人胚胎生殖细胞移植治疗猪急性心肌梗死的实验研究
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摘要
目的:1.建立猪急性心肌梗死模型;2.观察人胚胎生殖细胞(human embryonic germ cells,hEGCs)移植到猪急性心肌梗死模型心肌中的存活及分化;3.探讨人EG细胞移植心肌组织后血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达及意义。
方法:取5-10周流产人胚胎生殖腺嵴,采用组织块培养法培养人EG细胞。单独培养人EG细胞,同时将人EG细胞与胚胎心肌细胞共培养以模拟心肌微环境,分别检测二者第3天与第9天培养上清液中VEGF的表达水平。通过结扎冠状动脉左前降支的方法建立猪急性心肌梗死(AMI)模型,分为移植组、对照组。移植组为将体外扩增的人EG细胞注射到猪梗死心肌周边,对照组用同样方法注射无菌PBS。移植后2周处死猪,用免疫组织化学方法以鼠抗人细胞核抗体MAB1281作示踪剂,检测移植细胞的存活情况,并观察转录因子GATA-4、连接蛋白Cx43在移植细胞的表达。
结果:体外培养获得大量人EG细胞,人EG细胞与胚胎心肌细胞共培养组第3天与第9天上清液中VEGF含量分别较人EG细胞单独培养组高,差别有显著意义(P<0.05);成功建立猪急性心肌梗死模型,结扎冠脉后可见心电图典型的心肌梗死变化;免疫组化显示移植组抗人细胞核抗体MAB1281、转录因子GATA-4、连接蛋白Cx43均呈阳性表达,对照组为阴性表达。
结论:在猪急性心肌梗死模型建立后经心外膜移植人EG细胞,人EG细胞在宿主心肌存活且逐渐向心肌细胞分化,植入的人EG细胞可能通过分泌VEGF等细胞因子促进梗死区的心肌修复。
Objectives:1.To establish the model of acute myocardial infarction in the pig;2.To observe the differentiation and repair function of human embryonic germ cells in the infarcted pig heart;3.To investigate the expression and significance of VEGF after transplanting human EG cell into the myocardium.
Methods: Through tissue culture, the human EG cells were obtained by taking the gonadal ridges from human embryos aged 5～10 weeks. EG cells were isolated and cultured, at the same time, the human EG cells and human embryonic myocardial cells were cocultured to simulate cardiac micro-environment in vivo. The expression level of VEGF in culture supernatants on the third day and the ninth day of the both groups was detected respectively. The acute myocardial infarction (AMI) model was established by ligating the left anterior descending coronary artery in two pigs and they were divided into two groups:the transplanted group(hEGCs were injected into myocardium) and the control group(only PBS was injected). These pigs were killed respectively at the end of the second week after transplantation. Immunohistochemistry was used to detect the expression of anti–human cellular nuclei MAB1281 in order to locate the implanted cells and the expressions of transcription factor GATA-4 and connexin Cx43 were examined in the transplanted cells simultaneously.
Results: A large number of human EG cells were acquired in vitro.The supernatant VEGF levels on the third day and the ninth day in the human EG cells and myocardial cells co-culture group were higher than the human EG cells group and the difference between the two groups was statistically significant (P<0.05); Typical ECG changes of myocardial infarction after coronary artery ligation showed that the acute myocardial infarction (AMI) model of pigs was successfully established ; In the transplanted group, transcription factor GATA-4 and connexin Cx43 showed positive presentation in the anti-human nuclei antibody MAB1281-positive cells, the control group was negative.
Conclusion: Human EG cells transplanted into the myocardium of the pig through epicardium stay alive and differentiate into cardiomyocytes.The implanted Human EG cells can secrete cytokines such as VEGF to promote the repair of infarcted myocardium.
