【作者】 李红敏；

【导师】 赵学； 吴宗贵； 廖德宁； 梁春； 任雨笙； 张家友；

【作者基本信息】 第二军医大学 ， 内科学， 2010， 硕士

【摘要】 目的：通过细胞移植修复梗死心肌,诱导血管新生,改善心脏功能,成为当今倍受关注的课题。然而,移植细胞早期大量丧失的问题限制了其生物学功能的发挥。为此,通过动物实验,将雄性SD大鼠骨髓间充质干细胞(BMSCs)移植于雌性SD大鼠心肌梗死模型,观察移植后细胞存活的数量动态变化及其分布情况,初步探讨细胞移植后影响细胞长期存活的因素,在此基础上,提出在众多炎症因子中,诱导型一氧化氮合酶(iNOS)激活,生成高浓度的一氧化氮,可能是影响移植细胞长期存活的主要因素之一,并通过进一步的动物实验探讨心肌梗死后iNOS表达的变化规律。在此基础上,采用对照研究分析细胞移植后早期干预iNOS活性对移植细胞长期存活率的影响,为临床应用干细胞移植治疗心肌梗死与优化治疗方案提供必要的科学依据。方法：1、急性心肌梗死(AMI)模型制备：取清洁雌性SD大鼠,体重150-180g,结扎左冠状动脉前降支制备AMI模型,1小时后进行细胞移植,随机分组进行实验观察；2、骨髓间充质干细胞(BMSCs)制备：取100-130g雄性SD大鼠,分离、纯化、培养其BMSCs。分离纯化的大鼠BMSCs经流式细胞术鉴定及免疫组化检测；3、细胞移植：于AMI后1小时,直视下将1.2×106个培养的BMSCs注射植入到梗死区中心心外膜下；4、AMI后iNOS表达规律检测：免疫印迹法检测心肌梗死后1天、3天、7天、10天、14天梗死区的iNOS表达情况；5、移植细胞定位检测：以Brdu或Hoechst33342标记定位观察移植细胞,并与HE染色对照；6、移植细胞定量检测：采用Real-time PCR检测大鼠Y染色体雄性鉴别基因sry片段的表达,作为移植细胞存活率的定量比较指标。结果：1、方法学：结扎大鼠左冠状动脉前降支后,左室壁变苍白,收缩减弱,左心耳充血,与未缺血区界线清楚,表明AMI动物模型复制成功；分离纯化的大鼠BMSCs经流式细胞术鉴定为CD44+/CD34-,免疫组化检测有CD44表达,而无CD34表达,证明拟移植细胞为BMSCs。2、移植后不同时间的细胞存活观察：依据细胞移植后取材时间分为3组：一周组-细胞移植后1周取材；三周组-细胞移植后3周取材；六周组-细胞移植后6周取材。各组组织冰冻切片荧光显微镜下均可观察到大量Hoechst33342标记阳性细胞,三周组、六周组可见标记细胞部分聚集形成血管样结构；各组HE染色和Brdu免疫组织化学检测均可见有核呈棕色的阳性标记细胞集中分布；Real-time PCR检测六周组细胞存活率(8.73%),一周组(7.88%),三周组(7.82%)(n=8,p>0.05),提示移植后一周至六周移植细胞不再有进行性减少。3、AMI后不同时间心肌组织iNOS表达规律：AMI后不同时间点iNOS表达的强度变化规律为：3天>1天>7天>10天>14天(n=6,p<0.05),提示在第1至3天为iNOS表达高峰。4、选择性iNOS抑制剂对移植细胞长期存活的保护作用：实验组于移植后12h给予选择性iNOS抑制剂1400W,2mg/kg,腹腔注射,2次/天,维持4天,细胞移植后6周取材；对照组给予等量生理盐水,其余同实验组。结果可见干预iNOS活性的实验组中Brdu标记阳性细胞分布集中,周边未标记的炎症细胞明显减少,部分血管内皮上明显可见Brdu标记阳性细胞。相邻切片、相同部位的HE染色可见移植细胞的相似分布,核呈蓝紫色,核大,染色深。Real-time PCR检测显示实验组和对照组Y染色体sry序列的表达水平分别为8.75%、6.63%,提示抑制iNOS活性可明显增加移植细胞存活率(n=7,p<0.05)。结论：1.于AMI后1周至6周,移植细胞不再有进行性减少,早期相对集中分布,后期可见向周边游走现象。2. iNOS表达高峰在心肌梗死后第1至3天,随后下降；3.细胞移植后早期开始对iNOS活性进行干预能明显改善移植细胞的长期存活率。更多还原

