腺病毒载体介导C/ebp delta改善小鼠心脏缺血性损伤
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摘要
目的探讨腺病毒载体介导C/ebp delta对缺血性心脏病的治疗作用。方法构建腺病毒C/ebp delta增强型绿色荧光蛋白(Ad C/ebp delta-EGFP)载体,局部注射小鼠心脏左心室前壁,观察其对心肌细胞的转染效率。取C57BL/6小鼠,以腺病毒空载体局部注射心肌为对照组,Ad C/ebp delta-EGFP载体局部注射心肌为实验组。2组小鼠心肌局部注射腺病毒载体后行冠状动脉左前降支结扎,用心脏超声检测C/ebp delta对心脏功能的影响。无痛苦处死小鼠,收获心脏,TUNEL染色观察C/ebp delta对心肌细胞凋亡的影响;CD31免疫组化观察C/ebp delta对心肌梗死后梗死交界区毛细血管密度的影响;蛋白免疫印迹检测C/ebp delta对心肌梗死后心脏低氧诱导因子-1(HIF-1)、血红素加氧酶-1(HO-1)及血管内皮生长因子(VEGF)蛋白表达的影响。结果成功构建Ad C/ebp delta-EGFP载体。超声显示实验组小鼠心肌梗死后心脏左室短轴缩短分数(LVFS,0.323±0.031)和心脏左室射血分数(LVEF,0.605±0.085)较对照组(0.221±0.031和0.464±0.071)显著改善(P<0.05);心肌细胞凋亡率较对照组明显减少(20.36%±3.07%vs. 44.26%±6.25%,P<0.01);心肌梗死交界区毛细血管密度(毛细血管数目/每个视野)较对照组显著增加(145.41±15.52 vs. 77.60±5.19,P<0.01);HIF-1、HO-1和VEGF蛋白的表达较对照组均显著增加(P<0.01)。结论腺病毒介导的C/ebp delta可能通过激活HIF-1信号通路增加HO-1和VEGF表达,进而通过保护心肌细胞及促进心脏血管新生治疗缺血性心脏病。
Objective To investigate the therapeutic effect of adenoviral C/ebp delta vector on ischemic heart disease.Methods The Ad C/ebp delta-EGFP was constructed, and was injected into myocardium to observe the transductive efficiency to the cardiac myocytes. C57 BL/6 mice were used in the experiments. The mice were divided into two groups:control group and experimental group. In control group, the Ad-EGFP was injected into myocardium. In experimental group,the Ad C/ebp delta-EGFP was injected into myocardium. The left anterior descending coronary artery was ligated after Ad C/ebp delta-EGFP was injected into the left anterior wall of the left ventricle. The cardiac function of mice was examined by heart echocardiography. Mice were sacrificed without pain. The hearts were harvested, and the effect of C/ebp delta on myocardial apoptosis was observed with TUNEL staining. The effect of C/ebp delta on angiogenesis was performed by CD31 immunostaining. The effects of C/ebp delta on the expressions of HIF-1, HO-1 and VEGF proteins in myocardium after myocardial infarction were detected by immunoblotting.Results The Ad C/ebp delta-EGFP was successfully constructed.The expression level of C/ebp delta in the myocardium was significantly higher in experimental group than that of control group. Local injection of adenoviral vector carrying C/ebp delta significantly improved LVFS(0.323±0.031 in experimental group versus 0.221±0.031 in control group, P<0.05) and LVEF(0.605±0.085 in experimental group versus 0.464±0.071 in control group, P<0.05). The myocardial cell apoptotic rate was significantly reduced in experimental group(20.36%±3.07%) than that of control group(44.26%±6.25%, P<0.01). The vascular density at the border area of myocardial infarction was increased(145.41±15.52 number of capillaries/per view in experimental group versus 77.60±5.19 number of capillaries/per view in control group, P<0.01) after heart infarction. The expressions of HIF-1, HO-1, and VEGF proteins were significantly increased in experimental group compared with those of control group(P<0.01).Conclusion Adenoviralvector mediated C/ebp delta gene may activate HIF-1, which increases expressions of HO-1 and VEGF, resulting in protection of cardiomyocytes and promote angiogenesis in ischemic heart.
Objective To investigate the therapeutic effect of adenoviral C/ebp delta vector on ischemic heart disease.Methods The Ad C/ebp delta-EGFP was constructed, and was injected into myocardium to observe the transductive efficiency to the cardiac myocytes. C57 BL/6 mice were used in the experiments. The mice were divided into two groups:control group and experimental group. In control group, the Ad-EGFP was injected into myocardium. In experimental group,the Ad C/ebp delta-EGFP was injected into myocardium. The left anterior descending coronary artery was ligated after Ad C/ebp delta-EGFP was injected into the left anterior wall of the left ventricle. The cardiac function of mice was examined by heart echocardiography. Mice were sacrificed without pain. The hearts were harvested, and the effect of C/ebp delta on myocardial apoptosis was observed with TUNEL staining. The effect of C/ebp delta on angiogenesis was performed by CD31 immunostaining. The effects of C/ebp delta on the expressions of HIF-1, HO-1 and VEGF proteins in myocardium after myocardial infarction were detected by immunoblotting.Results The Ad C/ebp delta-EGFP was successfully constructed.The expression level of C/ebp delta in the myocardium was significantly higher in experimental group than that of control group. Local injection of adenoviral vector carrying C/ebp delta significantly improved LVFS(0.323±0.031 in experimental group versus 0.221±0.031 in control group, P<0.05) and LVEF(0.605±0.085 in experimental group versus 0.464±0.071 in control group, P<0.05). The myocardial cell apoptotic rate was significantly reduced in experimental group(20.36%±3.07%) than that of control group(44.26%±6.25%, P<0.01). The vascular density at the border area of myocardial infarction was increased(145.41±15.52 number of capillaries/per view in experimental group versus 77.60±5.19 number of capillaries/per view in control group, P<0.01) after heart infarction. The expressions of HIF-1, HO-1, and VEGF proteins were significantly increased in experimental group compared with those of control group(P<0.01).Conclusion Adenoviralvector mediated C/ebp delta gene may activate HIF-1, which increases expressions of HO-1 and VEGF, resulting in protection of cardiomyocytes and promote angiogenesis in ischemic heart.
