【作者】 陈湘琦；

【导师】 陈良龙；

【作者基本信息】 福建医科大学 ， 内科学， 2009， 博士

【摘要】 目的:本研究建立大鼠骨髓间充质干细胞(Bone-derived mesenchymal stem cells, BMSCs)体外分离、培养、传代及诱导分化的实验方法;构建携带促分泌信号肽FGF4碱性成纤维细胞生长因子(Basic fibroblast growth factor, BFGF)和增强型绿色荧光蛋白(Enhanced green fluorescent protein, EGFP)的慢病毒载体(Lentivirus vector, LVs);观察FGF4-BFGF&EGFP融合基因重组慢病毒载体修饰BMSCs修复大鼠心肌梗死(Myocardial infarction, MI)的作用及其相关机制,期望为心肌梗死的治疗提供新思路,探索一条新途径。方法:BMSCs体外分离、培养、传代及诱导分化:取SD大鼠股骨、胫骨,用低糖型DMEM细胞培养基反复冲洗骨髓腔,将冲洗液移入培养瓶培养,以贴壁筛选法分离、培养、传代扩增BMSCs,显微镜观察细胞的形态变化和生长分化情况,并收集第三代(P3)细胞进行流氏细胞仪技术鉴定;以携带EGFP的pGC-FU慢病毒载体系统作为基因转导的媒介,采用In-Fusion技术构建携带促分泌信号肽FGF4的BFGF基因重组慢病毒载体,通过聚合酶链反应(Polymerase chain reaction, PCR)、酶切和测序的方式鉴定所构建的载体;在脂质体Lipofectamine 2000作用下转染293T细胞,以实时荧光定量PCR(Real-time quantitative PCR, RT Q-PCR)检测慢病毒滴度,荧光表达、酶联免疫吸附试验(Enzyme linked immunosorbent assay, ELISA)确定最佳感染复数(Multiplicity of infection, MOI)及BFGF蛋白表达分泌水平;采用冠状动脉结扎法建立大鼠心肌梗死模型。120只大鼠随机均分为假手术组(Sham组)、溶剂对照组(Vehic组)、空载慢病毒组(EGFP/BMSCs组)、BFGF重组慢病毒组(BFGF/BMSCs组)和FGF4-BFGF4/BMSCs重组慢病毒组(FGF4-BFGF/BMSCs组),每组24只,按术后3天、7天、14天、28天均分为4个亚组,在心肌梗死模型建立后30-60min,分别以100ul 1×PBS溶液或2×106/只的细胞数散在注入相应实验动物局部心肌组织梗死区。伊红-苏木素(Hematoxylin Eosin, HE)染色观察细胞移植后心肌病理变化;应用激光共聚焦扫描显微镜(Laser scanning confocal microscope, LSCM)观察移植细胞存活及其分化;采用Western-blot检测各实验组大鼠心肌组织梗死区BFGF蛋白表达,免疫组织化学方法(Immunohistochemistry, IHC)检测VIII因子阳性表达;运用Western blot、Real Time Q-PCR分别检测凋亡调控基因BCL-2、BAX mRNA与蛋白表达,原位缺口末端标记法(TdT-mediated dUTP nick end labeling , TUNEL)检测凋亡指数变化;使用IHC检测MMP-9、TIMP-1蛋白表达;麦松(Masson)染色检测细胞移植后28天心肌组织梗死区胶原纤维改变,并行数字高帧频超声心动图检查,观察心功能状况。结果:原代培养的BMSCs 24小时内贴壁并伸展成多角形、梭形,前3天为相对抑制期,其后细胞增殖速度逐渐加快,呈对数形式,7天左右至平台期,细胞覆盖率达90%左右,约在2周内传至第3代时可到106个数量级细胞,能够满足细胞移植数量的需要,应用流式细胞仪检测BMSCs的标志,显示CD29、CD44、CD90阳性,而CD34、CD45阴性,符合BMSCs特征;采用In-Fusion技术构建FGF4-BFGF重组慢病毒,经PCR、酶切及测序鉴定完全正确,转染293T细胞能够正确表达,滴度为1×108TU/ml,慢病毒感染BMSCs最佳MOI值为10,且FGF4-BFGF重组慢病毒感染BMSCs能高效表达分泌BFGF。在心肌梗死模型建立后第3、7、14、28日,与Sham组对比,FGF4-BFGF/BMSCs组的心肌组织病理改变均明显轻于其他各组,且在冠脉结扎后第28日存活移植细胞的数量显著高于其他各组(P﹤0.05),但除少许移植细胞与宿主心肌细胞融合外未见明显分化为心肌样细胞的现象。此外,与Vehic组、EGFP/BMSCs组、BFGF/BMSCs组相比,FGF4-BFGF/BMSCs组心肌组织梗死区BFGF、BCL-2、TIMP-1表达上调,BAX、MMP-9水平下降,微血管密度明显增多,凋亡指数显著减少,差异均有统计学意义(P﹤0.05)。移植后第28天,FGF4-BFGF/BMSCs组心肌组织梗死区胶原纤维减少,心肌细胞增多,左室壁厚度增厚,心功能接近Sham组,较其他各组显著改善(P﹤0.05)。结论:采用贴壁筛选法可分离获得高纯度的BMSCs,该细胞体外增殖快,具有多项分化能力,是移植治疗心梗的良好种子细胞;In-Fusion技术构建的FGF4-BFGF&EGFP融合基因重组慢病毒载体能高效表达及分泌BFGF蛋白;其修饰大鼠BMSCs可促进新生血管生成,抗心肌细胞凋亡,抑制左室重塑,从多途径发挥作用而改善大鼠心肌梗死后心功能。更多还原

