重组人促红细胞生成素联合自体外周血干细胞移植治疗猪急性心肌梗死的实验研究
发布时间：2018-08-03 06:57
【摘要】：目的本实验旨在通过研究G-CSF动员后分离、采集自体外周血干细胞经皮经腔冠脉内移植联合EPO治疗猪急性心肌梗死,观察其对心肌细胞病理学改变及细胞凋亡的影响。方法健康小型猪30只,随机分为5组,每组6只。动物单纯干细胞移植组(SC)、单纯促红细胞生成素组(EPO)、EPO联合SC组(EPO+SC)、AMI对照组(control),假手术组(sham)。其中4组采用经皮穿刺球囊封堵冠状动脉左前降支的方法建立猪急性心肌梗死模型,1组行假手术。SC组建模后1h给予G-CSF连用7天,建模第8天采用血细胞分离机单采PBSC,通过IRA通道回输PBSC。EPO+SC组建模后1h给予G-CSF连用7天,rHu-EPO连续5天,并于建模后第8天以相同方法采集、标记、回输PBSC。EPO组建模后给予rHu-EPO注射连续5天,于第8天经IRA回输与SC组相同容量的生理盐水。Control组及sham组建模后仅经IRA回输相同容量的生理盐水。采用苏木精-依红(HE)染色观察心肌组织形态；TUNEL染色方法检测心肌细胞凋亡指数；Western-blot方法检测心肌梗死区域心肌组织中凋亡蛋白Bax、Caspase-3,抗凋亡蛋白Bcl-2的表达量。结果建模过程中因麻醉过量、室颤各死亡1只实验动物,剩余30只成功建模。HE染色：sham组可见心肌细胞排列整齐,心肌细胞结构完整；control组可见梗死区大片心肌细胞连续性坏死,梗死边缘区存在大量炎症细胞浸润及纤维组织增生；EPO组与SC组心肌梗死区心肌组织相似,纤维化程度及炎性细胞浸润情况较轻,期间散在分布正常心肌细胞；EPO+SC组心梗部位心肌组织可见更多正常心肌细胞,纤维化程度轻,边缘少量炎性细胞浸润。TUNEL结果：EPO+SC组凋亡指数显著低于对照组(37.93±12.60%vs75.70±15.50%,p0.01),EPO组凋亡指数与EPO+SC组相比较高(54.08±16.06%vs37.93±12.60%,p=0.047),EPO组与SC组凋亡指数均低于对照组(p0.01)Western blot结果：EPO+SC组Bcl-2蛋白表达显著高于EPO组、Control组及sham组(p0.01),Bax及Caspase-3蛋白表达量均减低(P0.05)。结论自体外周血干细胞移植联合给予重组人促红细胞生成素在急性心肌梗死的治疗中可减轻梗死区域心肌的病理学改变、抑制梗死区心肌细胞的凋亡,其机制可能是通过上调抗凋亡蛋白、下调凋亡蛋白的表达来实现的。
[Abstract]:Objective to investigate the effects of percutaneous transcatheter coronary transplantation of autologous peripheral blood stem cells (PBSCs) and EPO on myocardial cell pathological changes and apoptosis in porcine acute myocardial infarction (AMI) after G-CSF mobilization. Methods 30 healthy miniature pigs were randomly divided into 5 groups, 6 in each group. Simple stem cell transplantation group (SC), erythropoietin group (EPO) EPO + SC (EPO SC) control group (control), sham-operation group (sham). The left anterior descending coronary artery was occluded by percutaneous balloon in 4 groups. The model of acute myocardial infarction in pigs was established. Group 1 was sham-operated. Group SC was given G-CSF for 7 days after modeling. On the 8th day of modeling, the blood cell separator was used to collect PBSCs, and the PBSC.EPO SC group was given G-CSF for 7 days for 5 days after modeling, and collected and labeled by the same method on the 8th day after modeling. PBSC.EPO group was injected with rHu-EPO for 5 days after modeling. On the 8th day, normal saline of the same volume as SC group and sham group were injected with IRA for 8 days. Only saline of the same volume was injected through IRA after modeling in sham group. The expression of apoptosis protein (Baxton-Caspase-3) and anti-apoptotic protein (Bcl-2) in myocardial tissue of myocardial infarction area was detected by using hematoxylin and ired (HE) staining. Results during the course of modeling, one experimental animal died of ventricular fibrillation due to excessive anesthesia. The remaining 30 rats were successfully modeled. The myocardial cells were arranged neatly and the structure of myocardial cells was intact in the group of HE staining. Continuous necrosis of myocardial cells in infarct area was observed in control group. There were a large number of inflammatory cell infiltration and fibrous tissue proliferation in the margin of infarction. The degree of fibrosis and infiltration of inflammatory cells were less in control group than in SC group. In the EPO SC group, more normal cardiomyocytes were found in the myocardial tissue of the EPO SC group, and the degree of fibrosis was slight. The apoptotic index of EPO group was significantly lower than that of control group (37.93 卤12.60%vs75.70 卤15.50, p0.01). The apoptotic index of EPO group was significantly higher than that of EPO SC group (54.08 卤16.06%vs37.93 卤12.60p 0.047). The apoptotic index of EPO group and SC group were significantly lower than that of control group (p0.01) Western blot result). The expression of Bax and Caspase-3 in EPO group were significantly lower than those in control group and sham group (p0.01) (P0.05). Conclusion Autologous peripheral blood stem cell transplantation combined with recombinant human erythropoietin in the treatment of acute myocardial infarction can attenuate the pathological changes of myocardium and inhibit the apoptosis of myocardial cells in infarcted area. The mechanism may be by upregulating anti-apoptotic protein and down-regulating the expression of apoptotic protein.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R542.22;R457.7
