【摘要】 为了探讨成年动物骨髓基质细胞的体外培养和神经元诱导及其不同培养时程对外源基因的转染率,本实验采用直接和间接两种方法分离、培养成年大鼠和猴的骨髓基质细胞。在体外进行神经元诱导,用免疫细胞化学方法进行鉴定;用携带酪氨酸羟化酶基因的逆转录病毒载体转染体外培养第1～10代大鼠骨髓基质细胞,经免疫细胞化学鉴定并计算转染率。结果显示,成年大鼠和猴骨髓基质细胞以未分化状态至少可持续培养20余代,部分细胞表达干细胞的标志蛋白nestin,经BrdU孵育后呈BrdU抗体免疫阳性;经诱导后细胞具有典型的神经元形态,表达神经元的特异性蛋白NeuN;培养第3代至6代的骨髓基质细胞对外源性基因的转染效率较其他时程明显增高。实验结果说明,骨髓基质细胞可以在体外培养并被诱导成为神经元,它还可携带外源性治疗基因。更多还原

【Abstract】 The aim of the present study is to investigate the culture and neuronal induction of bone marrow stromal cells (MSCs) from adult rat and monkey and to explore their transfective efficiency of exogenous gene in different time-course of the culture. Both the direct and indirect methods were used to separate and to culture the MSCs. The neuronal induction of the cultured MSCs were done in vitro and this was identified by immunocytochemistry. The cultured passage 1-10 MSCs were trans-fected by a retrovirus vector which contained the gene of tyrosine hydroxylase (TH) and the efficiency of transfection was evaluated. The results show that the cultured MSCs were expanded as undifferentiated cells in vitro for at least 20 passages and they could express the nestin, a protein mark for the stem cells. In addition, they were shown to be BrdU immuno-positive after incubation in a medium containing BrdU. After induction, the cells were shown to have a classic pattern with neuronal morphology and to express NeuN, a specific protein for neurons. A relatively higher transfective efficiency of the retrovirus with TH gene was seen in the passage 3-6 among the whole time-course of the culture. Our results indicate that the MSCs can be cultured and induced into neurons in vitro and they can bring an exogenous gene for being used in gene therapy.更多还原