective To explore the effect of erythropoietin (EPO) on angiotensin Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway. Methods Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by Ang Ⅱ in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit. Results EPO (20 U/ml) significantly inhibited Ang Ⅱ induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P ＜0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P ＜ 0.05), increased the NO production (P ＜0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P ＜ 0.05). Conclusion EPO attenuates Ang Ⅱ induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.