转染Noggin基因的骨髓基质细胞在体外分化为神经元样细胞的初步研究

The study on bone marrow stromal cells transfected with Noggin gene differentiating into neuron-like cells in vitro

目的构建Noggin基因的表达载体,初步探讨转染Noggin基因的骨髓基质细胞(BMSCs)在体外分化为神经细胞的情况。方法应用RT-PCR技术,从正常人胎脑组织中扩增出Noggin基因,利用基因工程技术构建载体pCS2+Tα1-Noggin。用Percoll分离液分离出大鼠BMSCs,借助脂质体将Noggin基因转入BMSCs。观察细胞形态的改变,并利用免疫组化鉴定分化细胞是否表达神经细胞特异性烯醇化酶(NSE)、微管相关蛋白-2(MAP-2)以及胶质纤维酸性蛋白(GFAP)。结果成功构建Noggin基因的真核表达载体pCS2+Tα1-Noggin。Percoll分离液分离出BMSCs,转染Noggin基因表达载体后,形似神经细胞;免疫组化检测到NSE、MAP-2表达,未检测到GFAP表达。结论转染Noggin基因的BMSCs在体外可以分化为神经元样细胞。

Objective To construct an expression vector of Noggin and explore the differentiation of bone marrow derived stroma cells （BMSCs） transfected with this vector. Methods From the brain tissue of a normal human fetus, Noggin gene was amplified by RT- PCR By gene engineering techniques, the gene was recombinated to the new vector pCS2 ＋ [Tαl]-Noggin. The rats＇ bone marrow stromal stem cells were isolated by density gradient centrifugation with Percoll. The fourth and sixth generation of BMSCs were transfected the expression vector with Noggin by liposome. After culturation, the differentiated cells were detected on the expression of neurone specific enolase （NSE） , mierotubule-associated protein （MAP） -2 but no glial fibrillary acidic protein （GFAP） by immunocytochemistry. Results The full length of the fragment cloned from brain tissue of human fetal was correct and the recombinant pCS2 ＋ [ Tαl] -Noggin plasmid was successful- ly constructed. The BMSCs transfected the vector displayed the neuron morphologies with long and multipolar projections. It was confirmed that the differentiated cells expressed NSE, MAP-2 but no GFAP by immunocytochemistry. Conclusions The bone marrow stromal cells transfeeted with Noggin gene differentiate into neuron-like cells in vitro.