方法:取5-10周流产人胚胎生殖腺嵴,采用组织块培养法培养人EG细胞。单独培养人EG细胞,同时将人EG细胞与胚胎心肌细胞共培养以模拟心肌微环境,分别检测二者第3天与第9天培养上清液中VEGF的表达水平。通过结扎冠状动脉左前降支的方法建立猪急性心肌梗死(AMI)模型,分为移植组、对照组。移植组为将体外扩增的人EG细胞注射到猪梗死心肌周边,对照组用同样方法注射无菌PBS。移植后2周处死猪,用免疫组织化学方法以鼠抗人细胞核抗体MAB1281作示踪剂,检测移植细胞的存活情况,并观察转录因子GATA-4、连接蛋白Cx43在移植细胞的表达。
结果:体外培养获得大量人EG细胞,人EG细胞与胚胎心肌细胞共培养组第3天与第9天上清液中VEGF含量分别较人EG细胞单独培养组高,差别有显著意义(P<0.05);成功建立猪急性心肌梗死模型,结扎冠脉后可见心电图典型的心肌梗死变化;免疫组化显示移植组抗人细胞核抗体MAB1281、转录因子GATA-4、连接蛋白Cx43均呈阳性表达,对照组为阴性表达。
结论:在猪急性心肌梗死模型建立后经心外膜移植人EG细胞,人EG细胞在宿主心肌存活且逐渐向心肌细胞分化,植入的人EG细胞可能通过分泌VEGF等细胞因子促进梗死区的心肌修复。
Objectives:1.To establish the model of acute myocardial infarction in the pig;2.To observe the differentiation and repair function of human embryonic germ cells in the infarcted pig heart;3.To investigate the expression and significance of VEGF after transplanting human EG cell into the myocardium.
Methods: Through tissue culture, the human EG cells were obtained by taking the gonadal ridges from human embryos aged 5～10 weeks. EG cells were isolated and cultured, at the same time, the human EG cells and human embryonic myocardial cells were cocultured to simulate cardiac micro-environment in vivo. The expression level of VEGF in culture supernatants on the third day and the ninth day of the both groups was detected respectively. The acute myocardial infarction (AMI) model was established by ligating the left anterior descending coronary artery in two pigs and they were divided into two groups:the transplanted group(hEGCs were injected into myocardium) and the control group(only PBS was injected). These pigs were killed respectively at the end of the second week after transplantation. Immunohistochemistry was used to detect the expression of anti–human cellular nuclei MAB1281 in order to locate the implanted cells and the expressions of transcription factor GATA-4 and connexin Cx43 were examined in the transplanted cells simultaneously.
Results: A large number of human EG cells were acquired in vitro.The supernatant VEGF levels on the third day and the ninth day in the human EG cells and myocardial cells co-culture group were higher than the human EG cells group and the difference between the two groups was statistically significant (P<0.05); Typical ECG changes of myocardial infarction after coronary artery ligation showed that the acute myocardial infarction (AMI) model of pigs was successfully established ; In the transplanted group, transcription factor GATA-4 and connexin Cx43 showed positive presentation in the anti-human nuclei antibody MAB1281-positive cells, the control group was negative.
Conclusion: Human EG cells transplanted into the myocardium of the pig through epicardium stay alive and differentiate into cardiomyocytes.The implanted Human EG cells can secrete cytokines such as VEGF to promote the repair of infarcted myocardium.
引文
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2. Karciauskaite D , Grybauskiene R , Grybauskas P ,et al . Brain natriuretic peptide and other cardiac markers in predicting left ventricular remodeling inpatients with the first myocardial infarction [J] .Medicina, 2004,40(10):949 - 956.
3. Yanmei Wang,Hongmei Tang,Dong Wang,et al . Pretreatment With Transmyocardial Revascularization Might Improve Ischemic Myocardial Function Performed With Cell Transplantation[J].Circ J, 2006,70(5):625-630.
4. Changqing Xie,Jifeng Zhang,Yang Xiao,et al. Transplantation of Human Undifferentiated Embryonic Stem Cells into a Myocardial Infarction Rat Model[J].STEM CELLS AND DEVELOPMENT, 2007,16(1):25-29.
5. Singla DK,Hacker TA,Ma L,et al.Transplantation of embryonic stem cells into the infarcted mouse heart:formation of multiple cell types[J].J Mol Cell Cardiol,2006,40(1):195-200.
6. Min JY,Yang Y,Convemo KL,et al.Transplantation of embryonic stem cells improves cardiac function in postinfareted rats[J].J Appl Physiol, 2002,92 (1):288-296.
7. Min JY,Yang Y,Sullivan MF,et al. Long term improvement of cardiac function in rats after infarction by transplantation of embryonic stem cells[J].J ThoraC Cardiovasc surg, 2003,125(2):361-369.
8. Kolossov E,Bostani T,Roell W,et al.Engraftment of engineered ES cell–derived Cardiomyocytes but not BM cells restores contractile function to the infracted myocardium[J]. JEM. The Rockefeller University Press Vol. 203, No. 10, October 2, 2006,203(10):2315-2327.
9. Yang YK, Min JY, Ke QG, et al.Cardiogenesis and angiogenesis in infractedmyocardium by Introamyocardial transplantation of embryonic stem cells in mice [J]. Circulation, 2001(SuppleⅠ:Ⅱ) :262-270.
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