大鼠骨髓间充质干细胞的培养鉴定以及向神经干细胞分化的实验研究

Experimental study of cultivation and identification of rat bone marrow mesenchymal stem cells and their differentiation into neural stem cells

摘要:

目的：体外培养并鉴定大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs），研究其生物学特性，初步探索酸性成纤维细胞生长因子（acidic fibroblast growth factor，aFGF）诱导其向神经干细胞（neural stem cells，NSCs）分化的可行性。方法：全骨髓贴壁培养法分离培养大鼠BMSCs，倒置相差显微镜观察其形态特征，MTT法绘制生长曲线，流式细胞仪（flow cytometry，FCM）测定细胞周期并鉴定细胞表面标志物。用80 ng/ml aFGF的无血清DMEM/F12培养液诱导大鼠BMSCs向NSCs分化，镜下观察细胞形态特征，免疫荧光染色方法检测神经巢蛋白（Nestin）、神经元特异性烯醇化酶（neuron specific enolase，NSE）的表达。结果：大鼠BMSCs贴壁生长，呈梭形，多角形。第1、3、5代BMSCs的生长曲线均呈S形，活性无明显差异。细胞周期显示94.34% BMSCs处于G0/G1期，有较强的分裂增殖能力。FCM检测CD29、CD54、CD90表达阳性，CD45表达阴性。诱导6 h后细胞的胞体收缩成椭圆形或球形并伸出细长突起，免疫荧光染色显示Nestin表达阳性，继续诱导发现双极和多极细胞增多，诱导6 d后相互连接成网状，成典型的神经元样细胞，NSE表达阳性。结论：全骨髓贴壁培养法能培养出高纯度的BMSCs，细胞生长稳定、增殖快、可多次传代，具有干细胞特性。经aFGF诱导后具有向NSCs分化的潜能，NSCs能进一步分化为神经元样细胞。

Abstract:

Objective：To culture in vitro and identify rat bone marrow mesenchymal stem cells（BMSCs），to study their biological char－acteristics and to tentatively explore the feasibility of differentiation of BMSCs into neural stem cells（NSCs） induced by acidic fibrob－last growth factor（aFGF）. Methods：Rat BMSCs were isolated and cultured by the whole bone marrow adherent culture method. Mor－phological characteristics of BMSCs were observed under the inverted phase contrast microscope and cell growth curves were drawn by MTT method. Cell cycle and surface markers were detected with flow cytometry（FCM）. Rat BMSCs were induced to differentiate into NSCs by 80 ng/ml aFGF of DMEM/F12 medium. Cell morphological changes were continuously observed after using aFGF. Im－munofluorescence staining was used to detect the expressions of Nestin and neuron specific enolase（NSE）. Results：Rat BMSCs showed adherent growth and was spindle shaped or in polygonal appearance. Growth curves of the 1st，3rd，5th-generation BMSCs were in S shape and their activities were not significantly different. FCM showed that 94.34% BMSCs were in G0/G1 phase of the cell cycle，having strong proliferation ability. FCM detected that CD29，CD54 and CD90 were positively expressed，but CD45 was negative－ly expressed. At 6 h after the induction，cell bodies became to shrink into oval or sphere，and stretched out long and thin protrusions. Immunofluorescence staining showed that Nestin was positively expressed. Bipolar and multipolar cells were constantly increased after continuous induction. At 6 d after the induction，cells interconnected into a mesh and became typical neuron-like cells. NSE was posi－tively expressed. Conclusions：Whole bone marrow adherent culture method can cultivate highly purified rat BMSCs with stem cells’ characteristics：growing stably，proliferating rapidly and be multiple-passaged. By the induction of aFGF，BMSCs have the potentiality of differentiating into NSCs，which can further differentiate into neuron-like cells.