鼠骨髓间充质干细胞对神经元凋亡的影响

Effect of mouse bone marrow mesenchymal stem cells on apoptosis induced by glutamate in cultused rat cortical neuron

目的探讨体外培养鼠骨髓间充质干细胞对谷氨酸诱导大脑皮质神经元凋亡的影响。方法分离、培养、传代Wistar大鼠骨髓间充质干细胞,细胞融合达90%时更换培养基,继续培养24h,收集细胞培养液即为骨髓间充质干细胞条件培养基;培养新生Wistar大鼠大脑皮质神经元,第8d随机分为对照组、谷氨酸损伤组和骨髓间充质干细胞条件培养基处理组。采用台盼蓝染色计算神经元存活率,同时用流式细胞仪和透射电镜技术检测各组神经元凋亡情况。结果谷氨酸(0.8mmol/L)可诱导细胞凋亡,骨髓间充质干细胞条件培养基处理组较谷氨酸损伤组细胞成活率明显升高(P<0.001);流式细胞仪检测见骨髓间充质干细胞条件培养基处理组细胞凋亡率明显降低(P<0.001);而电镜检测发现对照组无明显的凋亡细胞,谷氨酸组有典型凋亡神经元,骨髓间充质干细胞条件培养基处理组凋亡细胞数明显减少。结论骨髓间充质干细胞条件培养基对谷氨酸神经元毒性具有拮抗作用。

Objective To explore the effects of rat bone marrow mesenchymal stem cells on apoptosis induced by glutamate in cultured rat cortical neurons in vitro. Methods When the bone marrow mesenchymal stem cells （BMMSC） grew to 90% confluence, the culture medium was substituted by a new one without serum. After cultured for 24 h, the medium was collected as BMMSC conditioned-medium （B-CM）. Cortical neurons of rats were cultured. On day 8, the cultured neurons were randomly divided into three groups： ①normal control group ; ② glutamate damage group ;③B-CM treatment group. The survival rates of neuron were detected by using trypan blue staining. Flow cytometry and transmission electron microscope were adopted to examine the cellular apoptosis. Results Glutamate （0. 8 mmol/L） could lead to the increasing of apoptosis of rat cortical neurons （P 〈0. 001 ）. The tyrpan blue staining demonstrated that cellular survival rates were （92.6 ± 2.30）%, （50. 2 ± 5.89）%and （82.4 ±3.20）% in normal control group, glutamate group and B-CM group, respectively. There was significant difference between cellular survival rate in glutamate group and that in B-CM group （P 〈0.001 ）. Flow cytometry showed there was a significant difference between cellular apoptotie rate in glutamate group and that in B-CM group （ P 〈 0. 001 ）. No apoptosis was found in the control group and the number of apoptotic cells in B-CM group was much fewer than that in glutamate group under transmission electron microscopy. Conclusion Bone marrow mesenhymal stem cells can reduce neuron apoptosis induced by glutamate.