大鼠骨髓间充质干细胞神经分化潜能的体内外研究

The study on the neural differentiation potential of rat Bone Marrow Mesenchymal Stem Cells in vitro and vivo.

目的体内外研究大鼠骨髓间充质于细胞向神经细胞分化的潜能。方法从大鼠骨髓中分离培养得到的BMSCs传至5代后分为:A组,无诱导剂组;B组,神经细胞诱导组;C组,成骨诱导组;D组,脂肪细胞诱导组。流式细胞仪检测细胞的免疫表型。A,B两组行神经细胞相关蛋白检测;C组行茜素红染色,D组行油红O染色。将Hoechst33258标记的第5代BMSCs立体定向植入(n=10只)大鼠顶叶皮质,4周后观测。结果BMSCs表面CD29、CD44、CD90阳性,CD31、CD34、CD45阴性。在体外,A组细胞仅Nestin阳性,B组细胞Nestin、MAP2、NSE、GFAP均呈阳性,且形态类似神经细胞。C组经诱导后可见矿化结节。D组细胞浆内出现脂肪滴。大鼠脑内移植部位可见Hoechst33258染色阳性的BMSCs。并呈Nestin,MAP2、NSE、GFAP阳性。结论体外BMSCs易于分离、培养及扩增。BMSCs具有多向分化潜能,可通过诱导向脂肪、成骨及神经细胞分化。在体外能自发表达神经干细胞相关蛋白,在大鼠脑内可自发向神经细胞分化。

Objective To evaluate the neural differentiation potential of rat bone marrow mesenchymal stem cells in vitro and vivo. Methods Rat BMSCs were isolated from rat bone marrow. Rat BMSCs at passage 5 were seeded and cultured in L-DMEM without inductors （group A） , with neural cell inductors （group B） , with osteogenic inductors （group C） and with inductors of adipocytes （group D）, respectively. The immune phenotype of BMSCs were identified by using flow cytometery. The markers of neural cells such as nestin, MAP2, GFAP, NSE were detected by using immunocytochem- istry in both A and B group. The biological features of the cells were detected by using alizarin red staining in group C and using rathonum O staining in group D, respectively rat BMSCs at passage 5 labled with Hoechst 33258 were stereotaxic transplanted into the parietal cortex of SD rats （ n = 10）, then observed and detected after 4 weeks of transplantation. Results Flow cytometry analysis showed that rat BMSCs expressed CD29 （99.83%）, CD44 （99.77%） and CD90 （99.86%）, but not CD31 （0. 83% ）, CD34（ 1.78% ） nor CIM5（2. 90% ）. Immunofluorescence examination showed that cytoplasm of rat BMSCs was nestin positive in A and B group,but MAP2,NSE,GFAP positive only in B group. Rat BMSCs differentiated into neuron-like cells in B group. Alizarin red staining showed that Rat BMSCs could form mineralized nodules in C group. In D group, lipid droplets were observed in cells by rathonum O staining. In vivo Hoechst 33258 staining positive cells were detected at graft site. Immunohistochemical staining showed that the transplanted cells were nestin,MAP2 ,NSE ,GFAP positive. Conclusions Rat BMSCs could be isolated, pured and expanded easily. Rat BMSCs have mutipotency which can be induced to differentiate into adipocytes, osteoblasts, neural-like cells. Rat BMSCs could express neural stem cells protein spontaneously in vitro,and could differentiate spontaneously into neurogennic cells in vivo.