细胞因子联合纹状体条件培养液定向诱导间充质干细胞体外分化为多巴胺能神经元

Cytokines combined with striatal conditioned medium to induce differentiation of mesenchymal stem cells into dopaminergic neurons in vitro

背景:在众多体外诱导间充质干细胞向多巴胺能神经元的诱导分化研究中,诱导阳性率仍不理想。目的:实验应用碱性成纤维细胞生长因子、表皮生长因子和纹状体条件培养液定向诱导大鼠骨髓间充质干细胞分化为多巴胺能神经元,拟探讨提高诱导阳性率的方法。设计、时间及地点:以细胞为对象的对照观察细胞学实验,于2006-07/2007-12在山东大学齐鲁儿童医院和山东大学第二医院血液实验室完成。材料:健康成年Wistar大鼠用于骨髓间充质干细胞的分离,新生Wistar大鼠用于纹状体条件培养液的制备。方法:采用贴壁法分离纯化健康成年Wistar大鼠骨髓间充质干细胞进行传代培养。取出生24h内新生Wistar大鼠,完整剥离其大脑组织制备纹状体条件培养液。取体外培养的第5代间充质干细胞,用含碱性成纤维细胞生长因子和表皮生长因子的预诱导液进行预诱导,24h后去除预诱导液,换用纹状体条件培养液进行诱导。主要观察指标:倒置显微镜下观察细胞形态变化,并应用细胞免疫化学技术检测细胞内神经元特异烯醇化酶和酪氨酸羟化酶表达。结果:大鼠骨髓间充质干细胞经碱性成纤维细胞生长因子、表皮生长因子和纹状体条件培养液诱导后细胞胞体逐渐回缩成团,形成梭形,部分细胞可见突起伸出,类似神经元。细胞免疫化学检测,诱导后细胞神经元特异烯醇化酶阳性表达率为(72.70±14.81)%,酪氨酸羟化酶阳性表达率为(34.50±15.93)%。结论:应用碱性成纤维细胞生长因子、表皮生长因子联合纹状体条件培养液诱导分化体系,获得了高比例的神经元,其中包括较多的多巴胺能神经元。

BACKGROUND： Induction rate of bone marrow mesenchymal stem cells （BMSCs） to differentiate into dopaminergic neurons in vitro is not satisfactory. OBJECTIVE： To explore the possibility of basic fibroblast growth factor, epidermal growth factor combined with striatal conditioned medium to promote directional differentiation of MSCs into dopaminergic neurons. DESIGN, TIME AND SETTING： Cell control observation. The experiment was performed at the Hematology Laboratory of Qilu Children＇s Hospital and Second Hospital of Shandong University from July 2006 to December 2007. MATERIALS： Healthy adult Wistar rats were selected for MSCs isolation, and newborn Wistar rats were selected for preparation of striatal conditioned medium. METHODS： BMSCs were harvested from healthy adult Wistar rats using attachment method for passage. Wistar rats born in 24 hours were selected, and their brain tissues were removed to prepare striatal conditioned medium. The 5th passage BMSCs were collected and pre-induced in L-DMEM containing basic fibroblast growth factor and epidermal growth factor. Twenty-four hours later, pre-induction liquor was replaced by striatal conditioned medium. MAIN OUTCOME MEASURES： Morphological changes of the stem cells were observed with the inverted phase microscope. The expressions of neuron specific enolase （NSE） and tyrosine hydroxylase （TH） were identified by immunocytochemical technique. RESULTS： The BMSC body contracted into round and spindle shape after induced by basic fibroblast growth factor and epidermal growth factor combined with striatal conditioned medium. Some neuron-like cells with prominence were seen. The immunocytochemical detection showed that the percentage of NSE and TH positive cells were （72.70±14.81）% and （34.50±15.93）%, respectively. CONCLUSION： BMSCs can be induced into dopaminergic neurons by basic fibroblast growth factor and epidermal growth factor combined with striatal conditioned medium in vitro.