【作者】 赵鸿；

【导师】 黄政德；

【作者基本信息】 湖南中医药大学 ， 中医内科学， 2012， 博士

【摘要】 目的：建立大鼠心肌缺血再灌注损伤(IRI)模型,观察加味丹参饮(JWDSY)含药血清体外诱导的骨髓间充质干细胞(BMSCs)移植对IRI大鼠的心脏保护作用,探讨其对心肌IRI大鼠血管新生的动态影响和作用机制。方法：实验一：采用全骨髓贴壁法培养大鼠BMSCs,并进行传代、扩增,对细胞形态学和生长特性进行观察,绘制生长曲线,通过流式细胞术对细胞表面抗原CD34、CD45、CD90进行表型鉴定。实验二：采用MTT法检测不同浓度加味丹参饮含药血清对BMSCs的毒性作用及增殖的影响。将全骨髓贴壁法培养的大鼠BMSCs分别加入加味丹参饮含药血清与空白血清,体外诱导分化3d。建立心肌IRI大鼠模型,随机分为四组：假手术组、模型组、空白血清诱导的BMSCs组和加味丹参饮含药血清诱导的BMSCs组。采用心肌内直接注射移植的方法,在动物造模成功后立即将诱导后的移植细胞分3点注射到梗死边缘区的心肌内,模型组和假手术组注射等量的L-DMEM。肉眼观察结扎部位的大体表现,分别于移植后3d、7d、14d三个时相点,TTC染色法测定大鼠左室心肌梗死面积的变化,HE染色光学显微镜观察心肌组织一般形态学改变,观察缺血区病理改变；应用全自动生化分析仪测定大鼠心肌酶CK、CK-MB水平；免疫组织化学染色法检测各组梗死部位边缘区血管内皮细胞特异性表面标志VⅢ因子和CD34的表达,计算和比较心肌微血管密度(MVD),评价其心脏保护和血管新生作用。实验三：诱导后的BMSCs移植方法同实验二。分别于移植后3d、7d、14d三个时相点,免疫组织化学染色法检测梗死心肌边缘区VEGF、 Ang-1、PDGF-β的蛋白表达,放射免疫方法检测血清IGF-1水平,Real-Time PCR法检测梗死边缘区VEGFmRNA、Ang-1mRNA的表达；评价加味丹参饮含药血清体外诱导的BMSCs心肌内移植促血管新生能力,探讨各种血管生长因子表达的变化与血管新生间的关系。结果：实验一：经流式细胞术鉴定,通过全骨髓贴壁法培养的BMSCs贴壁生长,传代3次后,细胞形态为较均匀一致的纺锤形,并呈平行样或漩涡样生长；纯度较高,细胞表面抗原CD90阳性细胞达到95%以上,CD34和CD45阳性细胞小于5%,符合实验要求。实验二：MTT结果显示,20%的加味丹参饮含药血清为诱导BMSCs的最佳工作浓度。大鼠结扎后ST段、T波异常增高,再灌注后ST段、T波快速回落,出现病理性Q波,提示IRI大鼠造模成功。BMSCs移植后不同时相点观察,与假手术组比较,模型组一般情况较差,心脏体积明显增大,血清心肌酶CK、CK-MB显著升高；BMSCs组治疗后大鼠一般情况有所改善,心脏外形、体积接近假手术组,心肌梗死面积缩小,含药血清组比空白血清组表现更为明显(P<0.05)；与模型组比较,加味丹参饮含药血清组、空白血清组的大鼠心肌酶CK、CK-MB水平均有所改善,而加味丹参饮含药血清诱导的BMSCs组改善更明显；加味丹参饮含药血清和空白血清诱导的BMSCs组梗死心肌边缘区的微血管密度高于模型组(P<0.05)和假手术组(P<0.05)；与空白血清诱导的BMSCs组相比,加味丹参饮含药血清诱导的BMSCs组增高更明显(P<0.05)。实验三：免疫组织化学染色法测定IRI大鼠梗死心肌边缘区的VEGF、Ang-1、PDGF-β蛋白表达的10D,放免法测定IGF-1水平,发现模型组上述生长因子的表达比假手术组明显增强(P<0.05),可以认为是机体对组织缺血缺氧的代偿机制导致,证明造模成功；加味丹参饮含药血清诱导的BMSCs组、空白血清诱导的BMSCs组的表达水平明显高于模型组(P<0.05),加味丹参饮含药血清诱导的BMSCs组升高更为显著(P<0.05),并普遍存在持续增高的趋势(P<0.05)。Real-TimePCR检测各组Ang-1mRNA的表达,发现各BMSCs组心脏组织在移植后3d、7d、14d均有VEGFmRNA、Ang-1mRNA表达,假手术组和模型组表达微弱；空白血清诱导的BMSCs组和含药血清诱导的BMSCs组的VEGFmRNA、 Ang-1mRNA表达高峰均在再灌注7d(P<0.05),以后逐渐减弱。加味丹参饮含药血清诱导的BMSCs组的VEGFmRNA、Ang-1mRNA表达明显强于同时相点的空白血清诱导的BMSCs组(P<0.05)。各生长因子的高表达与反映新生血管强度的微血管密度(MVD)显示了一定的相关性。结论：1、全骨髓贴壁法可以培养出纯度较高、活性较好的BMSCs。2、加味丹参饮含药血清诱导的BMSCs移植能改善心肌IRI大鼠的心功能,减小梗死面积,增加缺血区微血管密度,降低血清心肌酶CK、 CK-MB,比空白血清诱导的BMSCs移植改善更显著。3、加味丹参饮含药血清诱导的BMSCs移植可以明显促进梗死心肌边缘区的血管新生,其作用可能与持续稳定上调缺血心肌局部VEGF、Ang-1、 PDGF-β蛋白表达、血清IGF-1水平及VEGFmRNA、 Ang-1mRNA的表达有关,效果优于空白血清诱导的BMSCs移植。加味丹参饮体外诱导对BMSCs移植促心肌血管新生具有协同和促进作用。更多还原

【Abstract】 Objiective:Establish rat models of myocardial IRI, observe the improvement of heart function of myocardial IRI rats through BMSCs transplantation induced in vitro by JWDSY Medicated Serum, and investigate the dynamic effect and function mechanism of the angiogenesis of myocardial IRI rats.Methods:Test1:cultivate BMSCs of rats by the whole bone marrow stick wall of training, perform passage curture and amplification, observe the morphology and growth characteristics of cells, draw the growth curve, analysize phenotypes of CD34, CD45, CD90through the flow cytometry.Test2:detect toxic effects and proliferous influence of JWDSY Medicated Serum with different concentration on BMSCs by MTT method, mix BMSCs of rats cultivated by the whole bone marrow stick wall of training with JWDSY Medicated Serum and blank serum, induce in vitro to differentiate3d, establish rat models of