【作者】 董永强；

【导师】 易定华；

【作者基本信息】 中国人民解放军第四军医大学 ， 外科学， 2003， 博士

【摘要】 目前，冠心病已成为我国主要的心血管疾病，尽管对缺血性心肌病的治疗已取得了显著进展，如血管内溶栓、血管成形和冠脉搭桥术的广泛应用，但对于严重弥漫性心肌缺血的心脏，此类治疗并不能使心肌功能改善。因此如何恢复缺血区心肌血供、如何重建严重弥漫性缺血心肌结构以及恢复心肌功能成为近年来缺血性心脏病研究重点之一。我们研究目的是证实脐血中单个核细胞通过适当培养条件可以分化形成具有内皮细胞特征的细胞，并应用新生犬外周血单个核细胞模拟脐血单个核细胞移植进入缺血心肌组织，观察细胞活性及其对左室重构的影响，探讨细胞移植治疗心肌缺血的有效性。 第一部分 脐血单个核细胞培养及培养细胞特性观察 目的：研究能否从脐血中分离内皮前体细胞，并对此类细胞进行培养和鉴定。 方法：采集脐血后2h内梯度离心法分离单个核细胞。应用DMEM培养基，20％胎牛血清。接种于纤维素包被的6孔培养板，接种密度5×10~5／ml。观察细胞生长状况，MTT法绘制细胞生长曲线，以成年静脉血作为对照。贴壁细胞在培养10d时，流式细胞术检查KDR，VE-cadherin，CD34，和CD45表面分子表达，荧光显微镜观察细胞是否摄取乙酰化-LDL，RT-PCR研究培养细胞内是否存在eNOS和FLT-1／VEGF receptor-1 mRNA。 第四军医大学傅士学位论文一结果：梯度离心法获取的外周血单个核细胞活细胞百分率92土4 ％，脐血单个核细胞95土3％，两组无差别，外周血单个核细胞CD34阳性率仅为3 f 0．6％，脐血阳性率22 L 4％，后者明显高于前者。脐血单个核细胞48h后贴壁细胞生长良好，出现梭形细胞。培养早期可见圆形细胞类似于白细胞，但培养 10d后圆形细胞明显减少。培养细胞过程中可见细胞呈现梭形，部分细胞伸展。贴壁细胞可排列成条索状。高倍镜下这些细胞的核较大，部分细胞可见分裂相，贴壁细胞KDR，VE－。adherin，CD34阳性，仅有少量贴壁细胞表达CD45。超过95％的贴壁细胞能够摄取m－ac－LDL。RTPCR提示分离培养的单个核细胞含有eNOS和FLT－l／VEGF recCptor－IInRNAo茗二二淑分蔚生犬牟个麓纫赠说血’L哪彦技厉级撇度迷及真对左室互拇佚功肘——目的：应用新生犬外周血单个核细胞模拟脐血单个核细胞移植进人缺血心肌组织，观察细胞活性和其对左室重构的影响，探讨该细胞移植治疗心肌缺血的有效性。方法：健康杂种犬 16条，随机分为实验组和对照组。新生犬（出生后 sh）麻醉后开胸心内直接抽取血液，梯度离心法分离单个核细胞。用PKH26荧光细胞染料示踪。分离左冠状动脉左前降支，第二对角支远端结扎。lh后单个核细胞缺血区边缘直接注射。细胞总数 10‘，25个点，每点 0刀4ml，对照组直接注射无血清培养基。心脏标本观察缺血区域大小，常规检测缺血区血管和胶原含量变化。在术前和实验性心肌缺血后 Zd和 30d，经胸超声心动图GONOS 5500）检查心功能等指标，采用S4探头，应用组织多普勒技术定量分析局部收缩和舒张功能。结果：在缺血区域内可以发现PHK26－标记的活性细胞，该细胞在缺血组织内形成网状结构。缺血30d后心肌内血管密度移植组显著高于对照组，纤维化面积／左室面积在接受单个核细胞移植的动物较对照组低。心肌缺血后左室形态学改变：左室长轴示左室轮廓呈球形扩大，心尖圆钝，左室扩张增加。二维超声显 －3－ 第口刁军医大学博士学位论文一示术后30d实验组室壁节段性运动减弱较对照组程度轻。对照组A、LAT、LAF显著增大，而CO、MVCF、E及A／E显著降低，术后30d各指标较早期无明显变化。细胞移植组EDV、ESV及EF分别为术前的1．22、1．54及0石2倍，术后30d EDV、ESV及 EF分别为术前的 l．15、l．27及 0．86倍。CO、MVCF、E及A用较对照均有明显差异。细胞移植组室壁应力较对照组明显低，左房张力低。组织多普勒超声显示术后 30d二尖瓣环实验组 VS，、VE，、VE，／V＾，则有明显升高，V＾降低，两组相比实验组明显改善。实验组缺血区（左室前壁）30d组织多普勒各项指标较术后早期及对照组明显改善。人室前壁心肌缺血后其他各壁运动代偿性增强。室间隔则无明显升高。术后30d实验组较术后早期运动速度部分恢复。结论： 1．脐血中分离单个核细胞方法简单，CD34”细胞率较外周血高。 2．在有VEGF，bFGF和IGF－l存在的条件下培养脐血单个核细胞所获得的 大量贴壁细胞具有内皮细胞特性，生长速度和数量较外周血有明显优 势c 3．建立了犬结扎前降支心肌缺血模型，在术后早期及30d观察到严重心肌 缺血后出现。乙室扩张，左室轮廓呈球形，收缩和舒张功能降低，心肌应 力增加，心房出现代偿性收缩增强和张力增加，心肌出现纤维化，证实 心肌缺血后左室重构是一个慢性进行性过程。 4．首先应用新生犬（出生后sh）外周血单个核细胞进行缺血心肌内移植，更多还原

