【作者】 雷警输；

【导师】 王卫淑；

【作者基本信息】 山西医科大学 ， 老年医学， 2009， 硕士

【摘要】 研究背景:扩张性心肌病是一种重要的心血管病,死亡率很高,有创的机械循环辅助和心脏移植是以往治疗终末心衰的主要手段。细胞移植作为一种新的治疗方法已开始应用于心血管疾病,但目前的研究大多集中在改善心肌梗死区域的心功能和心肌组织的再生上,所使用的移植细胞包括了骨髓细胞、内皮祖细胞、心肌细胞和间充质干细胞等。特别是拥有多向分化潜能和易于扩增培养的间充质干细胞。心肌病发生心力衰竭的主要机制是心肌细胞大量丧失(死亡和凋亡)和间质重塑。骨髓间充质干细胞不仅可以归巢心衰大鼠心肌组织和分化为心肌细胞并改善心功能而且可以调节基质金属蛋白酶-9/基质金属蛋白酶抑制因子-1表达而影响心肌间质纤维重塑。目的:探讨骨髓间充质干细胞移植对阿霉素诱导的心衰大鼠心功能及心肌细胞基质金属蛋白酶-9及其抑制因子-1表达的影响。方法:1、取雌性Wister大鼠(n=38)先随机选取8只作为正常对照组,其余30只腹腔注射阿霉素(2.5mg.kg-1,每周一次,连续6周)建立心衰模型。6周后存活大鼠(n=21)随机分为细胞移植组(n=11)和移植对照组(n=10)。正常对照组大鼠腹腔注射等量的生理盐水。2、体外分离,纯化,增殖骨髓间充质干细胞(MSCs)并用含有12%胎牛血清的DAEM培养基培养,流式细胞仪对体外培养的MSCs行CD-44表型鉴定。取第三代MSCs进行移植,移植前MSCs用5-溴-2′脱氧尿苷(BrdU)进行标记。3、末次阿霉素注射后一周,将5×106个MSCs通过鼠尾静脉注射的方法移植到移植组大鼠。心衰对照组大鼠注射等量培养基,正常组注射等量生理盐水。移植后4周,采用超声测量心功能及心室重塑,心脏切片行病理和免疫组化检查了解移植细胞在受体心脏的存活情况及基质金属蛋白酶-9,基质金属蛋白酶抑制因子-1及cx-43表达情况。结果:体外培养的第三代MSCs流式细胞仪鉴定结果显示:CD44阳性细胞数占99.7%±0.9%,心衰MSCs移植组心肌组织中可以见5-溴-2′脱氧尿苷标记的骨髓间充质干细胞(MSCs)归巢,心衰细胞移植组间质基质金属蛋白酶阳性表达比心衰对照组和正常对照组差异有统计学意义(p<0.05)。骨髓间充质干细胞(MSCs)归巢并表达细胞缝隙连接蛋白-43阳性。左室射血分数细胞移植组与心衰对照组和正常对照组分别为(87.26±1.64) % , (72.42±2.46) % ,和(93.60+1.54)%, ( P < 0. 05)。细胞移植组相对于心衰对照组左室心功能差异有统计学意义(p<0.05)。结论:经静脉移植骨髓间充质干细胞不仅可以归巢心衰大鼠心肌组织改善心功能及而且可以调节基质金属蛋白酶-9/基质金属蛋白酶抑制因子-1表达影响间质纤维重塑。更多还原

【Abstract】 BackgroundHeart failure is one of the most important cardiovascular diseases, with high mortality, and invasive treatment such as mechanical circulatory support and cardiac transplantation is sometimes required for severe heart failure. Cell therapy is an attractive therapeutic strategies are used in clinical practice for cardiovascular diseases,But resecent studies have suggested benefits of stem cells transplantation for the regeneration of cardiac tissue and function improvement of regionally infracted myocardium.As a cell source, various kinds of stem cells such as bone marrow cells, endothelial progenitor cells, mesenchymal stem cells (MSC) and cardiac stem cells have been in transplantation. Especially, bone marrow-derived MSC possess multipotency and can be easily expanded in culture.Cardiomyocyte dropout (necrosis and apoptosis)and interstitial remodeling play a critical role in the progress of CHF.MSCs can not only migrate into the damaged heart anddifferentiate into specific cell types such as cardiomyocytes and improve the heart function but also induce the expression of Matrix metalloproteinase-9 and tissue inhibitors of metalloproteinase-1 on interstitial remodeling. ObjectiveTo investigate the effect of bone marrow mesenchymal stem cells transplantation on heart function and expression of Matrix metalloproteinase-9 and tissue inhibitors of metalloproteinase-1 in doxorubicin induced heart failure of Wister rats .MethodsTo establish a rat model of heart failure, Adr was given intraperitoneally to Wister rats (n=30) weekly at a dose of 2.5mg/kg body weight for 6 weeks.The surviving animal models (n=21) were then randomly divided into the cell transplantation group (n=11) and the transplantation control group (n=10).select normal control group (n=8) injected saline. MSCs were isolated、purified and amplified with Dulbecco’s modified Eagle’s medium (DMEM) containing 12 % fetal bovine serum (FBS) in ex vivo. Cells at the third passage were identified with a flow cytometer and were marked with 5-Bromo-2’-Dexuuridine (BrdU) before transplantation. One week after the last administration of Adr, BrdU-labeled MSCs (5×106 cells) were transplanted into heart failure models of cell transplantation group through tail vein. The transplantation control group was injected by equivalent volume culture medium (DAEM). Normal control group were injected by saline. Four weeks after the transplantation, the heart function and ventricular remodeling were evaluated by echocardiography. Pathological changes were measured and immunochemical analysis was performed .ResultsFlow cytometric analysis showed that the MSCs of third passage cultured in vitro expressed CD44 surface marker (99.7%±0.9%). Four weeks after transplantation, BrdU-labeled transplanted MSCs could be found in the hearts of the recipients. Immunochemical staining revealed that Matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of metalloproteinase-1 (TIMP-1) were significantly difference in transplantation group in contrast with The transplantation control group and normal control group(p<0.05).Immunochemical staining revealed that the engrafted stem cells expressed CX-43.The amount of collagen reduces in MSCs transplantation group. The LVEF of MSCs transplantation group and DMEM transplantation group and normal control group were (87.26±1.64) % , (72.42±2.46) % , and (93.60+1.54)%, respectively ( P < 0. 05) . MSCs significantly improved the heart function ( P < 0. 05)as compared with transplantation control group .ConclusionIntravenously transplanted MSCs can not only migrate into the damaged heart and improve the heart function but also induce the expression of Matrix metalloproteinase-9 and tissue inhibitors of metalloproteinase-1 on interstitial remodeling of rat with doxorubicin induced rats heart failure.更多还原