中文摘要：

目的：胆碱乙酰化转移酶是胆碱能神经系统功能变化的重要标记。观察大鼠骨髓间充质干细胞表达神经胆碱乙酰化转移酶的情况,探讨其定向诱导分化为特异胆碱能神经元的有效条件。方法：实验于2006-09/2007-05在暨南大学医学院血液病研究完成。①实验方法：SD大鼠颈椎脱臼处死,无菌条件下取股骨,从骨髓中分离间充质干细胞,待80%～90%融合后胰酶消化传代。体外培养传3代后,向胆碱乙酰化转移酶阳性神经细胞进行诱导分化。实验Ⅰ组：DMEM/F12＋0.1mmol/L2-巯基乙醇＋1mmol/L维甲酸诱导2d。实验Ⅱ组：体积分数为0.1的胎牛血清＋10μg/L碱性成纤维细胞生长因子＋0.1mmol/L2-巯基乙醇预诱导2d,换液后再加入1mmol/L维甲酸、10μg/L表皮生长因子、10μg/L神经生长因子继续诱导7d。实验Ⅲ组：在Ⅱ组方案的基础上加入1250u/mLHeparin。空白对照组不作任何处理。②实验评估：观察间充质干细胞诱导后的形态变化;诱导9d,采用RT-PCR检测及凝胶电泳分析实验Ⅱ组胆碱乙酰化转移酶mRNA的表达;诱导2,9d,以CY3间接免疫荧光法检测各组胆碱乙酰化转移酶阳性细胞分化情况。结果：①骨髓间充质干细胞诱导后形态学变化：诱导1d细胞形态逐渐由长梭形变为圆形或椭圆形,折光性及立体感增强,部分细胞可见突起开始形成;诱导2d细胞突起伸长,胞间或细胞与间质可形成连接;诱导9d细胞突起变粗,胞浆内分泌颗粒增加并见胞核。②胆碱乙酰化转移酶mRNA的表达：诱导后的细胞高表达胆碱乙酰化转移酶mRNA。③胆碱乙酰化转移酶阳性分化细胞检测：实验Ⅱ组胆碱乙酰化转移酶阳性分化细胞表达率为80%～90%,明显高于实验Ⅰ组的50%～70%,且细胞生长状态较好;实验Ⅲ组细胞脱壁死亡率最高,亦有较高的胆碱乙酰化转移酶阳性分化细胞表达率。结论：①鼠骨髓间充质干细胞能被转化为表达神经胆碱?

英文摘要：

AIM： Choline acetyl transferase （CHAT） is a significant labeling to indicate cholinergic neurological metergasis. This study is designed to observe the expression of CHAT in the mesenchymal stem cells （MSCs） from rat bone marrow, and detect the effective conditions of inducing MSCs to differentiate special cholinergic neuron. METHODS： The experimentation was accomplished in the Institute of Hematology, Medical College of Jinan University from September 2006 to May 2007.（1）Empirical methods： Under aseptic conditions, MSCs were isolated from the femurs of SD rats, which were executed by cervical vertebral dislocation, followed by the treatment with trypsin as 80%-90% MSCs confluence. After 3 passages, group Ⅰ ： MSCs were subcultured by treatment with DMEM/F12＋0.1 mmol/L 2-mercaptoethanol （ME）＋ 1 mmol/L retinoic acid （RA） for 2 days; group Ⅱ ： MSCs were force-induced by treatment with 10% fetal bovine serum＋10 μg/L basic fibroblast growth factor＋0.1 mmol/L 2-ME for 2 days, then the medium was replaced and added with 1 mmol/L RA, 10 μg/L epithermal growth factor and 10 μg/L nerve growth factor to induce for 7 days; group Ⅲ： Combined the treatment of group Ⅱ with 1 250 μ/mL heparin. Nothing treated for control group.（2） Experiment appraisal： The morphologic changes of the MSCs were observed; Nine days later, the expression of CHAT mRNA was detected by RT-PCR gel electrophoresis analysis; For the induction of 2 and 9 days, the differentiation of CHAT in the treated cells was examined by CY3 indirect immunological fluorescent staining. RESULTS： （1）The morphologic changes of induced MSCs： For 1 day, the shape of these induced cells gradually grew into round or ellipse from long fusiform shape. The cell trait of refraction and three-dimensional sense were getting distinguished, partly stretching out ecptoma; For 2 days, cell ecptoma got longer, parts of them connected with each other by ecptoma, others were conjunct to periplasm. Moreover, for 9 days, the e