本文编号：2160926
[Abstract]:Objective to investigate the effects of percutaneous transcatheter coronary transplantation of autologous peripheral blood stem cells (PBSCs) and EPO on myocardial cell pathological changes and apoptosis in porcine acute myocardial infarction (AMI) after G-CSF mobilization. Methods 30 healthy miniature pigs were randomly divided into 5 groups, 6 in each group. Simple stem cell transplantation group (SC), erythropoietin group (EPO) EPO + SC (EPO SC) control group (control), sham-operation group (sham). The left anterior descending coronary artery was occluded by percutaneous balloon in 4 groups. The model of acute myocardial infarction in pigs was established. Group 1 was sham-operated. Group SC was given G-CSF for 7 days after modeling. On the 8th day of modeling, the blood cell separator was used to collect PBSCs, and the PBSC.EPO SC group was given G-CSF for 7 days for 5 days after modeling, and collected and labeled by the same method on the 8th day after modeling. PBSC.EPO group was injected with rHu-EPO for 5 days after modeling. On the 8th day, normal saline of the same volume as SC group and sham group were injected with IRA for 8 days. Only saline of the same volume was injected through IRA after modeling in sham group. The expression of apoptosis protein (Baxton-Caspase-3) and anti-apoptotic protein (Bcl-2) in myocardial tissue of myocardial infarction area was detected by using hematoxylin and ired (HE) staining. Results during the course of modeling, one experimental animal died of ventricular fibrillation due to excessive anesthesia. The remaining 30 rats were successfully modeled. The myocardial cells were arranged neatly and the structure of myocardial cells was intact in the group of HE staining. Continuous necrosis of myocardial cells in infarct area was observed in control group. There were a large number of inflammatory cell infiltration and fibrous tissue proliferation in the margin of infarction. The degree of fibrosis and infiltration of inflammatory cells were less in control group than in SC group. In the EPO SC group, more normal cardiomyocytes were found in the myocardial tissue of the EPO SC group, and the degree of fibrosis was slight. The apoptotic index of EPO group was significantly lower than that of control group (37.93 卤12.60%vs75.70 卤15.50, p0.01). The apoptotic index of EPO group was significantly higher than that of EPO SC group (54.08 卤16.06%vs37.93 卤12.60p 0.047). The apoptotic index of EPO group and SC group were significantly lower than that of control group (p0.01) Western blot result). The expression of Bax and Caspase-3 in EPO group were significantly lower than those in control group and sham group (p0.01) (P0.05). Conclusion Autologous peripheral blood stem cell transplantation combined with recombinant human erythropoietin in the treatment of acute myocardial infarction can attenuate the pathological changes of myocardium and inhibit the apoptosis of myocardial cells in infarcted area. The mechanism may be by upregulating anti-apoptotic protein and down-regulating the expression of apoptotic protein.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R542.22;R457.7
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