【Abstract】 Coronary artery disease has currently become one of the major life-threatening cardiovascular diseases in China. Although many advances, such as intra-vascular thrombolysis, angioplasty, and coronary bypass grafting, have been achieved on the treatment of ischemic heart disease, the prognosis of severe diffused ischemic heart disease has not been improved. It has gained more and more attention on how to recover the blood supply to the ischemic regions and how to rebuild myocardial structure of severe diffused ischemic hearts. The experiment was designed to prove whether endothelial progenitor cells can be isolated and cultured from cord blood under certain culture conditions, and whether neonatal peripheral mononuclear cells, which have the same components as cord blood, can alleviate left ventricular remodeling after transplanted into ischemic myocardium.Part one Culture and characterizations of cord blood mononuclear cellsObjective: To investigate whether endothelial progenitor cells can be isolated and cultured from cord blood.Methods: Cord bloods were collected and mononuclear cells were isolated within 2 hours. Mononuclear cells with a density of 5×105/ml were cultured in DMEM medium with 20% fetal bovine serum and proper factors as supplement Growth statuses of cells were observed and growth curve was made by MTT method. At 10 days, KDR, VE-cadherin, CD34 and CD45 surface molecular expressions were investigated by flow cytometry and uptake of acetylated-LDL was analyzed by fiuoscent microscopy. RT-PCR was performed to detected eNOS and FLT-1/VEGF receptor-1 mRNA.Results: CD34 positive ratios were 3 ± 0.6% for adult peripheral bloods and 22 ?4% for cord bloods. After 48 hours, spindle-shaped cell sprouted from attached cells. At early phase, round shaped cells, which resembled leucocytes, could be observed, but the number of these cells decreased at late culture phase. At 10d, cultured cells showed spindle or polygon shapes. Some of the cultured cells grew in cord-like shape, and many of these cells were observed in dividing phase of cell cycle. AT cells expressed KDR, VE-cadherin, and CD34 but not CD45. More than 95% of attached cell could uptake Dil-ac-LDL with red fluorescence. After RT-PCR, Amplified eNOS and FLT-1/VEGF receptor-1 products were detected.Part two Study on Cell Viability and Impact on Left Ventricular Remodeling after Neonatal Mononuclear Cells Transplanting into Ischemic MyocardiumObjective: To investigate the validity of cell transplantation in the treatment of acute myocardial ischemia by neonatal dog peripheral mononuclear cells, which have the same composition and characteristic as cord blood counterparts.Methods: 16 mongrel dogs were divided into test and control groups randomly. Neonatal dog peripheral bloods were collected and mononuclear cells were separated by gradient centrifuge. PKH26 fluorescent dye was used to trace the transplantedcells. Left descending arteries were ligated at the distal side of the second diagonal branch. 1 hour later, mononuclear cells were injected into peri-ischemia regions with total number of 108 at 25 points, 0.04ml per point. Non-serum mediums were injected into ischemic myocardium of control group. At day 2 and day 30, trans-thoracic echocardiography (SONOS 5500) with S4 probe were undertaken to calculate heart function index and to investigate left ventricular morphology. Quantitative regional systolic and diastolic functions were analyzed with doppler tissue imaging. The sizes of ischemic region were calculated. Vascular intensity and collagen were studied in heart specimen.Result: PHK26 labeled cells were detected in the mononuclear cells transplanted myocardium. These cells incorporated into vascular network of ischemic tissue. Necropsy examination disclosed that capillary density was significantly greater in test group than in the control group. Moreover, the extent of left ventricular scarring was significantly less in dogs receiving monoclear cells than in controls. Left ventricular morphology changed af更多还原