myocardial IRI, which are randomly divided into four groups:Group SO (sham operated), Group model, Group BMSCs induced by blank serum, and Group BMSCs induced by JWDSY Medicated Serum. Use the approach of transplanting by direct injection in myocardium, inject the induced transplanting cells in the fringe area of myocardial infarction while Group model and Group SO are injected with the same amount of L-DMEM in three parts immediately after the animal model succeeding, and observe the general performance of the ligation part macroscopically. All observations are carried out by the three time points of3d,7d and14d respectively after transplantation. Assay the change of myocardial infarct size in the left ventricle of rats by TTC staining method, observe the morphologic change of cardiac muscular tissue with HE staining staining method, observe pathological changes of ischemia area, determine the level of cardiac enzyme CK and CK-MB by the automatic biochemistry analyzer, detect expressions of Ⅷ factor and CD34which are specific landmark of vascular endothelial cells in the edge of the infarction parts with the method of immunohistochemical staining, calculate and compare myocardial microessel-density (MVD), and then evaluate its heart protection and effect on angiogenesis.Test3:the transplanting method of induced BMSCs is the same with that in test2. All detections are processed by the three time points of3d,7d and14d respectively after transplantation. Detect protein expression of VEGF, Ang-1and PDGF-β with immunohistochemical method, Serum IGF-1level with radioimmunoassay, VEGFmRNA and Ang-1mRNA expression with Real-Time PCR in fringe area of myocardial infarction. Evaluate the ability of angiogenesis by intramyocardial transplantating of BMSCs that have been induced in vitro by JWDSY Medicated Serum, and investigate the relationship between the changes of various angiogenesis factors expression and angiogenesis.Results:Test1:according to the identification of FCM (flow cytometry), cells tends to have the shape of coincident spindle and grows in parallel or vortex after3times passage with BMSCs of rats cultivated by the whole bone marrow stick wall of training. The purity is high. Positive cells of CD90are above95%with CD34and CD45less than5%, which is in line with the requirements.Test2:the result of MTT method shows that20%of JWDSY Medicated Serum is the best concentration to induce BMSCs. After rats ligatured, ST segment and T wave increase abnormally. But after reperfusion, ST segment and T wave fall back quickly, and pathological Q wave appears, which indicates that IRI model of rat is successful. Observing by the three time points after BMSCs transplantation, it has found that the general situation of Group model is worse than Group SO, and the volume of heart increases obviously. Then serum myocardial enzymes CK and CK-MB also increase significantly. After the rat in Group BMSCs receives treating, its general situation has been improved with the shape and volume of heart being close to Group SO. The Group BMSCs induced by JWDSY Medicated Serum