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目的探讨骨髓干细胞(BMSCs)经谷氨酸培养后Smac基因表达情况及其是否参与了谷氨酸诱导的BMSCs凋亡调控。方法将原代培养的大鼠骨髓间充质干细胞,用流式细胞术鉴定细胞表型;加入30mmol/L谷氨酸培养后,采用DAPI荧光染色法,观察凋亡细胞的形态;用PI/Annexin-V双染法流式细胞仪计数各组(0、10、30、50mmol/L谷氨酸组)凋亡细胞的比例,通过逆转录聚合酶链反应(RT-PCR)观察经30mmol/L、50mmol/L谷氨酸作用24h后Smac基因mRNA的表达变化。结果培养的BMSCs经流式细胞术鉴定发现:CD29,CD44和CD105表达阳性,CD45,CD14和CD34表达阴性,具备BMSCs的特征;经谷氨酸培养后,可见BMSCs细胞核皱缩,边聚,双核状;谷氨酸诱导组较多细胞呈凋亡改变,且随着谷氨酸浓度的增加,凋亡细胞的百分率也相应地增加(分别为14.24%、30.72%、93.31%);RT-PCR法发现经30和50mmol/L谷氨酸作用后,BMSCs Smac基因表达较对照组升高(P<0.05),且随着谷氨酸浓度的增加,Smac基因表达也相应增加。结论BMSCs经谷氨酸培养后Smac基因存在高表达,它可能参与了谷氨酸诱导的BMSCs凋亡调控。
Objective To investigate the expression of Smac gene in bone marrow stem cells (BMSCs) cultured with glutamate and whether it is involved in the regulation of glutamate-induced apoptosis of BMSCs. Methods Primary cultured rat bone marrow mesenchymal stem cells were identified by flow cytometry. After adding 30mmol / L glutamic acid, the morphological changes of apoptotic cells were observed by DAPI fluorescent staining. The percentage of apoptotic cells in each group (0, 10, 30, 50mmol / L glutamate group) was counted by Annexin-V double staining flow cytometry. The percentage of apoptotic cells in each group was detected by reverse transcription polymerase chain reaction L, 50mmol / L glutamate 24h after Smac gene mRNA expression changes. Results The results of flow cytometry showed that the expression of CD29, CD44 and CD105 was positive, while the expression of CD45, CD14 and CD34 was negative, which was characteristic of BMSCs. After cultured with glutamate, , And the percentage of apoptotic cells increased with the increase of glutamate concentration (14.24%, 30.72%, 93.31%, respectively) in the glutamate-induced group; The results showed that Smac gene expression in BMSCs was significantly increased compared with control group (P <0.05) after 30 and 50 mmol / L glutamate treatment, and Smac gene expression increased with the increase of glutamate concentration. Conclusion The expression of Smac gene in BMSCs after glutamate culture is highly expressed, which may be involved in the regulation of glutamate-induced apoptosis of BMSCs.