大鼠骨髓间充质干细胞谷氨酸诱导后Smac基因的表达及其意义

Expression of Smac gene in bone mesenchymal stem cells cultured by glutamate induced in rats and Its Significance

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 explore the expression of Smac rat BMSCs cultured by glutamate , and whether Smac was involved in the apoptosis of BMSCs. Methods Primary cultured rat BMSCs were incubated. Cells phenotype was identified by flow cytometry; Apoptosis was detected using DAPI fluorescein staining after induce of 30 mmol/L glutamate. ; Apoptotic cells were counted by flow cytometry following PI/Annexin -V staining after induce of 0,10,30 and 50 mmol/L glutamate; RT- PCR was used to detect Smac mRNA levels in BMSCs at 24h after induce of 30mmol/L and 50mmol/L glutamate. Results Cells phenotype identification showed positive antigen expressions with CD29, CD44, CD105 and negative antigen expressions with CD45, CD14, CD34;Some of the cell showed apoptosis changes in glutamate induced group ; In comparison to the control group, the apoptotic rate of glutamate induced group increased （ P 〈 0.05 ） , and the percent of apoptotic cells increased with the increase of glutamate concentration（ 14.24% ,30.72% ,93.31% ） ; It was found by RT- PCR method that BMSCs Smac gene expression increased in comparison to the control group（ P 〈 0.05 ） and the expression of Smac gene increased with the increase of glutamate concentration. Conclusion The expression of Smac rat BMSCs cultured by glutamate are higher, Smac was involved in the apoptosis of BMSCs.