引文
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[12]Chang W,Parra M,Centrella M,et al.Interactions between CCAATenhancer binding protein delta and estrogen receptor alpha control insulin-like growth factor I(igf1)and estrogen receptor-dependent gene expression in osteoblasts[J].Gene,2005,345(2):225-235.doi:10.1016/j.gene.2004.11.017.
[13]Tsai HH,Lai HY,Chen YC,et al.Metformin promotes apoptosis in hepatocellular carcinoma through the CEBPD-induced autophagy pathway[J].Oncotarget,2017,8(8):13832-13845.doi:10.18632/oncotarget.14640.
[14]Pawar SA,Sarkar TR,Balamurugan K,et al.C/EBP{delta}targets cyclin D1 for proteasome-mediated degradation via induction of CDC27/APC3 expression[J].Proc Natl Acad Sci U S A,2010,107(20):9210-9215.doi:10.1073/pnas.0913813107.2018-10-282019-01-23
[2]Lee PJ,Rudenko D,Kuliszewski MA,et al.Survivin gene therapy attenuates left ventricular systolic dysfunction in doxorubicin cardiomyopathy by reducing apoptosis and fibrosis[J].Cardiovasc Res,2014,101(3):423-433.doi:10.1093/cvr/cvu001.
[3]Min Y,Li J,Qu P,et al.C/EBP-δpositively regulates MDSCexpansion and endothelial VEGFR2 expression in tumor development[J].Oncotarget,2017,8(31):50582-50593.doi:10.18632/oncotarget.16410.
[4]Min Y,Ghose S,Boelte K,et al.C/EBP-δregulates VEGF-Cautocrine signaling in lymphangiogenesis and metastasis of lung cancer through HIF-1α[J].Oncogene,2011,30(49):4901-4909.doi:10.1038/onc.2011.187.
[5]魏丽萍,齐新,孙旭森,等.心肌梗死小鼠microRNAs表达及干预后心功能变化的研究[J].天津医药,2018,46(8):851-855.Wei LP,Qi X,Sun XS,et al.The expression of microRNAs in mice with myocardial infarction and the evaluation of cardiac function after intervention[J].Tianjin Med J,2018,46(8):851-855.doi:10.11958/20180437.
[6]Lee SH,Wolf PL,Escudero R,et al.Early expression of angiogenesis factors in acute myocardial ischemia and infarction[J].N Engl J Med,2000,342(9):626-633.doi:10.1056/NEJM200003023420904.
[7]Jürgensen JS,Rosenberger C,Wiesener MS,et al.Persistent induction of HIF-1 alpha and-2 alpha in cardiomyocytes and stromal cells of ischemic myocardium[J].FASEB J,2004,18(12):1415-1417.doi:10.1096/fj.04-1605fje.
[8]Kido M,Du L,Sullivan CC,et al.Hypoxia-inducible factor 1-alpha reduces infarction and attenuates progression of cardiac dysfunction after myocardial infarction in the mouse[J].J Am Coll Cardiol,2005,46(11):2116-2124.doi:10.1016/j.jacc.2005.08.045.
[9]Li M,Cui Y,He W,et al.Effects of triple-mutated hypoxiainducible factor-1αon angiogenesis and cardiac function improvement in rats with myocardial infarction[J].Cell Physiol Biochem,2018,50(6):2329-2340.doi:10.1159/000495094.
[10]Du Y,Ge Y,Xu Z,et al.Hypoxia-inducible factor 1 alpha(HIF-1α)/vascular endothelial growth factor(VEGF)pathway participates in angiogenesis of myocardial infarction in muscone-treated mice:preliminary study[J].Med Sci Monit,2018,24:8870-8877.doi:10.12659/MSM.912051.
[11]Nerlov C.The C/EBP family of transcription factors:a paradigm for interaction between gene expression and proliferation control[J].Trends Cell Biol,2007,17(7):318-324.doi:10.1016/j.tcb.2007.07.004.
[12]Chang W,Parra M,Centrella M,et al.Interactions between CCAATenhancer binding protein delta and estrogen receptor alpha control insulin-like growth factor I(igf1)and estrogen receptor-dependent gene expression in osteoblasts[J].Gene,2005,345(2):225-235.doi:10.1016/j.gene.2004.11.017.
[13]Tsai HH,Lai HY,Chen YC,et al.Metformin promotes apoptosis in hepatocellular carcinoma through the CEBPD-induced autophagy pathway[J].Oncotarget,2017,8(8):13832-13845.doi:10.18632/oncotarget.14640.
[14]Pawar SA,Sarkar TR,Balamurugan K,et al.C/EBP{delta}targets cyclin D1 for proteasome-mediated degradation via induction of CDC27/APC3 expression[J].Proc Natl Acad Sci U S A,2010,107(20):9210-9215.doi:10.1073/pnas.0913813107.2018-10-282019-01-23