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背景:现有治疗手段不足以补充梗死心肌,研究表明干细胞移植有可能促使心肌和血管再生,改善心功能和预后。目的:观察急性心肌梗死区中心和周边移植胚胎干细胞后心肌组织形态学及血液动力学的变化。设计、时间及地点:随机对照动物观察,于2007-03/2008-10在中南大学湘雅医学院人体解剖学与神经生物学系神经生物学实验室完成。材料:SPF级Wistar大鼠40只,随机分为4组:正常对照组、梗死模型组、中心移植组、周边移植组,10只/组。胚胎干细胞-D3株(embryonicstemcells-D3,ES-D3)、布法罗大鼠肝细胞均由中国科学院上海细胞所提供。方法:ES-D3细胞复苏后,以(2.0~5.0)×107L-1种植于培养瓶中,加入含布法罗大鼠肝细胞的条件培养基体外培养分化。除正常对照组外,余3组结扎冠状动脉左前降支建立急性心肌梗死模型。造模后1周接受细胞移植,ES-D3细胞于移植前1d进行BrdU标记,以胚胎干细胞培养基调整密度至1×109L-1。中心移植组在梗死区选3个点,每点注入10μL细胞悬液(含104个细胞)至心室壁内;周边移植组在梗死区周边选3个点同法注入等量细胞悬液。主要观察指标:免疫组化及血流动力学指标检测结果。结果:布法罗大鼠肝细胞条件培养基体外培养的ES-D3细胞,呈相对规则的巢状集落样生长,分化8d即可见部分拟胚体出现自发的节律性收缩活动,心肌肌钙蛋白T免疫染色呈阳性,电镜下可见肌管、肌纤维等肌性结构,证实已分化为心肌细胞。周边移植组BrdU免疫荧光染色呈阳性,而中心移植组呈阴性,进一步对BrdU免疫荧光染色阳性细胞行双抗染色,可见心肌肌钙蛋白T呈阳性表达。移植后4周与正常对照组比较,梗死模型组左室收缩、dp/dtmax均减小(P<0.01),左室舒张末期压上升(P<0.01),左室质量、左室质量指数均升高(P<0.01)。与梗死模型组比较,中心移植组各项血流动力学指标无明显变化(P>0.05);周边移植组左室收缩压、±dp/dtmax均显著升高(P<0.01),左室舒张末期压显著减小(P<0.01),左室质量、左室质量指数、梗死面积均显著减小(P<0.01)。结论:急性心肌梗死后于梗死周边区移植胚胎干细胞可以阻止心室重构、减少瘢痕面积、改善心功能。
Background: Current therapies are not sufficient to replenish infarcted myocardium. Studies have shown that stem cell transplantation may promote myocardial and vascular regeneration and improve cardiac function and prognosis. OBJECTIVE: To observe the morphological and hemodynamic changes of myocardial tissue after transplantation of embryonic stem cells in and around the center of acute myocardial infarction. DESIGN, TIME AND SETTING: The randomized controlled animal observation was performed at the Department of Neurobiology, Department of Human Anatomy and Neurobiology, Xiangya School of Medicine, Central South University from March 2007 to October 2008. MATERIALS: Forty SPF Wistar rats were randomly divided into 4 groups: normal control group, infarction model group, central transplantation group and peripheral transplantation group, 10 rats / group. Embryonic stem cell-D3 strain (embryonic stem cells-D3, ES-D3), Buffalo rat liver cells by the Shanghai Institute of Cell. Methods: After ES-D3 cells were resuscitated, the conditioned medium of Buffalo rat hepatocytes was seeded into culture flasks with the volume of (2.0-5.0) × 107L-1 and cultured in vitro. Except normal control group, the other 3 groups were ligated with left anterior descending coronary artery to establish acute myocardial infarction model. One week after the model establishment, the cells were transplanted. ES-D3 cells were labeled with BrdU 1 day prior to transplantation, and the density was adjusted to 1 × 109 L-1 in embryonic stem cell culture medium. In the central transplantation group, 3 points were selected in the infarction area, and 10μL of cell suspension (104 cells) was injected into the ventricular wall at each point. Peripheral transplantation group was injected 3 times with the same amount of cell suspension in the same way around the infarct area. MAIN OUTCOME MEASURES: Results of immunohistochemistry and hemodynamic tests. Results: The ES-D3 cells cultured in vitro in Buffalo rat hepatocyte conditioned medium showed a relatively regular nested colony-like growth. After 8 days of differentiation, spontaneous rhythmic contractile activity of some embryoid bodies, cardiac troponin T immunostaining was positive, under the electron microscope myotube, muscle fibers and other muscular structures, confirmed that has been differentiated into cardiomyocytes. Peripheral transplantation group BrdU immunofluorescence staining was positive, while the central graft group was negative, and further BrdU immunofluorescence staining positive cells double-staining, showing cardiac troponin T was positive. Left ventricular systolic and dp / dtmax were decreased (P <0.01), left ventricular end-diastolic pressure was increased (P <0.01), left ventricular mass and left ventricular mass index Increased (P <0.01). Compared with the infarction model group, there was no significant change in the hemodynamic parameters in the central transplantation group (P> 0.05). Left ventricular systolic pressure and ± dp / dtmax in the peripheral transplantation group were significantly increased (P <0.01) (P <0.01), left ventricular mass, left ventricular mass index and infarct size were significantly decreased (P <0.01). CONCLUSION: Transplantation of embryonic stem cells in the peripheral infarction area after acute myocardial infarction can prevent ventricular remodeling, reduce scar area and improve cardiac function.