performs more obviously than the Group BMSCs induced by blank serum in narrowing fringe area of myocardial infarction (P<0.05). Comparing with Group model, Group BMSCs induced by JWDSY Medicated Serum and Group BMSCs induced by blank Serum have improved the level of CK and CK-MB, and Group BMSCs induced by JWDSY Medicated Serum performs more obviously. The Microvessel density (MVD) in fringe area of myocardial infarction of Group BMSCs that has been induced by JWDSY Medicated Serum and blank serum is higher than Group model (P<0.05) and Group SO (P<0.05). And Group BMSCs induced by JWDSY Medicated Serum increases more greatly than the Group BMSCs induced by blank serum (P<0.05) Test3:using immunohistochemical method to determine IRI rats VEGF, Ang-1and PDGF-(3protein expression of IOD in fringe area of myocardial infarction and radioimmunoassay to detect the level of IGF-1, it has found that the protein expression of the growth factor in Group model enhances more obviously than Group SO (P<0.05). Therefore, it can be considered that it arises from compensatory mechanism of organism on ischemia and anoxic of the tissue, proving that the model succeeds. The expression level of Group BMSCs induced by JWDSY Medicated Serum and blank serum is higher than Group model (P<0.05). Group BMSCs induced by JWDSY Medicated Serum increases more significantly (P<0.05), and it shows a general continuous trend (P<0.05). Applying Real-Time PCR to detect the expression of Ang-1mRNA, it has found that the heart tissue of Group BMSCs has the expression of VEGFmRNA and Ang-1mRNA in3d,7d and14d after transplantation. Group SO and Group model show weak expression. And Group BMSCs induced by blank serum and JWDSY Medicated Serum show a peak expression of VEGFmRNA and Ang-1mRNA in7d after reperfusion (P<0.05), then it decreases. The expression of VEGFmRNA and Ang-1mRNA in Group BMSCs induced by JWDSY Medicated Serum is stronger than Group BMSCs induced by blank serum at the same time point (P<0.05). The high expression of growth factor shows some relevance with MVD which reflects the strength of neovascularization.Conclusion:1. the method of the whole bone marrow stick wall of training can cultivate BMSCs with high purity and good activity.2. BMSCs transplantation induced by JWDSY Medicated Serum can improve heart function of myocardial IRI rats, reduce infarction area, increase MVD of ischemia area, and reduce serum myocardial enzymes CK and CK-MB. All the improvements are more significant than BMSCs transplantation induced by blank serum.3. BMSCs transplantation induced by JWDSY Medicated Serum can obviously promote angiogenesis in fringe area of myocardial infarction. The effects may be relevant with the protein expression of VEGF, Ang-1, PDGF-β and Serum IGF-1level and the expression of VEGFmRNA and Ang-1mRNA in local Ischemic Myocardium which is raised steadily and continuously. The effect is better than that of BMSCs transplantation induced by blank serum. BMSCs transplantation induced in vitro by JWDSY Medicated Serum has cooperative and stimulative role on angiogenesis.更多还原