【作者】 陈晓慧；

【导师】 刘明春； 石娇；

【作者基本信息】 沈阳农业大学 ， 基础兽医学， 2007， 硕士

【摘要】 脑源性神经营养因子(brain-derived neurotrophic factor，BDNF)作为神经生长因子家族成员，与神经系统发育有着密切关系，不仅对中枢神经元的发育、增殖分化、存活起重要作用，而且对损伤后神经元的修复与功能重塑也起重要作用。近年来神经生长因子家族一些成员纷纷被克隆并应用于中枢神经系统退行性疾病的防治。本试验目的是通过RT-PCR技术及基因克隆的方法，构建含大鼠脑源性神经营养因子(BDNF)基因的真核表达载体pEGFP(N1)-BDNF，以重组质粒pEGFP(N1)-BDNF作为外源性基因，与脂质体一起转染到骨髓基质干细胞(BMSCs)中，通过荧光显微镜观察，并结合免疫荧光化学染色方法鉴定基因工程细胞是否稳定表达BDNF蛋白，同时根据免疫细胞化学染色方法鉴定BMSCs细胞能否在其表达的BDNF蛋白诱导下转变为神经细胞或神经胶质细胞。BDNF真核表达载体pEGFP(N1)-BDNF的构建：从2-3日龄大鼠脑组织中抽提总RNA，应用RT-PCR技术扩增BDNF基因片段，将此片段克隆入PMD-18-T载体，酶切反应鉴定。双酶切BDNF-T载体和空质粒pEGFP(N1)，将所获目的片段和线性空载体用T4 DNA连接酶连接，构建真核表达载体pEGFP(N1)-BDNF。并进行酶切反应鉴定及DNA测序。测序结果与GenBank提供的已知序列比较，证明所克隆的BDNF基因从起始密码子ATG到终止密码子TAG全长共750bp，与报道的序列完全相同。pEGFP(N1)-BDNF在BMSCs中的表达：首先进行骨髓基质干细胞(BMSCs)的采集与贴壁法培养，并反复传代，使BMSCs得到扩增和纯化，获得高纯度的BMSCs。然后应用脂质体转染技术将pEGFP(N1)-BDNF转染到BMSCs中，对转染细胞进行荧光显微镜观察及BDNF免疫荧光化学染色鉴定。质粒转染BMSCs 18h后，在倒置荧光显微镜下，pEGFP(N1)-BDNF和pEGFP(N1)空载体组镜下可见细胞发出绿色荧光，GFP呈阳性的BMSCs细胞数约为60％。通过BDNF蛋白的免疫荧光化学方法鉴定成功转染pEGFP(N1)-BDNF组的BMSCs，阳性BMSCs细胞数约为30％，提示BDNF在细胞中表达。转染的细胞继续培养后，通过免疫细胞化学染色结果可以看出，阳性细胞逐渐表达神经元特异性烯醇化酶(NSE)。结果表明通过基因重组的方法将BDNF基因已克隆到质粒pEGFP(N1)上，采用限制性内切酶酶切和DNA序列分析鉴定表明成功构建成真核表达载体pEGFP(N1)-BDNF；采用脂质体介导的基因转染技术将pEGFP(N1)-BDNF转入BMSCs中，通过免疫荧光化学方法检测到转染后的BMSCs中有BDNF蛋白的表达；通过免疫细胞化学方法检测到转染后的BMSCs中表达神经元特异性烯醇化酶(NSE)，说明BMSCs在其自身分泌的BDNF蛋白作用下，逐渐向神经元样细胞分化。更多还原

【Abstract】 Brain-derived neurotrophic factor (BDNF) belongs to nerve growth factors.The relation between BDNF and development of the nerve system is very osculating. BDNF act on neuron’s growth, multiplication, deposit and repairment. A large number of members of nerve growth factor family are cloned and used to cure nerve system disease recently.The objective is that BDNF eukaryotic expression plasmid pEGFP (N1) -BDNF is constructed by RT-PCR and gene cloned. The recombined pEGFP (N1) -BDNF plasmid is transfected into bone marrow stromal cells (BMSCs) with lipofectamine. We check up constructed gene engineering cell by fluo-microscope and immunocytochemitry. And we observe if BMSCs can transform nerve cell or neuroglial cells by its expressed BDNF protein stably.The construction of pEGFP (N1) -BDNF plamid: extracting total RNA from the brain of 2-3 days mouse as templates, the BDNF gene was cloned using RT-PCR and T-Vector cloning methods, the positive clone was identified by the restriction enzymes. After recombinant plasmid BDNF-T and plasmid pEGFP (N1) were digested by two restriction enzymes, the BDNF fragments were linked to the linear pEGFP (N1) by T4 DNA ligase, the eukaryotic expression vector (pEGFP (N1) -BDNF) was constructed. The positive clone was identified by the restriction enzymes and sequenced. Compared the cloned BDNF gene with the GenBank sequence, we demonstrate that both of sequence were from the initiation codon ATG to the termination TAG identically. The cloned mouse BDNF gene is 750 bp.The expression of pEGFP (N1) -BDNF in BMSCs: At first, the isolation and attachment culture of rat bone marrow stromal stem cells, and the expansion and purification of rat bone marrow stromal stem cells to provide higher purification seed cells for experiment, the recombined pEGFP (N1)-BDNF plasmid was transfected into BMSCs with lipofectamine. To observe transfected BMSCs and identify BDNF by immunofluorescence. Eighteen hour after transfected plasmid into BMSCs, we could see green fluorescence in pEGFP (N1) - BDNF groups and pEGFP (N1) empty carrier. In reverse fluomicroscope, GFP positive BMSCs is about 60 percent, transfected BMSCs is green fluorescence by immunofluorescence. Positive BMSCs is about 30 percent, BDNF can express protein in transfected cell. The immunocytochemitry of the results, transfected cell can express NSE.Conclusion is that the eukaryotic expression vector pEGFP (N1) -BDNF are constructed and confirmed with the restriction enzymes and sequencing. The recombined pEGFP (N1) BDNF plasmids are transfected into BMSCs using lipofectamine. We prove that transfected BMSCs can express BDNF protein by immunofluorescence. At the same time, The BMSCs express NSE. It indicate BMSCs can differentiate towards nerve cell by its self secretary BDNF protein.更多还原