【Abstract】 Objective:Wide attention has been paid to the therapeutic strategy of cell transplantation which improves myocardial infarction through cell differentiation, angiogenesis and other mechanisms in recent years. However, only a very small number of transplanted cells can survive after the transplantation. Therefore,it is important to study the factors that influence the survival rate of transplanted cells.In this experiment, bone marrow-derived mesenchymal stem cells which were isolated from the male SD rats were transplanted into the female rats which were suffering from acute myocardial infarction.We investigated the dynamic state and distribution of the surviving rat mesenchymal stem cells engrafted in infarcted heart firstly. We hypothesis that the high concentration of NO is one of major contributors to the apoptosis of transplanted cells after myocardial infarction. So,some more work was made to study the changing expression of induced nitric oxide synthase (iNOS) after myocardial infarction.On this basis,we analysed the protected effect on the further survival rate of the engrafted mesenchymal stem cells after giving iNOS selective inhibitor 1400W.Methods:(1) Female SD rats weighing 150-180g were prepared to make acute myocardial infarction model. Acute myocardial infarction (AMI) was created by occluding the left anterior descending artery (LAD); The cells were transplanted 1 h after AMI and the animal models were randomly divided into several groups and detected. (2) BMSCs were isolated from the male SD rats weighing 100-130g, purified, proliferated and then they were detected by flow cytometry and immunohistochemical detection. (3) 1.2×106 BMSCs were injected into the centre zone of the infarct region at 1 h after MI. (4) at 1d、3d、7d、10d and 14d after AMI,the infarct regions were harvested and the expression of iNOS was analysed by western blot.(5) The BMSCs were labeled with Brdu or hoechst33342 previously and after BMSCs transplantation,the infarct regions were harvested and observed by fluorescence microscope or studied by HE staining and Brdu immunohistochemical examinations. (6)A Sry suquence of male rats Y chromatosome was analysed by the real time polymerase chain reaction.The results:1. After the anterior descending artery ligation, heart ischemic area became pale,the pulse was weakened and clear boundary appeared compared with no ischemia area; After isolation and purification, MSCs showed homogenous with CD44+/CD34-.Immunohistochemical detection demonstrated CD44 was positive, CD34 was negative and proved that the experimental cells were BMSCs.2. The dynamic state of the surviving cells engrafted in the infarcted heart:The cells labeled with hoechst33342 in different groups were viable in large numbers in the host hearts and a small quantity of them migrated from the center to the margin of the infarct region. Immunohistochemical staining revealed that Brdu-labelled BMSCs with oval nucleus were localized. The real time polymerase chain reaction demonstrated that the survival rate of engrafted BMSCs in three groups were 7.88%(1 week group)、7.82%(3 weeks group)、8.73%(6 weeks group).The result demonstrated that from one to six weeks there were no progressive decrease of the transplanted cells.3. Western blot detected the expression of iNOS: Different time myocardial infarction changed the intensity of iNOS expression:three days> 1 day> 7 days> 10 days> 14 days (n= 6, p<0.05);4. iNOS selective inhibitor 1400W contributed to further survival rate of the transplantated cells:The cells labeled with hoechst33342 in the test group and control group were both viable in large numbers in the host heart and a small quantity of them migrated from the center to the margin of the infarct region. Compared with control group,the cell density were higher in the test group. Immunohistochemical staining revealed that Brdu-labelled MSCs with oval nucleus were localized in both of the groups. The real time polymerase chain reaction demonstrated that the survival rate of engrafted BMSCs in two groups were 8.75%(the test roup)、6.63% (the control group);The survival rate of the engrafted cells in test group were significantly higher than the control group (n= 7, p<0.05).Conclusion:1. From one to six weeks after cell transplantation, BMSCs could survive stably in the host infarcted heart and there were no progressive decrease. The cells were localized firstly and later they migrated from the center to the margin of the infarct region.2.The expression peak of iNOS is the 1-3 day after myocardial infarction.3. iNOS selective inhibitor 1400w can significantly improved the further survival rate of transplanted cells.更多还原