【Abstract】 Objective This study was to establish the experimental method for isolating, culturing, proliferating and inducing rat bone marrow mesenchymal stem cells (BMSCs) in vitro, to construct portable FGF4 signal peptide for promoting secretion of basic fibroblast growth factor (BFGF) and to enhance green fluorescent protein (EGFP) of the lentiviral vector (LVs); to observe FGF4-BFGF & EGFP fusion gene recombinant lentiviral vector-modified BMSCs for repairing myocardial infarction (MI) and its related mechanism.Methods The method for isolating, cultivating, proliferating and inducing BMSCs: Bone marrow was taken out from the bilateral femora and tibiae, and cultured with L-DMEM culture solution in culture bottles. MSCs were isolated and purified by the wall-adhering method. The morphology and graph of every generation were observed under the phase-contrast microscope. And the third-generation (P3) cells were collected to assay by the flow cytometry technical appraisal. The pGC-FU lentiviral vector that carries the EGFP was chosen as a medium of gene transduction system. In-Fusion technology was adopted to construct the promoted secretion of FGF4 signal peptide and BFGF gene recombinant lentiviral vector, which was identified by the polymerase chain reaction (PCR), enzyme digestion and sequencing.. The 293T cells were transfected through the liposome Lipofectamine 2000. The lentiviral titer was detected by real-time fluorescence quantitative PCR (RT Q-PCR). The optimum infection plural (MOI) and the secretion of BFGF protein expression level were determined by expression of fluorescence and enzyme linked immunosorbent assay (ELISA). The rat model of myocardial infarction was set up by the coronary artery ligation . The 120 rats were randomly divided into sham operation group (Sham group), solvent control group (Vehic group), no-load lentiviral group (EGFP / BMSCs group), BFGF fusion gene lentivirus group (BFGF / BMSCs) and FGF4 - BFGF4 fusion gene lentivirus group (FGF4-BFGF/BMSCs group). The 24 rats of each group were divided into four sub-groups on the 3rd d, 7th d, 14th d, and 28th d after the operation. The local myocardial infarct area of the corresponding experimental animals was injected with 100ul 1×PBS solution or 2×106 cells after the model of myocardial infarction was set up for 30 - 60min. The Yihong - hematoxylin (Hematoxylin Eosin, HE) staining was used to observe the myocardial pathological changes after transplantation, and the laser scanning confocal microscope (LSCM) was used to observe the transplanted cell survival and differentiation. Western-blot was adopted to detect the infracted myocardial tissue BFGF protein expression of the experimental rats, and immunohistochemistry (IHC) was adopted to detect the positive expression of Factor VIII. Western-blot and Real Time Q-PCR were adopted to detect the apoptosis regulatory genes Bcl - 2, Bax mRNA and protein expression. In situ nick end labeling (TdT-mediated dUTP nick end labeling, TUNEL) was adopted to detect apoptotic index Detect Change. IHC was adopted to detect the protein expression of MMP-9, TIMP-1. Masson staining was adopted to detect the myocardial collagen fiber changes, and parallel digital high frame rate echocardiography was adopted to observe the conditions of cardiac function of the experimental rats after 28 days of the cell transplantation.Results Primary cultured BMSCs adhered to and stretched into polygon or spindle within 24 hours. The first 3 days was the relative inhibition period, and then the cell proliferation accelerated gradually in a pattern of logarithm and reached the period of plateau by Day 7 or so, with a 90% cell coverage. The number of P3 BMSCs can reach 106 2 weeks later, and that can satisfy the required number of cell transplantation. The signs of BMSCs with positive CD29, CD44, CD90 and negative CD34, CD45 were detected by the flow cytometry, and that is consisted with the characteristics of BMSCs. In-Fusion technology was adopted to construct the FGF4-BFGF fusion gene recombinant lentiviral vector. The lentiviral vector was identified entirely correct by PCR, enzyme digestion and sequencing, and the transfected 293T cells were identified to express correctly. The optimum MOI of BMSCs was 10 with the 1×10~8TU/ml lentiviral titer. BMSCs infected by FGF4-BFGF fusion gene recombinant lentiviral vector can express and secrete BFGF protein efficiently. Compared with Sham group, the myocardial tissue pathological changes were significantly lighter in the FGF4-BFGF/BMSCs group than in other groups, on the 3rd d, 7th d, 14th d, 28th d after the myocardial infarction model was set up. The survival number of transplanted cells was significantly larger in this group (P <0.05) than in other groups on the 28th d of the coronary ligation, but there were few cardiomyocyte-like cells visible except some transplanted cells with the host myocardial cell fusion. In addition, BFGF, Bcl-2, TIMP-1 were up-regulated, whereas Bax MMP-9 levels were decreased; the microvessel density was noticeably increased, whereas the apoptotic index was significantly reduced in the myocardial infarct areas of Vehic group, EGFP / BMSCs group, BFGF / BMSCs group, and FGF4-BFGF/BMSCs Group. These changes were of statistical significance (P <0.05). Compared with the other groups, collagen fibers in the myocardial infarct area decreased, myocardial cells increased and the left ventricular wall was thickened in FGF4-BFGF/BMSCs group. The cardiac function was significantly improved on the 28th d after transplantation (P <0.05), close to that of the shame group.Conclusion Highly purified BMSCs can be separated by adherent Screening method. BMSCs proliferate quickly in vitro. Due to their differentiations, BMSCs are good seed cells for treating myocardial infarction through transplantation. In-Fusion technology can be adopted to construct the FGF4-BFGF & EGFP fusion gene recombinant lentiviral vector, which can express and secrete BFGF protein efficiently. The modified BMSCs improve the rat cardiac function after myocardial infarction via angiogenesis promotion, anti-cardiomyocyte apoptosis and ventricular remodeling inhibition.更多还原